The interaction between the iron-responsive element binding protein and its cognate RNA is highly dependent upon both RNA sequence and structure.

PubMed

Jaffrey, S R; Haile, D J; Klausner, R D; Harford, J B

1993-09-25

To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

PubMed Central

Patton, J R; Habets, W; van Venrooij, W J; Pederson, T

1989-01-01

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins. Images PMID:2529425

Molecular mechanisms for the regulation of histone mRNA stem-loop–binding protein by phosphorylation

PubMed Central

Zhang, Jun; Tan, Dazhi; DeRose, Eugene F.; Perera, Lalith; Dominski, Zbigniew; Marzluff, William F.; Tong, Liang; Hall, Traci M. Tanaka

2014-01-01

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop–binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA. PMID:25002523

Degenerate RNA packaging signals in the genome of Satellite Tobacco Necrosis Virus: implications for the assembly of a T=1 capsid.

PubMed

Bunka, David H J; Lane, Stephen W; Lane, Claire L; Dykeman, Eric C; Ford, Robert J; Barker, Amy M; Twarock, Reidun; Phillips, Simon E V; Stockley, Peter G

2011-10-14

Using a recombinant, T=1 Satellite Tobacco Necrosis Virus (STNV)-like particle expressed in Escherichia coli, we have established conditions for in vitro disassembly and reassembly of the viral capsid. In vivo assembly is dependent on the presence of the coat protein (CP) N-terminal region, and in vitro assembly requires RNA. Using immobilised CP monomers under reassembly conditions with "free" CP subunits, we have prepared a range of partially assembled CP species for RNA aptamer selection. SELEX directed against the RNA-binding face of the STNV CP resulted in the isolation of several clones, one of which (B3) matches the STNV-1 genome in 16 out of 25 nucleotide positions, including across a statistically significant 10/10 stretch. This 10-base region folds into a stem-loop displaying the motif ACAA and has been shown to bind to STNV CP. Analysis of the other aptamer sequences reveals that the majority can be folded into stem-loops displaying versions of this motif. Using a sequence and secondary structure search motif to analyse the genomic sequence of STNV-1, we identified 30 stem-loops displaying the sequence motif AxxA. The implication is that there are many stem-loops in the genome carrying essential recognition features for binding STNV CP. Secondary structure predictions of the genomic RNA using Mfold showed that only 8 out of 30 of these stem-loops would be formed in the lowest-energy structure. These results are consistent with an assembly mechanism based on kinetically driven folding of the RNA. Copyright © 2011 Elsevier Ltd. All rights reserved.

Loss of G-A base pairs is insufficient for achieving a large opening of U4 snRNA K-turn motif.

PubMed

Cojocaru, Vlad; Klement, Reinhard; Jovin, Thomas M

2005-01-01

Upon binding to the 15.5K protein, two tandem-sheared G-A base pairs are formed in the internal loop of the kink-turn motif of U4 snRNA (Kt-U4). We have reported that the folding of Kt-U4 is assisted by protein binding. Unstable interactions that contribute to a large opening of the free RNA ('k-e motion') were identified using locally enhanced sampling molecular dynamics simulations, results that agree with experiments. A detailed analysis of the simulations reveals that the k-e motion in Kt-U4 is triggered both by loss of G-A base pairs in the internal loop and backbone flexibility in the stems. Essential dynamics show that the loss of G-A base pairs is correlated along the first mode but anti-correlated along the third mode with the k-e motion. Moreover, when enhanced sampling was confined to the internal loop, the RNA adopted an alternative conformation characterized by a sharper kink, opening of G-A base pairs and modified stacking interactions. Thus, loss of G-A base pairs is insufficient for achieving a large opening of the free RNA. These findings, supported by previously published RNA structure probing experiments, suggest that G-A base pair formation occurs upon protein binding, thereby stabilizing a selective orientation of the stems.

Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite

PubMed Central

Clingman, Carina C; Deveau, Laura M; Hay, Samantha A; Genga, Ryan M; Shandilya, Shivender MD; Massi, Francesca; Ryder, Sean P

2014-01-01

Gene expression and metabolism are coupled at numerous levels. Cells must sense and respond to nutrients in their environment, and specialized cells must synthesize metabolic products required for their function. Pluripotent stem cells have the ability to differentiate into a wide variety of specialized cells. How metabolic state contributes to stem cell differentiation is not understood. In this study, we show that RNA-binding by the stem cell translation regulator Musashi-1 (MSI1) is allosterically inhibited by 18–22 carbon ω-9 monounsaturated fatty acids. The fatty acid binds to the N-terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. We identify stearoyl-CoA desaturase-1 as a MSI1 target, revealing a feedback loop between ω-9 fatty acid biosynthesis and MSI1 activity. We propose that other RRM proteins could act as metabolite sensors to couple gene expression changes to physiological state. DOI: http://dx.doi.org/10.7554/eLife.02848.001 PMID:24935936

Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR

PubMed Central

Bonner, Eric R.; D’Elia, John N.; Billips, Benjamin K.; Switzer, Robert L.

2001-01-01

The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5′-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4–5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure. PMID:11726695

Chimeric RNase H–Competent Oligonucleotides Directed to the HIV-1 Rev Response Element

PubMed Central

Prater, Chrissy E.; Saleh, Anthony D.; Wear, Maggie P.; Miller, Paul S.

2007-01-01

Chimeric oligo-2′-O-methylribonucleotides containing centrally located patches of contiguous 2′-deoxyribonucleotides and terminating in a nuclease resistant 3′-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3′-side of HIV Rev response element (RRE) stem-loop IIB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop II RNA (KD approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop II RNA and have half lives exceeding 24 h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors. PMID:17566743

Robust Translation of the Nucleoid Protein Fis Requires a Remote Upstream AU Element and Is Enhanced by RNA Secondary Structure

PubMed Central

Nafissi, Maryam; Chau, Jeannette; Xu, Jimin

2012-01-01

Synthesis of the Fis nucleoid protein rapidly increases in response to nutrient upshifts, and Fis is one of the most abundant DNA binding proteins in Escherichia coli under nutrient-rich growth conditions. Previous work has shown that control of Fis synthesis occurs at transcription initiation of the dusB-fis operon. We show here that while translation of the dihydrouridine synthase gene dusB is low, unusual mechanisms operate to enable robust translation of fis. At least two RNA sequence elements located within the dusB coding region are responsible for high fis translation. The most important is an AU element centered 35 nucleotides (nt) upstream of the fis AUG, which may function as a binding site for ribosomal protein S1. In addition, a 44-nt segment located upstream of the AU element and predicted to form a stem-loop secondary structure plays a prominent role in enhancing fis translation. On the other hand, mutations close to the AUG, including over a potential Shine-Dalgarno sequence, have little effect on Fis protein levels. The AU element and stem-loop regions are phylogenetically conserved within dusB-fis operons of representative enteric bacteria. PMID:22389479

Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA.

PubMed Central

Honda, M; Brown, E A; Lemon, S M

1996-01-01

The initiation of translation on the positive-sense RNA genome of hepatitis C virus (HCV) is directed by an internal ribosomal entry site (IRES) that occupies most of the 341-nt 5' nontranslated RNA (5'NTR). Previous studies indicate that this IRES differs from picornaviral IRESs in that its activity is dependent upon RNA sequence downstream of the initiator AUG. Here, we demonstrate that the initiator AUG of HCV is located within a stem-loop (stem-loop IV) involving nt -12 to +12 (with reference to the AUG). This structure is conserved among HCV strains, and is present in the 5'NTR of the phylogenetically distant GB virus B. Mutant, nearly genome-length RNAs containing nucleotide substitutions predicted to enhance the stability of stem-loop IV were generally deficient in cap-independent translation both in vitro and in vivo. Additional mutations that destabilize the stem-loop restored translation to normal. Thus, the stability of the stem-loop is strongly but inversely correlated with the efficiency of internal initiation of translation. In contrast, mutations that stabilize this stem-loop had comparatively little effect on translation of 5' truncated RNAs by scanning ribosomes, suggesting that internal initiation of translation follows binding of the 40S ribosome directly at the site of stem-loop IV. Because stem-loop IV is not required for internal entry of ribosomes but is able to regulate this process, we speculate that it may be stabilized by interactions with a viral protein, providing a mechanism for feedback regulation of translation, which may be important for viral persistence. PMID:8849773

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine

PubMed Central

Zhang, Yan; Wang, Lei; Schultz, Peter G.; Wilson, Ian A.

2005-01-01

The Methanococcus jannaschii tRNATyr/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-l-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 Å, respectively, for comparison with the published structure of TyrRS complexed with tRNATyr and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257–263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through π-stacking and hydrogen bonding interactions. Loop 133–143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNATyr. Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133–143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over l-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids. PMID:15840835

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8

PubMed Central

Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R.; Kolb, Gaëlle; Wiley, Michael R.; Jozwick, Lucas; Kuhn, Jens H.; Palacios, Gustavo; Radoshitzky, Sheli R.; J. Le Grice, Stuart F.; Johnson, Reed F.

2016-01-01

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA–RNA and RNA–protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3′ stem-loop (nucleotides 1868–1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication. PMID:27651462

Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans.

PubMed

Matsunaga, James; Schlax, Paula J; Haake, David A

2013-11-01

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.

The C-terminal domain of Tetrahymena thermophila telomerase holoenzyme protein p65 induces multiple structural changes in telomerase RNA

PubMed Central

Akiyama, Benjamin M.; Loper, John; Najarro, Kevin; Stone, Michael D.

2012-01-01

The unique cellular activity of the telomerase reverse transcriptase ribonucleoprotein (RNP) requires proper assembly of protein and RNA components into a functional complex. In the ciliate model organism Tetrahymena thermophila, the La-domain protein p65 is required for in vivo assembly of telomerase. Single-molecule and biochemical studies have shown that p65 promotes efficient RNA assembly with the telomerase reverse transcriptase (TERT) protein, in part by inducing a bend in the conserved stem IV region of telomerase RNA (TER). The domain architecture of p65 consists of an N-terminal domain, a La-RRM motif, and a C-terminal domain (CTD). Using single-molecule Förster resonance energy transfer (smFRET), we demonstrate the p65CTD is necessary for the RNA remodeling activity of the protein and is sufficient to induce a substantial conformational change in stem IV of TER. Moreover, nuclease protection assays directly map the site of p65CTD interaction to stem IV and reveal that, in addition to bending stem IV, p65 binding reorganizes nucleotides that comprise the low-affinity TERT binding site within stem–loop IV. PMID:22315458

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walden, William E.; Selezneva, Anna I.; Dupuy, Jérôme

Iron regulatory protein 1 (IRP1) binds iron-responsive elements (IREs) in messenger RNAs (mRNAs), to repress translation or degradation, or binds an iron-sulfur cluster, to become a cytosolic aconitase enzyme. The 2.8 angstrom resolution crystal structure of the IRP1:ferritin H IRE complex shows an open protein conformation compared with that of cytosolic aconitase. The extended, L-shaped IRP1 molecule embraces the IRE stem-loop through interactions at two sites separated by {approx}30 angstroms, each involving about a dozen protein:RNA bonds. Extensive conformational changes related to binding the IRE or an iron-sulfur cluster explain the alternate functions of IRP1 as an mRNA regulator ormore » enzyme.« less

In Vitro Assembly of Human H/ACA Small Nucleolar RNPs Reveals Unique Features of U17 and Telomerase RNAs

PubMed Central

Dragon, François; Pogačić, Vanda; Filipowicz, Witold

2000-01-01

The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3′-terminal stem-loop followed by box ACA (U17/3′st) was sufficient to form an RNP, and U17/3′st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3′ moiety of human telomerase RNA (hTR), and its 3′-terminal stem-loop (hTR/3′st), also could form an RNP by binding H/ACA proteins. Hence, the 3′-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins. PMID:10757788

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8.

PubMed

Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R; Kolb, Gaëlle; Wiley, Michael R; Jozwick, Lucas; Kuhn, Jens H; Palacios, Gustavo; Radoshitzky, Sheli R; J Le Grice, Stuart F; Johnson, Reed F

2016-11-16

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2'-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3' stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation.

PubMed Central

McGregor, A; Rao, M V; Duckworth, G; Stockley, P G; Connolly, B A

1996-01-01

The preparation of a 4-thiouridine phosphoramidite suitable for RNA synthesis and its subsequent incorporation into oligoribonucleotides is described. The thiol group is protected with a 2-cyanoethyl group and the 2'-OH with a 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl function. Thiouridine-containing oligoribonucleotides were used as 350 nm UV crosslinking probes for the photoaffinity labelling of RNA binding proteins. Specific crosslinking was demonstrated between the Rev protein of HIV-1 (as a glutathione S-transferase fusion protein) and its RNA target, the Rev-responsive element. It was not possible to generate crosslinks between the RNA bacteriophage MS2 coat protein and the initiator stem-loop of the replicase gene, to which it binds. These results are consistent with the structural data available on both systems. PMID:8774897

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove.

PubMed

Filbin, Megan E; Kieft, Jeffrey S

2011-07-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem-loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem-loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.

Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations

PubMed Central

Hasenhindl, Christoph; Lai, Balder; Delgado, Javier; Traxlmayr, Michael W.; Stadlmayr, Gerhard; Rüker, Florian; Serrano, Luis; Oostenbrink, Chris; Obinger, Christian

2014-01-01

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules. PMID:24792385

Creating stable stem regions for loop elongation in Fcabs - insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations.

PubMed

Hasenhindl, Christoph; Lai, Balder; Delgado, Javier; Traxlmayr, Michael W; Stadlmayr, Gerhard; Rüker, Florian; Serrano, Luis; Oostenbrink, Chris; Obinger, Christian

2014-09-01

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules. Copyright © 2014. Published by Elsevier B.V.

Positive feedback between RNA-binding protein HuD and transcription factor SATB1 promotes neurogenesis.

PubMed

Wang, Feifei; Tidei, Joseph J; Polich, Eric D; Gao, Yu; Zhao, Huashan; Perrone-Bizzozero, Nora I; Guo, Weixiang; Zhao, Xinyu

2015-09-08

The mammalian embryonic lethal abnormal vision (ELAV)-like protein HuD is a neuronal RNA-binding protein implicated in neuronal development, plasticity, and diseases. Although HuD has long been associated with neuronal development, the functions of HuD in neural stem cell differentiation and the underlying mechanisms have gone largely unexplored. Here we show that HuD promotes neuronal differentiation of neural stem/progenitor cells (NSCs) in the adult subventricular zone by stabilizing the mRNA of special adenine-thymine (AT)-rich DNA-binding protein 1 (SATB1), a critical transcriptional regulator in neurodevelopment. We find that SATB1 deficiency impairs the neuronal differentiation of NSCs, whereas SATB1 overexpression rescues the neuronal differentiation phenotypes resulting from HuD deficiency. Interestingly, we also discover that SATB1 is a transcriptional activator of HuD during NSC neuronal differentiation. In addition, we demonstrate that NeuroD1, a neuronal master regulator, is a direct downstream target of SATB1. Therefore, HuD and SATB1 form a positive regulatory loop that enhances NeuroD1 transcription and subsequent neuronal differentiation. Our results here reveal a novel positive feedback network between an RNA-binding protein and a transcription factor that plays critical regulatory roles in neurogenesis.

Specific binding of a Pop6/Pop7 heterodimer to the P3 stem of the yeast RNase MRP and RNase P RNAs.

PubMed

Perederina, Anna; Esakova, Olga; Koc, Hasan; Schmitt, Mark E; Krasilnikov, Andrey S

2007-10-01

Pop6 and Pop7 are protein subunits of Saccharomyces cerevisiae RNase MRP and RNase P. Here we show that bacterially expressed Pop6 and Pop7 form a soluble heterodimer that binds the RNA components of both RNase MRP and RNase P. Footprint analysis of the interaction between the Pop6/7 heterodimer and the RNase MRP RNA, combined with gel mobility assays, demonstrates that the Pop6/7 complex binds to a conserved region of the P3 domain. Binding of these proteins to the MRP RNA leads to local rearrangement in the structure of the P3 loop and suggests that direct interaction of the Pop6/7 complex with the P3 domain of the RNA components of RNases MRP and P may mediate binding of other protein components. These results suggest a role for a key element in the RNase MRP and RNase P RNAs in protein binding, and demonstrate the feasibility of directly studying RNA-protein interactions in the eukaryotic RNases MRP and P complexes.

The 5′-tail of antisense RNAII of pMV158 plays a critical role in binding to the target mRNA and in translation inhibition of repB

PubMed Central

López-Aguilar, Celeste; Romero-López, Cristina; Espinosa, Manuel; Berzal-Herranz, Alfredo; del Solar, Gloria

2015-01-01

Rolling-circle replication of streptococcal plasmid pMV158 is controlled by the concerted action of two trans-acting elements, namely transcriptional repressor CopG and antisense RNAII, which inhibit expression of the repB gene encoding the replication initiator protein. The pMV158-encoded antisense RNAII exerts its activity of replication control by inhibiting translation of the essential repB gene. RNAII is the smallest and simplest among the characterized antisense RNAs involved in control of plasmid replication. Structure analysis of RNAII revealed that it folds into an 8-bp-long stem containing a 1-nt bulge and closed by a 6-nt apical loop. This hairpin is flanked by a 17-nt-long single-stranded 5′-tail and an 8-nt-long 3′-terminal U-rich stretch. Here, the 3′ and 5′ regions of the 5′-tail of RNAII are shown to play a critical role in the binding to the target mRNA and in the inhibition of repB translation, respectively. In contrast, the apical loop of the single hairpin of RNAII plays a rather secondary role and the upper stem region hardly contributes to the binding or inhibition processes. The entire 5′-tail is required for efficient inhibition of repB translation, though only the 8-nt-long region adjacent to the hairpin seems to be essential for rapid binding to the mRNA. These results show that a “kissing” interaction involving base-pairing between complementary hairpin loops in RNAII and mRNA is not critical for efficient RNA/RNA binding or repB translation inhibition. A singular binding mechanism is envisaged whereby initial pairing between complementary single-stranded regions in the antisense and sense RNAs progresses upwards into the corresponding hairpin stems to form the intermolecular duplex. PMID:26175752

Molecular determinants of Cytochrome C oxidase IV mRNA axonal trafficking

PubMed Central

Kar, Amar N.; Vargas, Jose Norberto S.; Chen, Cai-Yun; Kowalak, Jeffrey A; Gioio, Anthony E.; Kaplan, Barry B.

2017-01-01

In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3′ untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA. PMID:28161363

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

PubMed

Cho, Sang-Yun; Cho, Won Kyong; Sohn, Seong-Han; Kim, Kook-Hyung

2012-01-06

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP. Copyright © 2011 Elsevier Inc. All rights reserved.

Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops.

PubMed

Kolchinsky, P; Kiprilov, E; Bartley, P; Rubinstein, R; Sodroski, J

2001-04-01

The gp120 envelope glycoprotein of primary human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and the CCR5 chemokine receptor on the target cell. Previously, we adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for CD4-independent replication were limited to the V2 loop-V1/V2 stem. Here we show that elimination of a single glycosylation site at asparagine 197 in the V1/V2 stem is sufficient for CD4-independent gp120 binding to CCR5 and for HIV-1 entry into CD4-negative cells expressing CCR5. Deletion of the V1/V2 loops also allowed CD4-independent viral entry and gp120 binding to CCR5. The binding of the wild-type ADA gp120 to CCR5 was less dependent upon CD4 at 4 degrees C than at 37 degrees C. In the absence of the V1/V2 loops, neither removal of the N-linked carbohydrate at asparagine 197 nor lowering of the temperature increased the CD4-independent phenotypes. A CCR5-binding conformation of gp120, achieved by CD4 interaction or by modification of temperature, glycosylation, or variable loops, was preferentially recognized by the monoclonal antibody 48d. These results suggest that the CCR5-binding region of gp120 is occluded by the V1/V2 variable loops, the position of which can be modulated by temperature, CD4 binding, or an N-linked glycan in the V1/V2 stem.

Characterization of a Novel Association between Two Trypanosome-Specific Proteins and 5S rRNA

PubMed Central

Ciganda, Martin; Williams, Noreen

2012-01-01

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are essential and are involved in ribosome biogenesis. Here, we show that these proteins interact in vitro with the 5S rRNA with nearly identical binding characteristics in the absence of other cellular factors. The T. brucei 5S rRNA has a complex secondary structure and presents four accessible loops (A to D) for interactions with RNA-binding proteins. In other eukaryotes, loop C is bound by the L5 ribosomal protein and loop A mainly by TFIIIA. The binding of P34 and P37 to T. brucei 5S rRNA involves the LoopA region of the RNA, but these proteins also protect the L5 binding site located on LoopC. PMID:22253864

Structural basis for recognition of human 7SK long noncoding RNA by the La-related protein Larp7.

PubMed

Eichhorn, Catherine D; Yang, Yuan; Repeta, Lucas; Feigon, Juli

2018-06-26

The La and the La-related protein (LARP) superfamily is a diverse class of RNA binding proteins involved in RNA processing, folding, and function. Larp7 binds to the abundant long noncoding 7SK RNA and is required for 7SK ribonucleoprotein (RNP) assembly and function. The 7SK RNP sequesters a pool of the positive transcription elongation factor b (P-TEFb) in an inactive state; on release, P-TEFb phosphorylates RNA Polymerase II to stimulate transcription elongation. Despite its essential role in transcription, limited structural information is available for the 7SK RNP, particularly for protein-RNA interactions. Larp7 contains an N-terminal La module that binds UUU-3'OH and a C-terminal atypical RNA recognition motif (xRRM) required for specific binding to 7SK and P-TEFb assembly. Deletion of the xRRM is linked to gastric cancer in humans. We report the 2.2-Å X-ray crystal structure of the human La-related protein group 7 (hLarp7) xRRM bound to the 7SK stem-loop 4, revealing a unique binding interface. Contributions of observed interactions to binding affinity were investigated by mutagenesis and isothermal titration calorimetry. NMR 13 C spin relaxation data and comparison of free xRRM, RNA, and xRRM-RNA structures show that the xRRM is preordered to bind a flexible loop 4. Combining structures of the hLarp7 La module and the xRRM-7SK complex presented here, we propose a structural model for Larp7 binding to the 7SK 3' end and mechanism for 7SK RNP assembly. This work provides insight into how this domain contributes to 7SK recognition and assembly of the core 7SK RNP.

Interaction between the cellular protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis.

PubMed

Davis, William G; Blackwell, Jerry L; Shi, Pei-Yong; Brinton, Margo A

2007-09-01

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao

2010-07-20

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalentmore » intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.« less

RNA–protein binding interface in the telomerase ribonucleoprotein

PubMed Central

Bley, Christopher J.; Qi, Xiaodong; Rand, Dustin P.; Borges, Chad R.; Nelson, Randall W.; Chen, Julian J.-L.

2011-01-01

Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR–TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA–protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain. PMID:22123986

Terminal structures of West Nile virus genomic RNA and their interactions with viral NS5 protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong Hongping; Zhang Bo; Shi Peiyong

2008-11-10

Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5'-terminal 190 nucleotides and the 3'-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5'CS/3'CSI and 5'UAR/3'UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3'-terminal nucleotides of the genome (position - 8 to - 16) to be single-stranded. Viral NS5more » binds specifically to the 5'-terminal stem-loop (SL1) of the genomic RNA. The 5'SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5'SL1 RNA interaction facilitate NS5 binding to the 3' end of the genome for the initiation of viral minus-strand RNA synthesis.« less

RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem-loop structure, 5BSL3.2, and its negative strand.

PubMed

Kanamori, Hiroshi; Yuhashi, Kazuhito; Ohnishi, Shin; Koike, Kazuhiko; Kodama, Tatsuhiko

2010-05-01

The hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme involved in viral replication. Interaction between NS5B RdRp and the viral RNA sequence is likely to be an important step in viral RNA replication. The C-terminal half of the NS5B-coding sequence, which contains the important cis-acting replication element, has been identified as an NS5B-binding sequence. In the present study, we confirm the specific binding of NS5B to one of the RNA stem-loop structures in the region, 5BSL3.2. In addition, we show that NS5B binds to the complementary strand of 5BSL3.2 (5BSL3.2N). The bulge structure of 5BSL3.2N was shown to be indispensable for tight binding to NS5B. In vitro RdRp activity was inhibited by 5BSL3.2N, indicating the importance of the RNA element in the polymerization by RdRp. These results suggest the involvement of the RNA stem-loop structure of the negative strand in the replication process.

Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus type 1 Tat protein.

PubMed Central

Sumner-Smith, M; Roy, S; Barnett, R; Reid, L S; Kuperman, R; Delling, U; Sonenberg, N

1991-01-01

The human immunodeficiency virus type 1 Tat protein binds to an RNA stem-loop structure called TAR which is present at the 5' end of all human immunodeficiency virus type 1 transcripts. This binding is centered on a bulge within the stem of TAR and is an essential step in the trans-activation process which results in a dramatic increase in viral gene expression. By analysis of a series of TAR derivatives produced by transcription or direct chemical synthesis, we determined the structural and chemical requirements for Tat binding. Tat binds well to structures which have a bulge of two to at least five unpaired bases bounded on both sides by a double-stranded RNA stem. This apparent flexibility in bulge size is in contrast to an absolute requirement for an unpaired uridine (U) in the 5'-most position of the bulge (+23). Substitution of the U with either natural bases or chemical analogs demonstrated that the imido group at the N-3 position and, possibly, the carbonyl group at the C-4 position of U are critical for Tat binding. Cytosine (C), which differs from U at only these positions, is not an acceptable substitute. Furthermore, methylation at N-3 abolishes binding. While methylation of U at the C-5 position has little effect on binding, fluorination reduces it, possibly because of its effects on relative tautomer stability at the N-3 and C-4 positions. Thus, we have identified key moieties in the U residue that are of importance for the binding of Tat to TAR RNA. We hypothesize that the invariant U is involved in hydrogen bond interactions with either another part of TAR or the TAR-binding domain in Tat. Images PMID:1895380

Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC

PubMed Central

Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; Boudier, Christian; De Rocquigny, Hughes; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2011-01-01

An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5′ thymine interacts with residues of the N-terminal zinc knuckle and the 3′ guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA–protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids. PMID:21227929

Alternative Mode of E-Site tRNA Binding in the Presence of a Downstream mRNA Stem Loop at the Entrance Channel.

PubMed

Zhang, Yan; Hong, Samuel; Ruangprasert, Ajchareeya; Skiniotis, Georgios; Dunham, Christine M

2018-03-06

Structured mRNAs positioned downstream of the ribosomal decoding center alter gene expression by slowing protein synthesis. Here, we solved the cryo-EM structure of the bacterial ribosome bound to an mRNA containing a 3' stem loop that regulates translation. Unexpectedly, the E-site tRNA adopts two distinct orientations. In the first structure, normal interactions with the 50S and 30S E site are observed. However, in the second structure, although the E-site tRNA makes normal interactions with the 50S E site, its anticodon stem loop moves ∼54 Å away from the 30S E site to interact with the 30S head domain and 50S uL5. This position of the E-site tRNA causes the uL1 stalk to adopt a more open conformation that likely represents an intermediate state during E-site tRNA dissociation. These results suggest that structured mRNAs at the entrance channel restrict 30S subunit movement required during translation to slow E-site tRNA dissociation. Copyright © 2018 Elsevier Ltd. All rights reserved.

In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA.

PubMed

Osman, Toba A M; Coutts, Robert H A; Buck, Kenneth W

2006-11-01

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.

In Vitro Synthesis of Minus-Strand RNA by an Isolated Cereal Yellow Dwarf Virus RNA-Dependent RNA Polymerase Requires VPg and a Stem-Loop Structure at the 3′ End of the Virus RNA▿

PubMed Central

Osman, Toba A. M.; Coutts, Robert H. A.; Buck, Kenneth W.

2006-01-01

Cereal yellow dwarf virus (CYDV) RNA has a 5′-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3′-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3′ terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3′ end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3′-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg. PMID:16928757

The Murine Norovirus Core Subgenomic RNA Promoter Consists of a Stable Stem-Loop That Can Direct Accurate Initiation of RNA Synthesis

PubMed Central

Yunus, Muhammad Amir; Lin, Xiaoyan; Bailey, Dalan; Karakasiliotis, Ioannis; Chaudhry, Yasmin; Vashist, Surender; Zhang, Guo; Thorne, Lucy; Kao, C. Cheng

2014-01-01

ABSTRACT All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3′ of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter. IMPORTANCE Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase. PMID:25392209

The murine norovirus core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis.

PubMed

Yunus, Muhammad Amir; Lin, Xiaoyan; Bailey, Dalan; Karakasiliotis, Ioannis; Chaudhry, Yasmin; Vashist, Surender; Zhang, Guo; Thorne, Lucy; Kao, C Cheng; Goodfellow, Ian

2015-01-15

All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3' of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter. Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Distinct families of cis-acting RNA replication elements epsilon from hepatitis B viruses

PubMed Central

Chen, Augustine; Brown, Chris

2012-01-01

The hepadnavirus encapsidation signal, epsilon (ε), is an RNA structure located at the 5′ end of the viral pregenomic RNA. It is essential for viral replication and functions in polymerase protein binding and priming. This structure could also have potential regulatory roles in controlling the expression of viral replicative proteins. In addition to its structure, the primary sequence of this RNA element has crucial functional roles in the viral lifecycle. Although the ε elements in hepadnaviruses share common critical functions, there are some significant differences in mammalian and avian hepadnaviruses, which include both sequence and structural variations.   Here we present several covariance models for ε elements from the Hepadnaviridae. The model building included experimentally determined data from previous studies using chemical probing and NMR analysis. These models have sufficient similarity to comprise a clan. The clan has in common a highly conserved overall structure consisting of a lower-stem, bulge, upper-stem and apical-loop. The models differ in functionally critical regions—notably the two types of avian ε elements have a tetra-loop (UGUU) including a non-canonical UU base pair, while the hepatitis B virus (HBV) epsilon has a tri-loop (UGU). The avian epsilon elements have a less stable dynamic structure in the upper stem. Comparisons between these models and all other Rfam models, and searches of genomes, showed these structures are specific to the Hepadnaviridae. Two family models and the clan are available from the Rfam database. PMID:22418844

Protein-mediated loops in supercoiled DNA create large topological domains

PubMed Central

Yan, Yan; Ding, Yue; Leng, Fenfei; Dunlap, David; Finzi, Laura

2018-01-01

Abstract Supercoiling can alter the form and base pairing of the double helix and directly impact protein binding. More indirectly, changes in protein binding and the stress of supercoiling also influence the thermodynamic stability of regulatory, protein-mediated loops and shift the equilibria of fundamental DNA/chromatin transactions. For example, supercoiling affects the hierarchical organization and function of chromatin in topologically associating domains (TADs) in both eukaryotes and bacteria. On the other hand, a protein-mediated loop in DNA can constrain supercoiling within a plectonemic structure. To characterize the extent of constrained supercoiling, 400 bp, lac repressor-secured loops were formed in extensively over- or under-wound DNA under gentle tension in a magnetic tweezer. The protein-mediated loops constrained variable amounts of supercoiling that often exceeded the maximum writhe expected for a 400 bp plectoneme. Loops with such high levels of supercoiling appear to be entangled with flanking domains. Thus, loop-mediating proteins operating on supercoiled substrates can establish topological domains that may coordinate gene regulation and other DNA transactions across spans in the genome that are larger than the separation between the binding sites. PMID:29538766

A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency

PubMed Central

Le Blanc, Alexander; Mahrhold, Stefan; Piesker, Janett; Luppa, Peter B.

2018-01-01

The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin’s cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity. PMID:29718991

Energetics of codon-anticodon recognition on the small ribosomal subunit.

PubMed

Almlöf, Martin; Andér, Martin; Aqvist, Johan

2007-01-09

Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

PubMed Central

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

PubMed

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4*

PubMed Central

Dolezal, Olan; Cao, Benjamin; See, Heng B.; Pfleger, Kevin D. G.; Gorry, Paul R.; Pow, Andrew; Viduka, Katerina; Lim, Kevin; Lu, Bernadine G. C.; Chang, Denison H. C.; Murray-Rust, Thomas; Dogovski, Con; Doerflinger, Marcel; Zhang, Yuan; Parisi, Kathy; Casey, Joanne L.; Nuttall, Stewart D.; Foley, Michael

2016-01-01

CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an “i-body,” the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor. PMID:27036939

Disruption of a stem-loop structure located upstream of pseudoknot domain in Tobacco mosaic virus enhanced its infectivity and viral RNA accumulation.

PubMed

Guo, Song; Wong, Sek-Man

2018-06-01

A predicted stem-loop structure of 25 nucleotides, located in the coat protein (CP) gene and 3'-UTR sequences of Tobacco mosaic virus (TMV), was validated previously (Guo et al., 2015). In this study, both disrupted stem-loop and nucleotide deletion mutants of TMV replicated more rapidly in Nicotiana benthamiana protoplasts. The TMV mutant with a complete mirrored stem-loop structure showed similar level of viral RNA accumulation as TMV. Recovering the stem-loop structure also resulted in a similar replication level as TMV. All these mutants induced necrosis in N. benthamiana and assembled into typical rigid rod-shaped virions. TMV mutant without the stem-loop structure induced more local lesions in Chenopodium quinoa. When the putative stem-loop structure in Tomato mosaic virus (ToMV) was disrupted, the mutant also showed an enhanced virus replication. This suggests that the stem-loop structure of TMV is a new cis-acting element with a role in virus replication. Copyright © 2018 Elsevier Inc. All rights reserved.

Structural consequences of cutting a binding loop: two circularly permuted variants of streptavidin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Le Trong, Isolde; University of Washington, Box 357742, Seattle, WA 98195-7742; Chu, Vano

2013-06-01

The crystal structures of two circularly permuted streptavidins probe the role of a flexible loop in the tight binding of biotin. Molecular-dynamics calculations for one of the mutants suggests that increased fluctuations in a hydrogen bond between the protein and biotin are associated with cleavage of the binding loop. Circular permutation of streptavidin was carried out in order to investigate the role of a main-chain amide in stabilizing the high-affinity complex of the protein and biotin. Mutant proteins CP49/48 and CP50/49 were constructed to place new N-termini at residues 49 and 50 in a flexible loop involved in stabilizing themore » biotin complex. Crystal structures of the two mutants show that half of each loop closes over the binding site, as observed in wild-type streptavidin, while the other half adopts the open conformation found in the unliganded state. The structures are consistent with kinetic and thermodynamic data and indicate that the loop plays a role in enthalpic stabilization of the bound state via the Asn49 amide–biotin hydrogen bond. In wild-type streptavidin, the entropic penalties of immobilizing a flexible portion of the protein to enhance binding are kept to a manageable level by using a contiguous loop of medium length (six residues) which is already constrained by its anchorage to strands of the β-barrel protein. A molecular-dynamics simulation for CP50/49 shows that cleavage of the binding loop results in increased structural fluctuations for Ser45 and that these fluctuations destabilize the streptavidin–biotin complex.« less

The Basic Helix-Loop-Helix Transcription Factor MYC2 Directly Represses PLETHORA Expression during Jasmonate-Mediated Modulation of the Root Stem Cell Niche in Arabidopsis[W][OA

PubMed Central

Chen, Qian; Sun, Jiaqiang; Zhai, Qingzhe; Zhou, Wenkun; Qi, Linlin; Xu, Li; Wang, Bao; Chen, Rong; Jiang, Hongling; Qi, Jing; Li, Xugang; Palme, Klaus; Li, Chuanyou

2011-01-01

The root stem cell niche, which in the Arabidopsis thaliana root meristem is an area of four mitotically inactive quiescent cells (QCs) and the surrounding mitotically active stem cells, is critical for root development and growth. We report here that during jasmonate-induced inhibition of primary root growth, jasmonate reduces root meristem activity and leads to irregular QC division and columella stem cell differentiation. Consistently, jasmonate reduces the expression levels of the AP2-domain transcription factors PLETHORA1 (PLT1) and PLT2, which form a developmentally instructive protein gradient and mediate auxin-induced regulation of stem cell niche maintenance. Not surprisingly, the effects of jasmonate on root stem cell niche maintenance and PLT expression require the functioning of MYC2/JASMONATE INSENSITIVE1, a basic helix-loop-helix transcription factor that involves versatile aspects of jasmonate-regulated gene expression. Gel shift and chromatin immunoprecipitation experiments reveal that MYC2 directly binds the promoters of PLT1 and PLT2 and represses their expression. We propose that MYC2-mediated repression of PLT expression integrates jasmonate action into the auxin pathway in regulating root meristem activity and stem cell niche maintenance. This study illustrates a molecular framework for jasmonate-induced inhibition of root growth through interaction with the growth regulator auxin. PMID:21954460

bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression.

PubMed

Li, Xiaosu; Chen, Rui; Zhu, Sijun

2017-11-15

Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. Here, we demonstrate that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). We provide evidence that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. Our results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

PubMed Central

de Virgilio, Maddalena; Lombardi, Alessio; Caliandro, Rocco; Fabbrini, Maria Serena

2010-01-01

Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential. PMID:22069572

Selection of a platinum-binding sequence in a loop of a four-helix bundle protein.

PubMed

Yagi, Sota; Akanuma, Satoshi; Kaji, Asumi; Niiro, Hiroya; Akiyama, Hayato; Uchida, Tatsuya; Yamagishi, Akihiko

2018-02-01

Protein-metal hybrids are functional materials with various industrial applications. For example, a redox enzyme immobilized on a platinum electrode is a key component of some biofuel cells and biosensors. To create these hybrid materials, protein molecules are bound to metal surfaces. Here, we report the selection of a novel platinum-binding sequence in a loop of a four-helix bundle protein, the Lac repressor four-helix protein (LARFH), an artificial protein in which four identical α-helices are connected via three identical loops. We created a genetic library in which the Ser-Gly-Gln-Gly-Gly-Ser sequence within the first inter-helical loop of LARFH was semi-randomly mutated. The library was then subjected to selection for platinum-binding affinity by using the T7 phage display method. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Mapping the Laminin Receptor Binding Domains of Neisseria meningitidis PorA and Haemophilus influenzae OmpP2

PubMed Central

Mahdavi, Jafar; Oldfield, Neil J.; Wheldon, Lee M.; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

2012-01-01

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171–240 and 91–99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains. PMID:23049988

Mapping the laminin receptor binding domains of Neisseria meningitidis PorA and Haemophilus influenzae OmpP2.

PubMed

Abouseada, Noha M; Assafi, Mahde Saleh A; Mahdavi, Jafar; Oldfield, Neil J; Wheldon, Lee M; Wooldridge, Karl G; Ala'Aldeen, Dlawer A A

2012-01-01

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171-240 and 91-99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains.

The impact of viral RNA on the association free energies of capsid protein assembly: bacteriophage MS2 as a case study.

PubMed

ElSawy, Karim M

2017-02-01

A large number of single-stranded RNA viruses assemble their capsid and their genomic material simultaneously. The RNA viral genome plays multiple roles in this process that are currently only partly understood. In this work, we investigated the thermodynamic basis of the role of viral RNA on the assembly of capsid proteins. The viral capsid of bacteriophage MS2 was considered as a case study. The MS2 virus capsid is composed of 60 AB and 30 CC protein dimers. We investigated the effect of RNA stem loop (the translational repressor TR) binding to the capsid dimers on the dimer-dimer relative association free energies. We found that TR binding results in destabilization of AB self-association compared with AB and CC association. This indicates that the association of the AB and CC dimers is the most likely assembly pathway for the MS2 virus, which explains the experimental observation of alternating patterns of AB and CC dimers in dominant assembly intermediates of the MS2 virus. The presence of viral RNA, therefore, dramatically channels virus assembly to a limited number of pathways, thereby enhancing the efficiency of virus self-assembly process. Interestingly, Thr59Ser and Thr45Ala mutations of the dimers, in the absence of RNA stem loops, lead to stabilization of AB self-association compared with the AB and CC associations, thereby channelling virus assembly towards a fivefold (AB) 5 pentamer intermediate, providing a testable hypothesis of our thermodynamic arguments.

Stem-Loop V of Varkud Satellite RNA Exhibits Characteristics of the Mg2+ Bound Structure in the Presence of Monovalent Ions

PubMed Central

2015-01-01

The Varkud Satellite RNA contains a self-cleaving ribozyme that has been shown to function independently of its surroundings. This 160 nucleotide ribozyme adopts a catalytically active tertiary structure that includes a kissing hairpin complex formed by stem-loop I and stem-loop V (SLV). The five-nucleotide 5′-rUGACU loop of the isolated SLV has been shown to adopt a Mg2+-dependent U-turn structure by solution NMR. This U-turn hairpin is examined here by molecular dynamics simulations in the presence of monovalent and divalent ions. Simulations confirm on an all-atom level the hypotheses for the role of the Mg2+ ions in stabilizing the loop, as well as the role of the solvent exposed U700 base. Additionally, these simulations suggest the Mg2+-free stem-loop adopts a wide range of structures, including energetically favorable structures similar to the Mg2+-bound loop structure. We propose this structure is a “gatekeeper” or precursor to Mg2+ binding when those ions are present. PMID:26328924

Intrinsic Nucleic Acid Dynamics Modulates HIV-1 Nucleocapsid Protein Binding to Its Targets

PubMed Central

Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; De Rocquigny, Hugues; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2012-01-01

HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using 13C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome. PMID:22745685

Synthesis of aspartyl-tRNA(Asp) in Escherichia coli--a snapshot of the second step.

PubMed Central

Eiler, S; Dock-Bregeon, A; Moulinier, L; Thierry, J C; Moras, D

1999-01-01

The 2.4 A crystal structure of the Escherichia coli aspartyl-tRNA synthetase (AspRS)-tRNA(Asp)-aspartyl-adenylate complex shows the two substrates poised for the transfer of the aspartic acid moiety from the adenylate to the 3'-hydroxyl of the terminal adenosine of the tRNA. A general molecular mechanism is proposed for the second step of the aspartylation reaction that accounts for the observed conformational changes, notably in the active site pocket. The stabilization of the transition state is mediated essentially by two amino acids: the class II invariant arginine of motif 2 and the eubacterial-specific Gln231, which in eukaryotes and archaea is replaced by a structurally non-homologous serine. Two archetypal RNA-protein modes of interactions are observed: the anticodon stem-loop, including the wobble base Q, binds to the N-terminal beta-barrel domain through direct protein-RNA interactions, while the binding of the acceptor stem involves both direct and water-mediated hydrogen bonds in an original recognition scheme. PMID:10562565

Insights into the regioselectivity and RNA-binding affinity of HIV-1 nucleocapsid protein from linear-scaling quantum methods.

PubMed

Khandogin, Jana; Musier-Forsyth, Karin; York, Darrin M

2003-07-25

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) plays several important roles in the viral life-cycle and presents an attractive target for rational drug design. Here, the macromolecular reactivity of NC and its binding to RNA is characterized through determination of electrostatic and chemical descriptors derived from linear-scaling quantum calculations in solution. The computational results offer a rationale for the experimentally observed susceptibility of the Cys49 thiolate toward small-molecule electrophilic agents, and support the recently proposed stepwise protonation mechanism of the C-terminal Zn-coordination complex. The distinctive binding mode of NC to SL2 and SL3 stem-loops of the HIV-1 genomic RNA packaging signal is studied on the basis of protein side-chain contributions to the electrostatic binding energies. These results indicate the importance of several basic residues in the 3(10) helical region and the N-terminal zinc finger, and rationalize the presence of several evolutionarily conserved residues in NC. The combined reactivity and RNA-binding study provides new insights that may contribute toward the structure-based design of anti-HIV therapies.

Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

PubMed

Lamping, Erwin; Niimi, Masakazu; Cannon, Richard D

2013-07-29

A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%. We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.

Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

PubMed Central

2013-01-01

Background A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5′ UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Results Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5′ UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = −15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = −4.4 kcal/mol) inhibited Cdr1p expression by ~50%. Conclusion We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression. PMID:23895661

Specific RNA-protein interactions detected with saturation transfer difference NMR.

PubMed

Harris, Kimberly A; Shekhtman, Alexander; Agris, Paul F

2013-08-01

RNA, at the forefront of biochemical research due to its central role in biology, is recognized by proteins through various mechanisms. Analysis of the RNA-protein interface provides insight into the recognition determinants and function. As such, there is a demand for developing new methods to characterize RNA-protein interactions. Saturation transfer difference (STD) NMR can identify binding ligands for proteins in a rather short period of time, with data acquisitions of just a few hours. Two RNA-protein systems involved in RNA modification were studied using STD NMR. The N (6)-threonylcarbamoyltransferase, YrdC, with nucleoside-specific recognition, was shown to bind the anticodon stem-loop of tRNA(Lys)UUU. The points of contact on the RNA were assigned and a binding interface was identified. STD NMR was also applied to the interaction of the archaeal ribosomal protein, L7Ae, with the box C/D K-turn RNA. The distinctiveness of the two RNA-protein interfaces was evident. Both RNAs exhibited strong STD signals indicative of direct contact with the respective protein, but reflected the nature of recognition. Characterization of nucleic acid recognition determinants traditionally involves cost and time prohibitive methods. This approach offers significant insight into interaction interfaces fairly rapidly, and complements existing structural methods.

Structures of apo IRF-3 and IRF-7 DNA binding domains: effect of loop L1 on DNA binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Ioannes, Pablo; Escalante, Carlos R.; Aggarwal, Aneel K.

2013-11-20

Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-{beta} (IFN-{beta}) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-{beta} promoter elements revealed flexibility in the loops (L1-L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures ofmore » IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding.« less

Looping and clustering model for the organization of protein-DNA complexes on the bacterial genome

NASA Astrophysics Data System (ADS)

Walter, Jean-Charles; Walliser, Nils-Ole; David, Gabriel; Dorignac, Jérôme; Geniet, Frédéric; Palmeri, John; Parmeggiani, Andrea; Wingreen, Ned S.; Broedersz, Chase P.

2018-03-01

The bacterial genome is organized by a variety of associated proteins inside a structure called the nucleoid. These proteins can form complexes on DNA that play a central role in various biological processes, including chromosome segregation. A prominent example is the large ParB-DNA complex, which forms an essential component of the segregation machinery in many bacteria. ChIP-Seq experiments show that ParB proteins localize around centromere-like parS sites on the DNA to which ParB binds specifically, and spreads from there over large sections of the chromosome. Recent theoretical and experimental studies suggest that DNA-bound ParB proteins can interact with each other to condense into a coherent 3D complex on the DNA. However, the structural organization of this protein-DNA complex remains unclear, and a predictive quantitative theory for the distribution of ParB proteins on DNA is lacking. Here, we propose the looping and clustering model, which employs a statistical physics approach to describe protein-DNA complexes. The looping and clustering model accounts for the extrusion of DNA loops from a cluster of interacting DNA-bound proteins that is organized around a single high-affinity binding site. Conceptually, the structure of the protein-DNA complex is determined by a competition between attractive protein interactions and loop closure entropy of this protein-DNA cluster on the one hand, and the positional entropy for placing loops within the cluster on the other. Indeed, we show that the protein interaction strength determines the ‘tightness’ of the loopy protein-DNA complex. Thus, our model provides a theoretical framework for quantitatively computing the binding profiles of ParB-like proteins around a cognate (parS) binding site.

Rewriting nature's assembly manual for a ssRNA virus.

PubMed

Patel, Nikesh; Wroblewski, Emma; Leonov, German; Phillips, Simon E V; Tuma, Roman; Twarock, Reidun; Stockley, Peter G

2017-11-14

Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1-127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes. Copyright © 2017 the Author(s). Published by PNAS.

Analyte-Triggered DNA-Probe Release from a Triplex Molecular Beacon for Nanopore Sensing.

PubMed

Guo, Bingyuan; Sheng, Yingying; Zhou, Ke; Liu, Quansheng; Liu, Lei; Wu, Hai-Chen

2018-03-26

A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex-forming molecular beacon and a stem-forming DNA component that is modified with a host-guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α-hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point-of-care diagnostics with a portable nanopore kit in the future. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Activation Domain of the Bovine Papillomavirus E2 Protein Mediates Association of DNA-Bound Dimers to form DNA Loops

NASA Astrophysics Data System (ADS)

Knight, Jonathan D.; Li, Rong; Botchan, Michael

1991-04-01

The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, performed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on te DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and D8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition.

PubMed

Kumari, Pooja; Aeschimann, Florian; Gaidatzis, Dimos; Keusch, Jeremy J; Ghosh, Pritha; Neagu, Anca; Pachulska-Wieczorek, Katarzyna; Bujnicki, Janusz M; Gut, Heinz; Großhans, Helge; Ciosk, Rafal

2018-04-19

RNA-binding proteins regulate all aspects of RNA metabolism. Their association with RNA is mediated by RNA-binding domains, of which many remain uncharacterized. A recently reported example is the NHL domain, found in prominent regulators of cellular plasticity like the C. elegans LIN-41. Here we employ an integrative approach to dissect the RNA specificity of LIN-41. Using computational analysis, structural biology, and in vivo studies in worms and human cells, we find that a positively charged pocket, specific to the NHL domain of LIN-41 and its homologs (collectively LIN41), recognizes a stem-loop RNA element, whose shape determines the binding specificity. Surprisingly, the mechanism of RNA recognition by LIN41 is drastically different from that of its more distant relative, the fly Brat. Our phylogenetic analysis suggests that this reflects a rapid evolution of the domain, presenting an interesting example of a conserved protein fold that acquired completely different solutions to RNA recognition.

Negative feedback regulation of wild-type p53 biosynthesis.

PubMed Central

Mosner, J; Mummenbrauer, T; Bauer, C; Sczakiel, G; Grosse, F; Deppert, W

1995-01-01

When growth-arrested mouse fibroblasts re-entered the cell-cycle, the rise in tumour suppressor p53 mRNA level markedly preceded the rise in expression of the p53 protein. Furthermore, gamma-irradiation of such cells led to a rapid increase in p53 protein biosynthesis even in the presence of the transcription inhibitor actinomycin D. Both findings strongly suggest that p53 biosynthesis in these cells is regulated at the translational level. We present evidence for an autoregulatory control of p53 expression by a negative feed-back loop: p53 mRNA has a predicted tendency to form a stable stem-loop structure that involves the 5'-untranslated region (5'-UTR) plus some 280 nucleotides of the coding sequence. p53 binds tightly to the 5'-UTR region and inhibits the translation of its own mRNA, most likely mediated by the p53-intrinsic RNA re-annealing activity. The inhibition of p53 biosynthesis requires wild-type p53, as it is not observed with MethA mutant p53, p53-catalysed translational inhibition is selective; it might be restricted to p53 mRNA and a few other mRNAs that are able to form extensive stem-loop structures. Release from negative feed-back regulation of p53 biosynthesis, e.g. after damage-induced nuclear transport of p53, might provide a means for rapidly increasing p53 protein levels when p53 is required to act as a cell-cycle checkpoint determinant after DNA damage. Images PMID:7556087

RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii.

PubMed

Yamasaki, Tomohito; Onishi, Masayuki; Kim, Eun-Jeong; Cerutti, Heriberto; Ohama, Takeshi

2016-09-20

Canonical microRNAs (miRNAs) are embedded in duplexed stem-loops in long precursor transcripts and are excised by sequential cleavage by DICER nuclease(s). In this miRNA biogenesis pathway, dsRNA-binding proteins play important roles in animals and plants by assisting DICER. However, these RNA-binding proteins are poorly characterized in unicellular organisms. Here we report that a unique RNA-binding protein, Dull slicer-16 (DUS16), plays an essential role in processing of primary-miRNA (pri-miRNA) transcripts in the unicellular green alga Chlamydomonas reinhardtii In animals and plants, dsRNA-binding proteins involved in miRNA biogenesis harbor two or three dsRNA-binding domains (dsRBDs), whereas DUS16 contains one dsRBD and also an ssRNA-binding domain (RRM). The null mutant of DUS16 showed a drastic reduction in most miRNA species. Production of these miRNAs was complemented by expression of full-length DUS16, but the expression of RRM- or dsRBD-truncated DUS16 did not restore miRNA production. Furthermore, DUS16 is predominantly localized to the nucleus and associated with nascent (unspliced form) pri-miRNAs and the DICER-LIKE 3 protein. These results suggest that DUS16 recognizes pri-miRNA transcripts cotranscriptionally and promotes their processing into mature miRNAs as a component of a microprocessor complex. We propose that DUS16 is an essential factor for miRNA production in Chlamydomonas and, because DUS16 is functionally similar to the dsRNA-binding proteins involved in miRNA biogenesis in animals and land plants, our report provides insight into this mechanism in unicellular eukaryotes.

SRY-box-containing Gene 2 Regulation of Nuclear Receptor Tailless (Tlx) Transcription in Adult Neural Stem Cells*

PubMed Central

Shimozaki, Koji; Zhang, Chun-Li; Suh, Hoonkyo; Denli, Ahmet M.; Evans, Ronald M.; Gage, Fred H.

2012-01-01

Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs. PMID:22194602

SRY-box-containing gene 2 regulation of nuclear receptor tailless (Tlx) transcription in adult neural stem cells.

PubMed

Shimozaki, Koji; Zhang, Chun-Li; Suh, Hoonkyo; Denli, Ahmet M; Evans, Ronald M; Gage, Fred H

2012-02-17

Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs.

The allosteric switching mechanism in bacteriophage MS2

NASA Astrophysics Data System (ADS)

Perkett, Matthew R.; Mirijanian, Dina T.; Hagan, Michael F.

2016-07-01

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.

The allosteric switching mechanism in bacteriophage MS2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perkett, Matthew R.; Mirijanian, Dina T.; Hagan, Michael F., E-mail: hagan@brandeis.edu

2016-07-21

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we usemore » all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.« less

The allosteric switching mechanism in bacteriophage MS2

PubMed Central

Perkett, Matthew R.; Mirijanian, Dina T.

2016-01-01

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates. PMID:27448905

Engineering Encodable Lanthanide-Binding Tags (LBTs) into Loop Regions of Proteins

PubMed Central

Barthelmes, Katja; Reynolds, Anne M.; Peisach, Ezra; Jonker, Hendrik R. A.; DeNunzio, Nicholas J.; Allen, Karen N.; Imperiali, Barbara; Schwalbe, Harald

2011-01-01

Lanthanide-binding-tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β and varied the length of the spacer between the LBT and the protein (denoted 1-3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing 1H-15N-HSQC NMR spectra with wild-type IL1β. Additionally, binding of LBT-loop IL1β proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop-constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated based on RDCs and pseudocontact shifts (PCSs). A structural model of the IL1β-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modelling. PMID:21182275

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein.

PubMed

Eberini, Ivano; Guerini Rocco, Alessandro; Ientile, Anna Rita; Baptista, António M; Gianazza, Elisabetta; Tomaselli, Simona; Molinari, Henriette; Ragona, Laura

2008-06-01

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family. (c) 2008 Wiley-Liss, Inc.

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA.

PubMed

Stoltenburg, Regina; Krafčiková, Petra; Víglaský, Viktor; Strehlitz, Beate

2016-09-21

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5'-end also contributes essentially to the aptameric function.

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA

PubMed Central

Stoltenburg, Regina; Krafčiková, Petra; Víglaský, Viktor; Strehlitz, Beate

2016-01-01

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5′-end also contributes essentially to the aptameric function. PMID:27650576

Role of the Pepino mosaic virus 3'-untranslated region elements in negative-strand RNA synthesis in vitro.

PubMed

Osman, Toba A M; Olsthoorn, René C L; Livieratos, Ioannis C

2014-09-22

Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element. Copyright © 2014 Elsevier B.V. All rights reserved.

Stem loop recognition by DDX17 facilitates miRNA processing and antiviral defense

PubMed Central

Moy, Ryan H.; Cole, Brian S.; Yasunaga, Ari; Gold, Beth; Shankarling, Ganesh; Varble, Andrew; Molleston, Jerome M.; tenOever, Benjamin R.; Lynch, Kristen W.; Cherry, Sara

2014-01-01

DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using cross-linking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing, and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous miRNA biogenesis and in the cytoplasm for surveillance against structured non-self elements. PMID:25126784

Identification and characterization of the sodium-binding site of activated protein C.

PubMed

He, X; Rezaie, A R

1999-02-19

Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221-225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221-225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (Kd(app)) = 44.1 +/- 8.6 mM. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5-6-fold with Kd(app) = 2.3 +/- 0.3 mM. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221-225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme.

Structural dynamics of the lac repressor-DNA complex revealed by a multiscale simulation.

PubMed

Villa, Elizabeth; Balaeff, Alexander; Schulten, Klaus

2005-05-10

A multiscale simulation of a complex between the lac repressor protein (LacI) and a 107-bp-long DNA segment is reported. The complex between the repressor and two operator DNA segments is described by all-atom molecular dynamics; the size of the simulated system comprises either 226,000 or 314,000 atoms. The DNA loop connecting the operators is modeled as a continuous elastic ribbon, described mathematically by the nonlinear Kirchhoff differential equations with boundary conditions obtained from the coordinates of the terminal base pairs of each operator. The forces stemming from the looped DNA are included in the molecular dynamics simulations; the loop structure and the forces are continuously recomputed because the protein motions during the simulations shift the operators and the presumed termini of the loop. The simulations reveal the structural dynamics of the LacI-DNA complex in unprecedented detail. The multiple domains of LacI exhibit remarkable structural stability during the simulation, moving much like rigid bodies. LacI is shown to absorb the strain from the looped DNA mainly through its mobile DNA-binding head groups. Even with large fluctuating forces applied, the head groups tilt strongly and keep their grip on the operator DNA, while the remainder of the protein retains its V-shaped structure. A simulated opening of the cleft of LacI by 500-pN forces revealed the interactions responsible for locking LacI in the V-conformation.

Solution structure of an ATP-binding RNA aptamer reveals a novel fold.

PubMed Central

Dieckmann, T; Suzuki, E; Nakamura, G K; Feigon, J

1996-01-01

In vitro selection has been used to isolate several RNA aptamers that bind specifically to biological cofactors. A well-characterized example in the ATP-binding RNA aptamer family, which contains a conserved 11-base loop opposite a bulged G and flanked by regions of double-stranded RNA. The nucleotides in the consensus sequence provide a binding pocket for ATP (or AMP), which binds with a Kd in the micromolar range. Here we present the three-dimensional solution structure of a 36-nucleotide ATP-binding RNA aptamer complexed with AMP, determined from NMR-derived distance and dihedral angle restraints. The conserved loop and bulged G form a novel compact, folded structure around the AMP. The backbone tracing of the loop nucleotides can be described by a Greek zeta (zeta). Consecutive loop nucleotides G, A, A form a U-turn at the bottom of the zeta, and interact with the AMP to form a structure similar to a GNRA tetraloop, with AMP standing in for the final A. Two asymmetric G. G base pairs close the stems flanking the internal loop. Mutated aptamers support the existence of the tertiary interactions within the consensus nucleotides and with the AMP found in the calculated structures. PMID:8756406

Saturation mutagenesis reveals manifold determinants of exon definition.

PubMed

Ke, Shengdong; Anquetil, Vincent; Zamalloa, Jorge Rojas; Maity, Alisha; Yang, Anthony; Arias, Mauricio A; Kalachikov, Sergey; Russo, James J; Ju, Jingyue; Chasin, Lawrence A

2018-01-01

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins. © 2018 Ke et al.; Published by Cold Spring Harbor Laboratory Press.

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2.

PubMed

Bubenik, Jodi L; Miniard, Angela C; Driscoll, Donna M

2014-01-01

Selenium, a micronutrient, is primarily incorporated into human physiology as selenocysteine (Sec). The 25 Sec-containing proteins in humans are known as selenoproteins. Their synthesis depends on the translational recoding of the UGA stop codon to allow Sec insertion. This requires a stem-loop structure in the 3' untranslated region of eukaryotic mRNAs known as the Selenocysteine Insertion Sequence (SECIS). The SECIS is recognized by SECIS-binding protein 2 (SBP2) and this RNA:protein interaction is essential for UGA recoding to occur. Genetic mutations cause SBP2 deficiency in humans, resulting in a broad set of symptoms due to differential effects on individual selenoproteins. Progress on understanding the different phenotypes requires developing robust tools to investigate SBP2 structure and function. In this study we demonstrate that SBP2 protein produced by in vitro translation discriminates among SECIS elements in a competitive UGA recoding assay and has a much higher specific activity than bacterially expressed protein. We also show that a purified recombinant protein encompassing amino acids 517-777 of SBP2 binds to SECIS elements with high affinity and selectivity. The affinity of the SBP2:SECIS interaction correlated with the ability of a SECIS to compete for UGA recoding activity in vitro. The identification of a 250 amino acid sequence that mediates specific, selective SECIS-binding will facilitate future structural studies of the SBP2:SECIS complex. Finally, we identify an evolutionarily conserved core cysteine signature in SBP2 sequences from the vertebrate lineage. Mutation of multiple, but not single, cysteines impaired SECIS-binding but did not affect protein localization in cells.

Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNALysUUU species.

PubMed

Yarian, C; Marszalek, M; Sochacka, E; Malkiewicz, A; Guenther, R; Miskiewicz, A; Agris, P F

2000-11-07

Nucleoside modifications are important to the structure of all tRNAs and are critical to the function of some tRNA species. The transcript of human tRNA(Lys3)(UUU) with a UUU anticodon, and the corresponding anticodon stem and loop domain (ASL(Lys3)(UUU)), are unable to bind to poly-A programmed ribosomes. To determine if specific anticodon domain modified nucleosides of tRNA(Lys) species would restore ribosomal binding and also affect thermal stability, we chemically synthesized ASL(Lys) heptadecamers and site-specifically incorporated the anticodon domain modified nucleosides pseudouridine (Psi(39)), 5-methylaminomethyluridine (mnm(5)U(34)) and N6-threonylcarbamoyl-adenosine (t(6)A(37)). Incorporation of t(6)A(37) and mnm(5)U(34) contributed structure to the anticodon loop, apparent by increases in DeltaS, and significantly enhanced the ability of ASL(Lys3)(UUU) to bind poly-A programmed ribosomes. Neither ASL(Lys3)(UUU)-t(6)A(37) nor ASL(Lys3)(UUU)-mnm(5)U(34) bound AAG programmed ribosomes. Only the presence of both t(6)A(37) and mnm(5)U(34) enabled ASL(Lys3)(UUU) to bind AAG programmed ribosomes, as well as increased its affinity for poly-A programmed ribosomes to the level of native Escherichia coli tRNA(Lys). The completely unmodified anticodon stem and loop of human tRNA(Lys1,2)(CUU) with a wobble position-34 C bound AAG, but did not wobble to AAA, even when the ASL was modified with t(6)A(37). The data suggest that tRNA(Lys)(UUU) species require anticodon domain modifications in the loop to impart an ordered structure to the anticodon for ribosomal binding to AAA and require a combination of modified nucleosides to bind AAG.

A SELEX-Screened Aptamer of Human Hepatitis B Virus RNA Encapsidation Signal Suppresses Viral Replication

PubMed Central

Feng, Hui; Beck, Jürgen; Nassal, Michael; Hu, Kang-hong

2011-01-01

Background The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the ε RNA stem-loop on pregenomic (pg) RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. Methodology/Principal Findings Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP), to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA. Conclusions/Significance This study demonstrates the first successful identification of human HBV ε aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B. PMID:22125633

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

PubMed

Figueroa-Angulo, Elisa E; Calla-Choque, Jaeson S; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

2015-11-26

Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

Functional anticodon architecture of human tRNALys3 includes disruption of intraloop hydrogen bonding by the naturally occurring amino acid modification, t6A.

PubMed

Stuart, J W; Gdaniec, Z; Guenther, R; Marszalek, M; Sochacka, E; Malkiewicz, A; Agris, P F

2000-11-07

The structure of the human tRNA(Lys3) anticodon stem and loop domain (ASL(Lys3)) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t(6)A(37)) to tRNA(Lys3) functions. The t(6)A(37)-modified anticodon stem and loop domain of tRNA(Lys3)(UUU) (ASL(Lys3)(UUU)- t(6)A(37)) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASL(Lys3)(UUU) is not [Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390-13395]. The structure, determined to an average rmsd of 1.57 +/- 0.33 A (relative to the mean structure) by NMR spectroscopy and restrained molecular dynamics, is the first reported of an RNA in which a naturally occurring hypermodified nucleoside was introduced by automated chemical synthesis. The ASL(Lys3)(UUU)-t(6)A(37) loop is significantly different than that of the unmodified ASL(Lys3)(UUU), although the five canonical base pairs of both ASL(Lys3)(UUU) stems are in the standard A-form of helical RNA. t(6)A(37), 3'-adjacent to the anticodon, adopts the form of a tricyclic nucleoside with an intraresidue H-bond and enhances base stacking on the 3'-side of the anticodon loop. Critically important to ribosome binding, incorporation of the modification negates formation of an intraloop U(33).A(37) base pair that is observed in the unmodified ASL(Lys3)(UUU). The anticodon wobble position U(34) nucleobase in ASL(Lys3)(UUU)-t(6)A(37) is significantly displaced from its position in the unmodified ASL and directed away from the codon-binding face of the loop resulting in only two anticodon bases for codon binding. This conformation is one explanation for ASL(Lys3)(UUU) tendency to prematurely terminate translation and -1 frame shift. At the pH 5.6 conditions of our structure determination, A(38) is protonated and positively charged in ASL(Lys3)(UUU)-t(6)A(37) and the unmodified ASL(Lys3)(UUU). The ionized carboxylic acid moiety of t(6)A(37) possibly neutralizes the positive charge of A(+)(38). The protonated A(+)(38) can base pair with C(32), but t(6)A(37) may weaken the interaction through steric interference. From these results, we conclude that ribosome binding cannot simply be an induced fit of the anticodon stem and loop, otherwise the unmodified ASL(Lys3)(UUU) would bind as well as ASL(Lys3)(UUU)-t(6)A(37). t(6)A(37) and other position 37 modifications produce the open, structured loop required for ribosomal binding.

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

PubMed

Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of interaction with several enzymes and adaptor proteins.

Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

PubMed Central

Liu, Yuanjie; Mao, Richeng; Mitra, Bidisha; Cai, Dawei; Yan, Ran; Guo, Ju-Tao; Block, Timothy M.; Mechti, Nadir

2017-01-01

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general. PMID:28399146

Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA.

PubMed

Liu, Yuanjie; Nie, Hui; Mao, Richeng; Mitra, Bidisha; Cai, Dawei; Yan, Ran; Guo, Ju-Tao; Block, Timothy M; Mechti, Nadir; Guo, Haitao

2017-04-01

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general.

The diversity of H3 loops determines the antigen-binding tendencies of antibody CDR loops.

PubMed

Tsuchiya, Yuko; Mizuguchi, Kenji

2016-04-01

Of the complementarity-determining regions (CDRs) of antibodies, H3 loops, with varying amino acid sequences and loop lengths, adopt particularly diverse loop conformations. The diversity of H3 conformations produces an array of antigen recognition patterns involving all the CDRs, in which the residue positions actually in contact with the antigen vary considerably. Therefore, for a deeper understanding of antigen recognition, it is necessary to relate the sequence and structural properties of each residue position in each CDR loop to its ability to bind antigens. In this study, we proposed a new method for characterizing the structural features of the CDR loops and obtained the antigen-binding ability of each residue position in each CDR loop. This analysis led to a simple set of rules for identifying probable antigen-binding residues. We also found that the diversity of H3 loop lengths and conformations affects the antigen-binding tendencies of all the CDR loops. © 2016 The Protein Society.

Noncanoncial signal recognition particle RNAs in a major eukaryotic phylum revealed by purification of SRP from the human pathogen Cryptococcus neoformans

PubMed Central

Dumesic, Phillip A.; Rosenblad, Magnus A.; Samuelsson, Tore; Nguyen, Tiffany; Moresco, James J.; Yates, John R.; Madhani, Hiten D.

2015-01-01

Despite conservation of the signal recognition particle (SRP) from bacteria to man, computational approaches have failed to identify SRP components from genomes of many lower eukaryotes, raising the possibility that they have been lost or altered in those lineages. We report purification and analysis of SRP in the human pathogen Cryptococcus neoformans, providing the first description of SRP in basidiomycetous yeast. The C. neoformans SRP RNA displays a predicted structure in which the universally conserved helix 8 contains an unprecedented stem-loop insertion. Guided by this sequence, we computationally identified 152 SRP RNAs throughout the phylum Basidiomycota. This analysis revealed additional helix 8 alterations including single and double stem-loop insertions as well as loop diminutions affecting RNA structural elements that are otherwise conserved from bacteria to man. Strikingly, these SRP RNA features in Basidiomycota are accompanied by phylum-specific alterations in the RNA-binding domain of Srp54, the SRP protein subunit that directly interacts with helix 8. Our findings reveal unexpected fungal SRP diversity and suggest coevolution of the two most conserved SRP features—SRP RNA helix 8 and Srp54—in basidiomycetes. Because members of this phylum include important human and plant pathogens, these noncanonical features provide new targets for antifungal compound development. PMID:26275773

Defining scaffold geometries for interacting with proteins: geometrical classification of secondary structure linking regions.

PubMed

Tran, Tran T; Kulis, Christina; Long, Steven M; Bryant, Darryn; Adams, Peter; Smythe, Mark L

2010-11-01

Medicinal chemists synthesize arrays of molecules by attaching functional groups to scaffolds. There is evidence suggesting that some scaffolds yield biologically active molecules more than others, these are termed privileged substructures. One role of the scaffold is to present its side-chains for molecular recognition, and biologically relevant scaffolds may present side-chains in biologically relevant geometries or shapes. Since drug discovery is primarily focused on the discovery of compounds that bind to proteinaceous targets, we have been deciphering the scaffold shapes that are used for binding proteins as they reflect biologically relevant shapes. To decipher the scaffold architecture that is important for binding protein surfaces, we have analyzed the scaffold architecture of protein loops, which are defined in this context as continuous four residue segments of a protein chain that are not part of an α-helix or β-strand secondary structure. Loops are an important molecular recognition motif of proteins. We have found that 39 clusters reflect the scaffold architecture of 89% of the 23,331 loops in the dataset, with average intra-cluster and inter-cluster RMSD of 0.47 and 1.91, respectively. These protein loop scaffolds all have distinct shapes. We have used these 39 clusters that reflect the scaffold architecture of protein loops as biological descriptors. This involved generation of a small dataset of scaffold-based peptidomimetics. We found that peptidomimetic scaffolds with reported biological activities matched loop scaffold geometries and those peptidomimetic scaffolds with no reported biologically activities did not. This preliminary evidence suggests that organic scaffolds with tight matches to the preferred loop scaffolds of proteins, implies the likelihood of the scaffold to be biologically relevant.

Defining scaffold geometries for interacting with proteins: geometrical classification of secondary structure linking regions

NASA Astrophysics Data System (ADS)

Tran, Tran T.; Kulis, Christina; Long, Steven M.; Bryant, Darryn; Adams, Peter; Smythe, Mark L.

2010-11-01

Medicinal chemists synthesize arrays of molecules by attaching functional groups to scaffolds. There is evidence suggesting that some scaffolds yield biologically active molecules more than others, these are termed privileged substructures. One role of the scaffold is to present its side-chains for molecular recognition, and biologically relevant scaffolds may present side-chains in biologically relevant geometries or shapes. Since drug discovery is primarily focused on the discovery of compounds that bind to proteinaceous targets, we have been deciphering the scaffold shapes that are used for binding proteins as they reflect biologically relevant shapes. To decipher the scaffold architecture that is important for binding protein surfaces, we have analyzed the scaffold architecture of protein loops, which are defined in this context as continuous four residue segments of a protein chain that are not part of an α-helix or β-strand secondary structure. Loops are an important molecular recognition motif of proteins. We have found that 39 clusters reflect the scaffold architecture of 89% of the 23,331 loops in the dataset, with average intra-cluster and inter-cluster RMSD of 0.47 and 1.91, respectively. These protein loop scaffolds all have distinct shapes. We have used these 39 clusters that reflect the scaffold architecture of protein loops as biological descriptors. This involved generation of a small dataset of scaffold-based peptidomimetics. We found that peptidomimetic scaffolds with reported biological activities matched loop scaffold geometries and those peptidomimetic scaffolds with no reported biologically activities did not. This preliminary evidence suggests that organic scaffolds with tight matches to the preferred loop scaffolds of proteins, implies the likelihood of the scaffold to be biologically relevant.

Negative Factor from SIV Binds to the Catalytic Subunit of the V-ATPase to Internalize CD4 and to Increase Viral Infectivity

PubMed Central

Mandic, Robert; Fackler, Oliver T.; Geyer, Matthias; Linnemann, Thomas; Zheng, Yong-Hui; Peterlin, B. Matija

2001-01-01

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239 for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity. PMID:11179428

Structural insights into the neuroprotective-acting carbonyl reductase Sniffer of Drosophila melanogaster.

PubMed

Sgraja, Tanja; Ulschmid, Julia; Becker, Katja; Schneuwly, Stephan; Klebe, Gerhard; Reuter, Klaus; Heine, Andreas

2004-10-01

In vivo studies with the fruit-fly Drosophila melanogaster have shown that the Sniffer protein prevents age-dependent and oxidative stress-induced neurodegenerative processes. Sniffer is a NADPH-dependent carbonyl reductase belonging to the enzyme family of short-chain dehydrogenases/reductases (SDRs). The crystal structure of the homodimeric Sniffer protein from Drosophila melanogaster in complex with NADP+ has been determined by multiple-wavelength anomalous dispersion and refined to a resolution of 1.75 A. The observed fold represents a typical dinucleotide-binding domain as detected for other SDRs. With respect to the cofactor-binding site and the region referred to as substrate-binding loop, the Sniffer protein shows a striking similarity to the porcine carbonyl reductase (PTCR). This loop, in both Sniffer and PTCR, is substantially shortened compared to other SDRs. In most enzymes of the SDR family this loop adopts a well-defined conformation only after substrate binding and remains disordered in the absence of any bound ligands or even if only the dinucleotide cofactor is bound. In the structure of the Sniffer protein, however, the conformation of this loop is well defined, although no substrate is present. Molecular modeling studies provide an idea of how binding of substrate molecules to Sniffer could possibly occur.

Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD.

PubMed

Neupane, Durga P; Avalos, Dante; Fullam, Stephanie; Roychowdhury, Hridindu; Yukl, Erik T

2017-10-20

Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of a Csr system in Serratia marcescens 2170.

PubMed

Ito, Manabu; Nomura, Kazuki; Sugimoto, Hayuki; Watanabe, Takeshi; Suzuki, Kazushi

2014-01-01

The carbon storage regulator (Csr) global regulatory system is conserved in many eubacteria and coordinates the expression of various genes that facilitate adaptation during the major physiological growth phase. The Csr system in Escherichia coli comprises an RNA-binding protein, CsrA; small non-coding RNAs, CsrB and CsrC; and a decay factor for small RNAs, CsrD. In this study, we identified the Csr system in Serratia marcescens 2170. S. marcescens CsrA was 97% identical to E. coli CsrA. CsrB and CsrC RNAs had typical stem-loop structures, including a GGA motif that is the CsrA binding site. CsrD was composed of N-terminal two times transmembrane region and HAMP-like, GGDEF, and EAL domains. Overexpression of S. marcescens csr genes complemented the phenotype of E. coli csr mutants. S. marcescens CsrD affected the decay of CsrB and CsrC RNAs in E. coli. These results suggest that the Csr system in S. marcescens is composed of an RNA-binding protein, two Csr small RNAs, and a decay factor for Csr small RNAs.

Additional hydrogen bonds and base-pair kinetics in the symmetrical AMP-DNA aptamer complex.

PubMed Central

Nonin-Lecomte, S; Lin, C H; Patel, D J

2001-01-01

The solution structure of an adenosine monophosphate (AMP)-DNA aptamer complex has been determined previously [Lin, C. H., and Patel, D. J. (1997) Chem. Biol. 4:817-832]. On a symmetrical aptamer complex containing the same binding loop, but with better resolved spectra, we have identified two additional hydrogen bond-mediated associations in the binding loop. One of these involves a rapidly exchanging G imino proton. The phosphate group of the AMP ligand was identified as the acceptor by comparison with other aptamer complexes. Imino proton exchange measurements also yielded the dissociation constants of the stem and binding loop base pairs. This study shows that nuclear magnetic resonance-based imino proton exchange is a good probe for detection of weak hydrogen-bond associations. PMID:11721004

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

PubMed

Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-05-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

PubMed Central

Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-01-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding. PMID:11350033

Artificial sRNAs activating the Gac/Rsm signal transduction pathway in Pseudomonas fluorescens.

PubMed

Valverde, Claudio

2009-04-01

In Pseudomonas fluorescens CHA0, the synthesis of antifungal compounds is post-transcriptionally activated by the Gac/Rsm cascade. The two-component system GacS/GacA promotes transcription of three small regulatory RNAs (i.e., sRNAs), RsmX, RsmY, and RsmZ, which remove the regulatory proteins RsmA and RsmE from the ribosome-binding sites of exoproduct-related mRNAs. The GacS/GacA-dependent accumulation of RsmX/Y/Z and formation of RsmX/Y/Z-RsmA/E complexes relieve mRNA translational repression. Other bacteria as E. coli and Vibrio spp. utilize similar sRNA-protein based systems to adjust mRNA translation (e.g., the E. coli Csr system for carbon storage, motility and biofilm regulation). The Rsm/Csr sRNAs are remarkably similar in that they contain several stem-loops with an invariant GGA trinucleotide exposed in the hairpin loop that would be the characteristic structural-sequence motifs relevant for sRNA activity and stability. Here it is shown that the dysfunctional Gac/Rsm cascade of P. fluorescens DeltarsmXYZ mutants could be restored by appropriate transcription levels of artificial genes encoding RNAs with unrelated primary sequence but with two or more hairpins displaying the RsmA/E binding motifs. The results support the hypothesis that the molecular mimicry of Rsm/Csr sRNAs is based on proper secondary structures that expose critical binding motifs irrespective of their overall sequence.

Structure and thermodynamics of effector molecule binding to the nitrogen signal transduction PII protein GlnZ from Azospirillum brasilense.

PubMed

Truan, Daphné; Bjelić, Saša; Li, Xiao-Dan; Winkler, Fritz K

2014-07-29

The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Imino proton exchange rates imply an induced-fit binding mechanism for the VEGF165-targeting aptamer, Macugen

PubMed Central

Lee, Joon-Hwa; Jucker, Fiona; Pardi, Arthur

2008-01-01

The 2′-fluoro/2′-O-methyl modified RNA aptamer Macugen is a potent inhibitor of the angiogenic regulatory protein, VEGF165. Macugen binds with high affinity to the heparin-binding domain (HBD) of VEGF165. Hydrogen exchange rates of the imino protons were measured for free Macugen and Macugen bound to the HBD or full-length VEGF to better understand the mechanism for high affinity binding. The results here show that the internal loop and hairpin loop of Macugen are highly dynamic in the free state and are greatly stabilized and/or protected from solvent upon protein binding. PMID:18485899

Identification and analysis of host proteins that interact with the 3'-untranslated region of tick-borne encephalitis virus genomic RNA.

PubMed

Muto, Memi; Kamitani, Wataru; Sakai, Mizuki; Hirano, Minato; Kobayashi, Shintaro; Kariwa, Hiroaki; Yoshii, Kentaro

2018-04-02

Tick-borne encephalitis virus (TBEV) causes severe neurological disease, but the pathogenetic mechanism is unclear. The conformational structure of the 3'-untranslated region (UTR) of TBEV is associated with its virulence. We tried to identify host proteins interacting with the 3'-UTR of TBEV. Cellular proteins of HEK293T cells were co-precipitated with biotinylated RNAs of the 3'-UTR of low- and high-virulence TBEV strains and subjected to mass spectrometry analysis. Fifteen host proteins were found to bind to the 3'-UTR of TBEV, four of which-cold shock domain containing-E1 (CSDE1), spermatid perinuclear RNA binding protein (STRBP), fragile X mental retardation protein (FMRP), and interleukin enhancer binding factor 3 (ILF3)-bound specifically to that of the low-virulence strain. An RNA immunoprecipitation and pull-down assay confirmed the interactions of the complete 3'-UTRs of TBEV genomic RNA with CSDE1, FMRP, and ILF3. Partial deletion of the stem loop (SL) 3 to SL 5 structure of the variable region of the 3'-UTR did not affect interactions with the host proteins, but the interactions were markedly suppressed by deletion of the complete SL 3, 4, and 5 structures, as in the high-virulence TBEV strain. Further analysis of the roles of host proteins in the neurologic pathogenicity of TBEV is warranted. Copyright © 2018 Elsevier B.V. All rights reserved.

Highly sensitive self-complementary DNA nanoswitches triggered by polyelectrolytes.

PubMed

Wu, Jincai; Yu, Feng; Zhang, Zheng; Chen, Yong; Du, Jie; Maruyama, Atsushi

2016-01-07

Dimerization of two homologous strands of genomic DNA/RNA is an essential feature of retroviral replication. Herein we show that a cationic comb-type copolymer (CCC), poly(L-lysine)-graft-dextran, accelerates the dimerization of self-complementary stem-loop DNA, frequently found in functional DNA/RNA molecules, such as aptamers. Furthermore, an anionic polymer poly(sodium vinylsulfonate) (PVS) dissociates CCC from the duplex shortly within a few seconds. Then single stem-loop DNA spontaneously transforms from its dimer. Thus we can easily control the dimer and stem-loop DNA by switching on/off CCC activity. Both polyelectrolytes and DNA concentrations are in the nanomole per liter range. The polyelectrolyte-assisted transconformation and sequences design strategy ensures the reversible state control with rapid response and effective switching under physiologically relevant conditions. A further application of this sensitive assembly is to construct an aptamer-type drug delivery system, bind or release functional molecules responding to its transconformation.

Cellular mRNA decay protein AUF1 negatively regulates enterovirus and human rhinovirus infections.

PubMed

Cathcart, Andrea L; Rozovics, Janet M; Semler, Bert L

2013-10-01

To successfully complete their replication cycles, picornaviruses modify several host proteins to alter the cellular environment to favor virus production. One such target of viral proteinase cleavage is AU-rich binding factor 1 (AUF1), a cellular protein that binds to AU-rich elements, or AREs, in the 3' noncoding regions (NCRs) of mRNAs to affect the stability of the RNA. Previous studies found that, during poliovirus or human rhinovirus infection, AUF1 is cleaved by the viral proteinase 3CD and that AUF1 can interact with the long 5' NCR of these viruses in vitro. Here, we expand on these initial findings to demonstrate that all four isoforms of AUF1 bind directly to stem-loop IV of the poliovirus 5' NCR, an interaction that is inhibited through proteolytic cleavage of AUF1 by the viral proteinase 3CD. Endogenous AUF1 was observed to relocalize to the cytoplasm of infected cells in a viral protein 2A-driven manner and to partially colocalize with the viral protein 3CD. We identify a negative role for AUF1 in poliovirus infection, as AUF1 inhibited viral translation and, ultimately, overall viral titers. Our findings also demonstrate that AUF1 functions as an antiviral factor during infection by coxsackievirus or human rhinovirus, suggesting a common mechanism that targets these related picornaviruses.

Cellular mRNA Decay Protein AUF1 Negatively Regulates Enterovirus and Human Rhinovirus Infections

PubMed Central

Cathcart, Andrea L.; Rozovics, Janet M.

2013-01-01

To successfully complete their replication cycles, picornaviruses modify several host proteins to alter the cellular environment to favor virus production. One such target of viral proteinase cleavage is AU-rich binding factor 1 (AUF1), a cellular protein that binds to AU-rich elements, or AREs, in the 3′ noncoding regions (NCRs) of mRNAs to affect the stability of the RNA. Previous studies found that, during poliovirus or human rhinovirus infection, AUF1 is cleaved by the viral proteinase 3CD and that AUF1 can interact with the long 5′ NCR of these viruses in vitro. Here, we expand on these initial findings to demonstrate that all four isoforms of AUF1 bind directly to stem-loop IV of the poliovirus 5′ NCR, an interaction that is inhibited through proteolytic cleavage of AUF1 by the viral proteinase 3CD. Endogenous AUF1 was observed to relocalize to the cytoplasm of infected cells in a viral protein 2A-driven manner and to partially colocalize with the viral protein 3CD. We identify a negative role for AUF1 in poliovirus infection, as AUF1 inhibited viral translation and, ultimately, overall viral titers. Our findings also demonstrate that AUF1 functions as an antiviral factor during infection by coxsackievirus or human rhinovirus, suggesting a common mechanism that targets these related picornaviruses. PMID:23903828

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

PubMed Central

Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

2015-01-01

Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

Functions of Replication Protein A as a Sensor of R Loops and a Regulator of RNaseH1

PubMed Central

Nguyen, Hai Dang; Yadav, Tribhuwan; Giri, Sumanprava; Saez, Borja; Graubert, Timothy A.; Zou, Lee

2017-01-01

R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here, we show that replication protein A (RPA), a ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability. PMID:28257700

Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus.

PubMed Central

Kanai, Akio; Oida, Hanako; Matsuura, Nana; Doi, Hirofumi

2003-01-01

We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic. PMID:12614195

N -Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hilimire, Thomas A.; Bennett, Ryan P.; Stewart, Ryan A.

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stemmore » loop with low nanomolar afinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.« less

N -Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

DOE PAGES

Hilimire, Thomas A.; Bennett, Ryan P.; Stewart, Ryan A.; ...

2015-10-23

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stemmore » loop with low nanomolar afinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.« less

A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5′ End or within the Genome of Feline Parvovirus and Can Modify Virus Replication

PubMed Central

Wang, Dai; Parrish, Colin R.

1999-01-01

Phage display of cDNA clones prepared from feline cells was used to identify host cell proteins that bound to DNA-containing feline panleukopenia virus (FPV) capsids but not to empty capsids. One gene found in several clones encoded a heterogeneous nuclear ribonucleoprotein (hnRNP)-related protein (DBP40) that was very similar in sequence to the A/B-type hnRNP proteins. DBP40 bound specifically to oligonucleotides representing a sequence near the 5′ end of the genome which is exposed on the outside of the full capsid but did not bind most other terminal sequences. Adding purified DBP40 to an in vitro fill-in reaction using viral DNA as a template inhibited the production of the second strand after nucleotide (nt) 289 but prior to nt 469. DBP40 bound to various regions of the viral genome, including a region between nt 295 and 330 of the viral genome which has been associated with transcriptional attenuation of the parvovirus minute virus of mice, which is mediated by a stem-loop structure of the DNA and cellular proteins. Overexpression of the protein in feline cells from a plasmid vector made them largely resistant to FPV infection. Mutagenesis of the protein binding site within the 5′ end viral genome did not affect replication of the virus. PMID:10438866

Conformational Control of the Binding of the Transactivation Domain of the MLL Protein and c-Myb to the KIX Domain of CREB

PubMed Central

Korkmaz, Elif Nihal; Nussinov, Ruth; Haliloğlu, Türkan

2012-01-01

The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL) transcription factor lead to the biologically active ternary MLL∶KIX∶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD) simulations for the MLL∶KIX∶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX∶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX∶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events. PMID:22438798

A Positive Autoregulatory BDNF Feedback Loop via C/EBPβ Mediates Hippocampal Memory Consolidation

PubMed Central

Bambah-Mukku, Dhananjay; Travaglia, Alessio; Chen, Dillon Y.; Pollonini, Gabriella

2014-01-01

Little is known about the temporal progression and regulation of the mechanisms underlying memory consolidation. Brain-derived-neurotrophic-factor (BDNF) has been shown to mediate the maintenance of memory consolidation, but the mechanisms of this regulation remain unclear. Using inhibitory avoidance (IA) in rats, here we show that a hippocampal BDNF-positive autoregulatory feedback loop via CCAAT-enhancer binding protein β (C/EBPβ) is necessary to mediate memory consolidation. At training, a very rapid, learning-induced requirement of BDNF accompanied by rapid de novo translation controls the induction of a persistent activation of cAMP-response element binding-protein (CREB) and C/EBPβ expression. The latter, in turn, controls an increase in expression of bdnf exon IV transcripts and BDNF protein, both of which are necessary and, together with the initial BDNF requirement, mediate memory consolidation. The autoregulatory loop terminates by 48 h after training with decreased C/EBPβ and pCREB and increased methyl-CpG binding protein-2, histone-deacetylase-2, and switch-independent-3a binding at the bdnf exon IV promoter. PMID:25209292

Vital Roles of the Second DNA-binding Site of Rad52 Protein in Yeast Homologous Recombination*

PubMed Central

Arai, Naoto; Kagawa, Wataru; Saito, Kengo; Shingu, Yoshinori; Mikawa, Tsutomu; Kurumizaka, Hitoshi; Shibata, Takehiko

2011-01-01

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a “recombination mediator” to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing. PMID:21454474

Functional significance of the E loop, a novel motif conserved in the lantibiotic immunity ATP-binding cassette transport systems.

PubMed

Okuda, Ken-ichi; Yanagihara, Sae; Sugayama, Tomomichi; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

2010-06-01

Lantibiotics are peptide-derived antibacterial substances produced by some Gram-positive bacteria and characterized by the presence of unusual amino acids, like lanthionines and dehydrated amino acids. Because lantibiotic producers may be attacked by self-produced lantibiotics, they express immunity proteins on the cytoplasmic membrane. An ATP-binding cassette (ABC) transport system mediated by the LanFEG protein complex is a major system in lantibiotic immunity. Multiple-sequence alignment analysis revealed that LanF proteins contain the E loop, a variant of the Q loop, which is a well-conserved motif in the nucleotide-binding domains (NBDs) of general ABC transporters. To elucidate E loop function, we introduced a mutation in the NukF protein, which is involved in the nukacin-ISK-1 immunity system. Amino acid replacement of glutamic acid in the E loop with glutamine (E85Q) resulted in slight decreases in the immunity level and transport activity. Additionally, the E85A mutation severely impaired the immunity level and transport activity. On the other hand, ATPase activities of purified E85Q and E85A mutants were almost similar to that of the wild type. These results suggested that the E loop found in ABC transporters involved in lantibiotic immunity plays a significant role in the function of these transporters, especially in the structural change of transmembrane domains.

Inhibition of Influenza H7 Hemagglutinin-Mediated Entry

PubMed Central

Antanasijevic, Aleksandar; Cheng, Han; Wardrop, Duncan J.; Rong, Lijun; Caffrey, Michael

2013-01-01

The recent outbreak of H7N9 influenza in China is of high concern to public health. H7 hemagglutinin (HA) plays a critical role in influenza entry and thus HA presents an attractive target for antivirals. Previous studies have suggested that the small molecule tert-butyl hydroquinone (TBHQ) inhibits the entry of influenza H3 HA by binding to the stem loop of HA and stabilizing the neutral pH conformation of HA, thereby disrupting the membrane fusion step. Based on amino acid sequence, structure and immunogenicity, H7 is a related Group 2 HA. In this work we show, using a pseudovirus entry assay, that TBHQ inhibits H7 HA-mediated entry, as well as H3 HA-mediated entry, with an IC50∼6 µM. Using NMR, we show that TBHQ binds to the H7 stem loop region. STD NMR experiments indicate that the aromatic ring of TBHQ makes extensive contact with the H7 HA surface. Limited proteolysis experiments indicate that TBHQ inhibits influenza entry by stabilizing the H7 HA neutral pH conformation. Together, this work suggests that the stem loop region of H7 HA is an attractive target for therapeutic intervention and that TBHQ, which is a widely used food preservative, is a promising lead compound. PMID:24194835

hLARP7 C-terminal domain contains an xRRM that binds the 3' hairpin of 7SK RNA

DOE PAGES

Eichhorn, Catherine D.; Chug, Rahul; Feigon, Juli

2016-09-26

The 7SK small nuclear ribonucleoprotein (snRNP) sequesters and inactivates the positive transcription elongation factor b (P-TEFb), an essential eukaryotic mRNA transcription factor. The human La-related protein group 7 (hLARP7) is a constitutive component of the 7SK snRNP and localizes to the 3' terminus of the 7SK long noncoding RNA. hLARP7, and in particular its C-terminal domain (CTD), is essential for 7SK RNA stability and assembly with P-TEFb. The hLARP7 N-terminal Lamodule binds and protects the 3' end from degradation, but the structural and functional role of its CTD is unclear.We report the solution NMR structure of the hLARP7 CTD andmore » show that this domain contains an xRRM, a class of atypical RRM first identified in the Tetrahymena thermophila telomerase LARP7 protein p65. The xRRM binds the 3' end of 7SK RNA at the top of stem-loop 4 (SL4) and interacts with both unpaired and base-paired nucleotides. This study thus confirms that the xRRM is general to the LARP7 family of proteins and defines the binding site for hLARP7 on the 7SK RNA, providing insight into function.« less

hLARP7 C-terminal domain contains an xRRM that binds the 3' hairpin of 7SK RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eichhorn, Catherine D.; Chug, Rahul; Feigon, Juli

The 7SK small nuclear ribonucleoprotein (snRNP) sequesters and inactivates the positive transcription elongation factor b (P-TEFb), an essential eukaryotic mRNA transcription factor. The human La-related protein group 7 (hLARP7) is a constitutive component of the 7SK snRNP and localizes to the 3' terminus of the 7SK long noncoding RNA. hLARP7, and in particular its C-terminal domain (CTD), is essential for 7SK RNA stability and assembly with P-TEFb. The hLARP7 N-terminal Lamodule binds and protects the 3' end from degradation, but the structural and functional role of its CTD is unclear.We report the solution NMR structure of the hLARP7 CTD andmore » show that this domain contains an xRRM, a class of atypical RRM first identified in the Tetrahymena thermophila telomerase LARP7 protein p65. The xRRM binds the 3' end of 7SK RNA at the top of stem-loop 4 (SL4) and interacts with both unpaired and base-paired nucleotides. This study thus confirms that the xRRM is general to the LARP7 family of proteins and defines the binding site for hLARP7 on the 7SK RNA, providing insight into function.« less

Molecular dynamics simulations of human tRNA Lys,3 UUU: the role of modified bases in mRNA recognition.

PubMed

McCrate, Nina E; Varner, Mychel E; Kim, Kenneth I; Nagan, Maria C

2006-01-01

Accuracy in translation of the genetic code into proteins depends upon correct tRNA-mRNA recognition in the context of the ribosome. In human tRNA(Lys,3)UUU three modified bases are present in the anticodon stem-loop--2-methylthio-N6-threonylcarbamoyladenosine at position 37 (ms2t6A37), 5-methoxycarbonylmethyl-2-thiouridine at position 34 (mcm5s2U34) and pseudouridine (psi) at position 39--two of which, ms2t6A37 and mcm5s2U34, are required to achieve wild-type binding activity of wild-type human tRNA(Lys,3)UUU [C. Yarian, M. Marszalek, E. Sochacka, A. Malkiewicz, R. Guenther, A. Miskiewicz and P. F. Agris (2000) Biochemistry, 39, 13390-13395]. Molecular dynamics simulations of nine tRNA anticodon stem-loops with different combinations of nonstandard bases were performed. The wild-type simulation exhibited a canonical anticodon stair-stepped conformation. The ms2t6 modification at position 37 is required for maintenance of this structure and reduces solvent accessibility of U36. Ms2t6A37 generally hydrogen bonds across the loop and may prevent U36 from rotating into solution. A water molecule does coordinate to psi39 most of the simulation time but weakly, as most of the residence lifetimes are <40 ps.

Toxin MqsR Cleaves Single-Stranded mRNA with Various 5 Ends

DTIC Science & Technology

2016-08-24

either protein ORIGINAL RESEARCH Toxin MqsR cleaves single- stranded mRNA with various 5’ ends Nityananda Chowdhury1,*, Brian W. Kwan1,*, Louise C...in which a single 5′- GCU site was predicted to be single- stranded (ssRNA), double- stranded (dsRNA), in the loop of a stem - loop (slRNA), or in a...single- stranded 5′- GCU sites since cleavage was approximately 20- fold higher than cleavage seen with the 5′- GCU site in the stem - loop and

Autoregulatory mechanisms controlling the Microprocessor.

PubMed

Triboulet, Robinson; Gregory, Richard I

2010-01-01

The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature 22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part ofa newly identified regulatory mechanism controlling Microprocessor activity.

Identification and Functional Characterization of a Novel Acetylcholine-binding Protein from the Marine Annelid Capitella teleta

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCormack, T.; Petrovich,; Mercier, K

2010-01-01

We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) {alpha} subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has beenmore » implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and {alpha}-bungarotoxin bind to ct-AChBP with high affinity, with KD values of 28.7 {micro}M, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K{sub D} = 163 {micro}M). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.« less

α -Actinin TvACTN3 of Trichomonas vaginalis is an RNA-binding protein that could participate in its posttranscriptional iron regulatory mechanism.

PubMed

Calla-Choque, Jaeson Santos; Figueroa-Angulo, Elisa Elvira; Ávila-González, Leticia; Arroyo, Rossana

2014-01-01

Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

α-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism

PubMed Central

Calla-Choque, Jaeson Santos; Figueroa-Angulo, Elisa Elvira; Ávila-González, Leticia; Arroyo, Rossana

2014-01-01

Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system. PMID:24719864

Functional and structural analysis of the sialic acid-binding domain of rotaviruses.

PubMed Central

Isa, P; López, S; Segovia, L; Arias, C F

1997-01-01

The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8. To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase. Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA. Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay. However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs). These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule. The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9. The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8. PMID:9261399

The RNA-binding protein repertoire of embryonic stem cells.

PubMed

Kwon, S Chul; Yi, Hyerim; Eichelbaum, Katrin; Föhr, Sophia; Fischer, Bernd; You, Kwon Tae; Castello, Alfredo; Krijgsveld, Jeroen; Hentze, Matthias W; Kim, V Narry

2013-09-01

RNA-binding proteins (RBPs) have essential roles in RNA-mediated gene regulation, and yet annotation of RBPs is limited mainly to those with known RNA-binding domains. To systematically identify the RBPs of embryonic stem cells (ESCs), we here employ interactome capture, which combines UV cross-linking of RBP to RNA in living cells, oligo(dT) capture and MS. From mouse ESCs (mESCs), we have defined 555 proteins constituting the mESC mRNA interactome, including 283 proteins not previously annotated as RBPs. Of these, 68 new RBP candidates are highly expressed in ESCs compared to differentiated cells, implicating a role in stem-cell physiology. Two well-known E3 ubiquitin ligases, Trim25 (also called Efp) and Trim71 (also called Lin41), are validated as RBPs, revealing a potential link between RNA biology and protein-modification pathways. Our study confirms and expands the atlas of RBPs, providing a useful resource for the study of the RNA-RBP network in stem cells.

RTVP-1 promotes mesenchymal transformation of glioma via a STAT-3/IL-6-dependent positive feedback loop

PubMed Central

Giladi, Nis David; Ziv-Av, Amotz; Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Ben-Asher, Hiba Waldman; deCarvalho, Ana; Mikkelsen, Tom; Poisson, Laila; Brodie, Chaya

2015-01-01

Glioblastomas (GBMs), the most aggressive primary brain tumors, exhibit increased invasiveness and resistance to anti-tumor treatments. We explored the role of RTVP-1, a glioma-associated protein that promotes glioma cell migration, in the mesenchymal transformation of GBM. Analysis of The Cancer Genome Atlas (TCGA) demonstrated that RTVP-1 expression was higher in mesenchymal GBM and predicted tumor recurrence and poor clinical outcome. ChiP analysis revealed that the RTVP-1 promoter binds STAT3 and C/EBPβ, two master transcription factors that regulate mesenchymal transformation of GBM. In addition, IL-6 induced RTVP-1 expression in a STAT3-dependent manner. RTVP-1 increased the migration and mesenchymal transformation of glioma cells. Similarly, overexpression of RTVP-1 in human neural stem cells induced mesenchymal differentiation, whereas silencing of RTVP-1 in glioma stem cells (GSCs) decreased the mesenchymal transformation and stemness of these cells. Silencing of RTVP-1 also increased the survival of mice bearing GSC-derived xenografts. Using gene array analysis of RTVP-1 silenced glioma cells we identified IL-6 as a mediator of RTVP-1 effects on the mesenchymal transformation and migration of GSCs, therefore acting in a positive feedback loop by upregulating RTVP-1 expression via the STAT3 pathway. Collectively, these results implicate RTVP-1 as a novel prognostic marker and therapeutic target in GBM. PMID:26267319

Rhodopsin TM6 Can Interact with Two Separate and Distinct Sites on Arrestin: Evidence for Structural Plasticity and Multiple Docking Modes in Arrestin–Rhodopsin Binding

PubMed Central

2015-01-01

Various studies have implicated the concave surface of arrestin in the binding of the cytosolic surface of rhodopsin. However, specific sites of contact between the two proteins have not previously been defined in detail. Here, we report that arrestin shares part of the same binding site on rhodopsin as does the transducin Gα subunit C-terminal tail, suggesting binding of both proteins to rhodopsin may share some similar underlying mechanisms. We also identify two areas of contact between the proteins near this region. Both sites lie in the arrestin N-domain, one in the so-called “finger” loop (residues 67–79) and the other in the 160 loop (residues 155–165). We mapped these sites using a novel tryptophan-induced quenching method, in which we introduced Trp residues into arrestin and measured their ability to quench the fluorescence of bimane probes attached to cysteine residues on TM6 of rhodopsin (T242C and T243C). The involvement of finger loop binding to rhodopsin was expected, but the evidence of the arrestin 160 loop contacting rhodopsin was not. Remarkably, our data indicate one site on rhodopsin can interact with multiple structurally separate sites on arrestin that are almost 30 Å apart. Although this observation at first seems paradoxical, in fact, it provides strong support for recent hypotheses that structural plasticity and conformational changes are involved in the arrestin–rhodopsin binding interface and that the two proteins may be able to interact through multiple docking modes, with arrestin binding to both monomeric and dimeric rhodopsin. PMID:24724832

Rhodopsin TM6 can interact with two separate and distinct sites on arrestin: evidence for structural plasticity and multiple docking modes in arrestin-rhodopsin binding.

PubMed

Sinha, Abhinav; Jones Brunette, Amber M; Fay, Jonathan F; Schafer, Christopher T; Farrens, David L

2014-05-27

Various studies have implicated the concave surface of arrestin in the binding of the cytosolic surface of rhodopsin. However, specific sites of contact between the two proteins have not previously been defined in detail. Here, we report that arrestin shares part of the same binding site on rhodopsin as does the transducin Gα subunit C-terminal tail, suggesting binding of both proteins to rhodopsin may share some similar underlying mechanisms. We also identify two areas of contact between the proteins near this region. Both sites lie in the arrestin N-domain, one in the so-called "finger" loop (residues 67-79) and the other in the 160 loop (residues 155-165). We mapped these sites using a novel tryptophan-induced quenching method, in which we introduced Trp residues into arrestin and measured their ability to quench the fluorescence of bimane probes attached to cysteine residues on TM6 of rhodopsin (T242C and T243C). The involvement of finger loop binding to rhodopsin was expected, but the evidence of the arrestin 160 loop contacting rhodopsin was not. Remarkably, our data indicate one site on rhodopsin can interact with multiple structurally separate sites on arrestin that are almost 30 Å apart. Although this observation at first seems paradoxical, in fact, it provides strong support for recent hypotheses that structural plasticity and conformational changes are involved in the arrestin-rhodopsin binding interface and that the two proteins may be able to interact through multiple docking modes, with arrestin binding to both monomeric and dimeric rhodopsin.

Drosophila stem loop binding protein coordinates accumulation of mature histone mRNA with cell cycle progression

PubMed Central

Sullivan, Eileen; Santiago, Carlos; Parker, Emily D.; Dominski, Zbigniew; Yang, Xiaocui; Lanzotti, David J.; Ingledue, Tom C.; Marzluff, William F.; Duronio, Robert J.

2001-01-01

Replication-associated histone genes encode the only metazoan mRNAs that lack polyA tails, ending instead in a conserved 26-nt sequence that forms a stem–loop. Most of the regulation of mammalian histone mRNA is posttranscriptional and mediated by this unique 3′ end. Stem–loop–binding protein (SLBP) binds to the histone mRNA 3′ end and is thought to participate in all aspects of histone mRNA metabolism, including cell cycle regulation. To examine SLBP function genetically, we have cloned the gene encoding Drosophila SLBP (dSLBP) by a yeast three-hybrid method and have isolated mutations in dSLBP. dSLBP function is required both zygotically and maternally. Strong dSLBP alleles cause zygotic lethality late in development and result in production of stable histone mRNA that accumulates in nonreplicating cells. These histone mRNAs are cytoplasmic and have polyadenylated 3′ ends like other polymerase II transcripts. Hypomorphic dSLBP alleles support zygotic development but cause female sterility. Eggs from these females contain dramatically reduced levels of histone mRNA, and mutant embryos are not able to complete the syncytial embryonic cycles. This is in part because of a failure of chromosome condensation at mitosis that blocks normal anaphase. These data demonstrate that dSLBP is required in vivo for 3′ end processing of histone pre-mRNA, and that this is an essential function for development. Moreover, dSLBP-dependent processing plays an important role in coupling histone mRNA production with the cell cycle. PMID:11157774

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting.

PubMed

Herbert, Kristina M; Sarkar, Susanta K; Mills, Maria; Delgado De la Herran, Hilda C; Neuman, Keir C; Steitz, Joan A

2016-02-01

During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem-loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins. © 2016 Herbert et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Crystal structures of the archaeal RNase P protein Rpp38 in complex with RNA fragments containing a K-turn motif.

PubMed

Oshima, Kosuke; Gao, Xuzhu; Hayashi, Seiichiro; Ueda, Toshifumi; Nakashima, Takashi; Kimura, Makoto

2018-01-01

A characteristic feature of archaeal ribonuclease P (RNase P) RNAs is that they have extended helices P12.1 and P12.2 containing kink-turn (K-turn) motifs to which the archaeal RNase P protein Rpp38, a homologue of the human RNase P protein Rpp38, specifically binds. PhoRpp38 from the hyperthermophilic archaeon Pyrococcus horikoshii is involved in the elevation of the optimum temperature of the reconstituted RNase P by binding the K-turns in P12.1 and P12.2. Previously, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was determined at 3.4 Å resolution. In this study, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was improved to 2.1 Å resolution and the structure of PhoRpp38 in complex with the K-turn in P12.1 was also determined at a resolution of 3.1 Å. Both structures revealed that Lys35, Asn38 and Glu39 in PhoRpp38 interact with characteristic G·A and A·G pairs in the K-turn, while Thr37, Asp59, Lys84, Glu94, Ala96 and Ala98 in PhoRpp38 interact with the three-nucleotide bulge in the K-turn. Moreover, an extended stem-loop containing P10-P12.2 in complex with PhoRpp38, as well as PhoRpp21 and PhoRpp29, which are the archaeal homologues of the human proteins Rpp21 and Rpp29, respectively, was affinity-purified and crystallized. The crystals thus grown diffracted to a resolution of 6.35 Å. Structure determination of the crystals will demonstrate the previously proposed secondary structure of stem-loops including helices P12.1 and P12.2 and will also provide insight into the structural organization of the specificity domain in P. horikoshii RNase P RNA.

Highly sensitive self-complementary DNA nanoswitches triggered by polyelectrolytes

NASA Astrophysics Data System (ADS)

Wu, Jincai; Yu, Feng; Zhang, Zheng; Chen, Yong; Du, Jie; Maruyama, Atsushi

2015-12-01

Dimerization of two homologous strands of genomic DNA/RNA is an essential feature of retroviral replication. Herein we show that a cationic comb-type copolymer (CCC), poly(l-lysine)-graft-dextran, accelerates the dimerization of self-complementary stem-loop DNA, frequently found in functional DNA/RNA molecules, such as aptamers. Furthermore, an anionic polymer poly(sodium vinylsulfonate) (PVS) dissociates CCC from the duplex shortly within a few seconds. Then single stem-loop DNA spontaneously transforms from its dimer. Thus we can easily control the dimer and stem-loop DNA by switching on/off CCC activity. Both polyelectrolytes and DNA concentrations are in the nanomole per liter range. The polyelectrolyte-assisted transconformation and sequences design strategy ensures the reversible state control with rapid response and effective switching under physiologically relevant conditions. A further application of this sensitive assembly is to construct an aptamer-type drug delivery system, bind or release functional molecules responding to its transconformation.Dimerization of two homologous strands of genomic DNA/RNA is an essential feature of retroviral replication. Herein we show that a cationic comb-type copolymer (CCC), poly(l-lysine)-graft-dextran, accelerates the dimerization of self-complementary stem-loop DNA, frequently found in functional DNA/RNA molecules, such as aptamers. Furthermore, an anionic polymer poly(sodium vinylsulfonate) (PVS) dissociates CCC from the duplex shortly within a few seconds. Then single stem-loop DNA spontaneously transforms from its dimer. Thus we can easily control the dimer and stem-loop DNA by switching on/off CCC activity. Both polyelectrolytes and DNA concentrations are in the nanomole per liter range. The polyelectrolyte-assisted transconformation and sequences design strategy ensures the reversible state control with rapid response and effective switching under physiologically relevant conditions. A further application of this sensitive assembly is to construct an aptamer-type drug delivery system, bind or release functional molecules responding to its transconformation. Electronic supplementary information (ESI) available: I. Sequences of DIS25, DIS25-2a and DIS25-3a. II. Structural formula of poly(l-lysine)-graft-dextran (PLL-g-Dex). 1H-NMR spectra of PLL-g-Dex in D2O. III. Gel electrophoretic analysis of dimerization of DIS25 with various N/P ratios. IV. The effect of polyelectrolyte on the fluorescence polarity of TAMRA-labeled duplex. V. UV absorption/Tm profiles of DIS25. VI. Arrhenius plots for spontaneous dissociation of the DIS25 dimer and PLL-g-Dex-assisted dimerization of DIS25.VII. Switching between double stem-loop DIS42 and extended multiplex drived by PLL-g-Dex and PVS. See DOI: 10.1039/c5nr05193b

Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12.

PubMed

Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Amaro, Itzel; Ortíz, Ernesto; Becerril, Baltazar; Ibarra, Jorge E; Bravo, Alejandra; Soberón, Mario

2015-04-01

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reverse Transcription of a Self-Primed Retrotransposon Requires an RNA Structure Similar to the U5-IR Stem-Loop of Retroviruses

PubMed Central

Lin, Jia-Hwei; Levin, Henry L.

1998-01-01

An inverted repeat (IR) within the U5 region of the Rous sarcoma virus (RSV) mRNA forms a structure composed of a 7-bp stem and a 5-nucleotide (nt) loop. This U5-IR structure has been shown to be required for the initiation of reverse transcription. The mRNA of Tf1, long terminal repeat-containing retrotransposon from fission yeast (Schizosaccharomyces pombe) contains nucleotides with the potential to form a U5-IR stem-loop that is strikingly similar to that of RSV. The putative U5-IR stem-loop of Tf1 consists of a 7-bp stem and a 25-nt loop. Results from mutagenesis studies indicate that the U5-IR stem-loop in the mRNA of Tf1 does form and that it is required for Tf1 transposition. Although the loop is required for transposition, we were surprised that the specific sequence of the nucleotides within the loop was unimportant for function. Additional investigation indicates that the loss of transposition activity due to a reduction in the loop size to 6 nt could be rescued by increasing the GC content of the stem. This result indicates that the large loop in the Tf1 mRNA relative to that of the RSV allows the formation of the relatively weak U5-IR stem. The levels of Tf1 proteins expressed and the amounts of Tf1 RNA packaged into the virus-like particles were not affected by mutations in the U5-IR structure. However, all of the mutations in the U5-IR structure that caused defects in transposition produced low amounts of reverse transcripts. A unique feature in the initiation of Tf1 reverse transcription is that, instead of a tRNA, the first 11 nt of the Tf1 mRNA serve as the minus-strand primer. Analysis of the 5′ end of Tf1 mRNA revealed that the mutations in the U5-IR stem-loop that resulted in defects in reverse transcription caused a reduction in the cleavage activity required to generate the Tf1 primer. Our results indicate that the U5-IR stems of Tf1 and RSV are conserved in size, position, and function. PMID:9774699

Heat Capacity Changes and Disorder-to-Order Transitions in Allosteric Activation.

PubMed

Cressman, William J; Beckett, Dorothy

2016-01-19

Allosteric coupling in proteins is ubiquitous but incompletely understood, particularly in systems characterized by coupling over large distances. Binding of the allosteric effector, bio-5'-AMP, to the Escherichia coli biotin protein ligase, BirA, enhances the protein's dimerization free energy by -4 kcal/mol. Previous studies revealed that disorder-to-order transitions at the effector binding and dimerization sites, which are separated by 33 Å, are integral to functional coupling. Perturbations to the transition at the ligand binding site alter both ligand binding and coupled dimerization. Alanine substitutions in four loops on the dimerization surface yield a range of energetic effects on dimerization. A glycine to alanine substitution at position 142 in one of these loops results in a complete loss of allosteric coupling, disruption of the disorder-to-order transitions at both functional sites, and a decreased affinity for the effector. In this work, allosteric communication between the effector binding and dimerization surfaces in BirA was further investigated by performing isothermal titration calorimetry measurements on nine proteins with alanine substitutions in three dimerization surface loops. In contrast to BirAG142A, at 20 °C all variants bind to bio-5'-AMP with free energies indistinguishable from that measured for wild-type BirA. However, the majority of the variants exhibit altered heat capacity changes for effector binding. Moreover, the ΔCp values correlate with the dimerization free energies of the effector-bound proteins. These thermodynamic results, combined with structural information, indicate that allosteric activation of the BirA monomer involves formation of a network of intramolecular interactions on the dimerization surface in response to bio-5'-AMP binding at the distant effector binding site.

The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constantsmore » for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5–α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5–α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.« less

Structure of adenovirus bound to cellular receptor car

DOEpatents

Freimuth, Paul I.

2007-01-02

Disclosed is a mutant CAR-DI-binding adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have a significantly weakened binding affinity for CAR-DI relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type.

Search for Functional Flexible Regions in the G-protein Family: New Reading of the FoldUnfold Program.

PubMed

Galzitskaya, Oxana; Deryusheva, Eugenia; Machulin, Andrey; Nemashkalova, Ekaterina; Glyakina, Anna

2018-06-21

High prediction accuracy of flexible loops in different protein families is a challenge because of the crucial functions associated with these regions. Results of the currently available programs for prediction of loops vary from protein to protein. For prediction of flexible regions in the G-domain for 23 representatives of G-proteins with the known 3D structure we have used eight programs. The results of predictions demonstrate that the FoldUnfold program predicts better loop positions than the PONDR, RОNN, DisEMBL, IUPred, GlobPlot 2, FoldIndex, and MobiDB programs. When classifying the predicted loops (rigid/flexible) according to the Debye-Waller fluctuation factors, our data reveal the existing weak correlation between the B-factors and the average number of closed residues according to the FoldUnfold program; the percentage of overlapping characteristics (residue fold/unfold status) of the protein residues from the two methods is about 60-70%. According to the FoldUnfold program, for G-proteins with the posttranslational modifications, the surrounding binding site residues by disordered-promoting glycine and alanine residues conduces to a more flexible position of the binding sites for fatty acid, while methionine, cysteine and isoleucine residues provide more rigid binding sites. Thus, our research demonstrates additional possibilities of the FoldUnfold program for prediction of flexible regions and characteristics of individual residues in a different protein family. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase.

PubMed

Titushin, Maxim S; Markova, Svetlana V; Frank, Ludmila A; Malikova, Natalia P; Stepanyuk, Galina A; Lee, John; Vysotski, Eugene S

2008-02-01

The Renilla bioluminescent system in vivo is comprised of three proteins--the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca(2+)-binding superfamily of proteins with only three of the EF-hand loops having the Ca(2+)-binding consensus sequences. There is weak sequence homology with the Ca(2+)-regulated photoproteins but only as a result of the necessary Ca(2+)-binding loop structure. In combination with Renilla luciferase, addition of only one Ca(2+) is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.

Disturbances of Ligand Potency and Enhanced Degradation of the Human Glycine Receptor at Affected Positions G160 and T162 Originally Identified in Patients Suffering from Hyperekplexia

PubMed Central

Atak, Sinem; Langlhofer, Georg; Schaefer, Natascha; Kessler, Denise; Meiselbach, Heike; Delto, Carolyn; Schindelin, Hermann; Villmann, Carmen

2015-01-01

Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GlyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GlyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GlyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, T162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof. PMID:26733802

Mechanism of Disruption of the Amt-GlnK Complex by PII-Mediated Sensing of 2-Oxoglutarate

PubMed Central

Maier, Sarah; Schleberger, Paula; Lü, Wei; Wacker, Tobias; Pflüger, Tobias; Litz, Claudia; Andrade, Susana L. A.

2011-01-01

GlnK proteins regulate the active uptake of ammonium by Amt transport proteins by inserting their regulatory T-loops into the transport channels of the Amt trimer and physically blocking substrate passage. They sense the cellular nitrogen status through 2-oxoglutarate, and the energy level of the cell by binding both ATP and ADP with different affinities. The hyperthermophilic euryarchaeon Archaeoglobus fulgidus possesses three Amt proteins, each encoded in an operon with a GlnK ortholog. One of these proteins, GlnK2 was recently found to be incapable of binding 2-OG, and in order to understand the implications of this finding we conducted a detailed structural and functional analysis of a second GlnK protein from A. fulgidus, GlnK3. Contrary to Af-GlnK2 this protein was able to bind both ATP/2-OG and ADP to yield inactive and functional states, respectively. Due to the thermostable nature of the protein we could observe the exact positioning of the notoriously flexible T-loops and explain the binding behavior of GlnK proteins to their interaction partner, the Amt proteins. A thermodynamic analysis of these binding events using microcalorimetry evaluated by microstate modeling revealed significant differences in binding cooperativity compared to other characterized PII proteins, underlining the diversity and adaptability of this class of regulatory signaling proteins. PMID:22039461

Dissecting binding of a β-barrel membrane protein by phage display.

PubMed

Meneghini, Luz M; Tripathi, Sarvind; Woodworth, Marcus A; Majumdar, Sudipta; Poulos, Thomas L; Weiss, Gregory A

2017-07-25

Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal. Here, we apply a new approach for the expedited protein characterization of the TBDT ShuA from Shigella dysenteriae, and elucidate the protein's initial steps during heme-uptake. ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable MP variants for rapid characterization of binding to various ligands. Site-directed mutagenesis studies targeting each extracellular loop region of ShuA demonstrate no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. The results identify a loop susceptible to antibody binding, and also a small molecule motif capable of disrupting ShuA from S. dysenteriae. The approach is generalizable to the dissection of other phage-displayed TBDTs and MPs.

Isolation and characterization of an RNA aptamer for the HPV-16 E7 oncoprotein.

PubMed

Toscano-Garibay, Julia D; Benítez-Hess, María L; Alvarez-Salas, Luis M

2011-02-01

Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer. Copyright © 2011 IMSS. Published by Elsevier Inc. All rights reserved.

bicoid RNA localization requires specific binding of an endosomal sorting complex

PubMed Central

Irion, Uwe; St Johnston, Daniel

2007-01-01

Summary paragraph: bicoid mRNA localises to the anterior of the Drosophila egg, where it is translated to form a morphogen gradient of Bicoid protein that patterns the head and thorax of the embryo. Although bicoid was the first identified localised cytoplasmic determinant1-4, little is known about how the mRNA is coupled to the microtubule-dependent transport pathway that targets it to the anterior, and it has been proposed that it is recognised by a complex of many redundant proteins, each of which binds to the localisation element in its 3'UTR with little or no specificity5. Indeed, the only known RNA-binding protein that co-localises with bicoid mRNA is Staufen, which binds non-specifically to dsRNA in vitro6, 7. Here we show that mutants in all subunits of the ESCRT-II complex (Vps22, Vps25 and Vps36) abolish the final Staufen-dependent step in bcd RNA localisation. ESCRT-II is a highly conserved component of the pathway that sorts ubiquitinated endosomal proteins into internal vesicles8, 9, and functions as a tumour-suppressor by removing activated receptors from the cytoplasm10, 11. However, the role of ESCRT-II in bicoid localisation appears to be independent of endosomal sorting, because mutations in ESCRT-I and III components have no effect of the targeting of bicoid mRNA. Instead, Vps36 functions by binding directly and specifically to stem-loop V of the bicoid 3'UTR through its N-terminal GLUE domain12, making it the first example of a sequence specific RNA-binding protein that recognises the bicoid localisation signal. Furthermore, Vps36 localises to the anterior of the oocyte in a bicoid mRNA-dependent manner, and is required for the subsequent recruitment of Staufen to the bicoid complex. This novel function of ESCRT-II as an RNA-binding complex is conserved in vertebrates, and may explain some of its roles that are independent of endosomal sorting. PMID:17268469

Translational control of ribosomal protein S15.

PubMed

Portier, C; Philippe, C; Dondon, L; Grunberg-Manago, M; Ebel, J P; Ehresmann, B; Ehresmann, C

1990-08-27

The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene. Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger. In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes. The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon. Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot. Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15. A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.

Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

PubMed

Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J

2014-01-07

Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. Copyright © 2014 Elsevier Ltd. All rights reserved.

R-spondin1/Wnt-enhanced Ascl2 autoregulation controls the self-renewal of colorectal cancer progenitor cells.

PubMed

Ye, Jun; Liu, Shanxi; Shang, Yangyang; Chen, Haoyuan; Wang, Rongquan

2018-06-25

The Wnt signaling pathway controls stem cell identity in the intestinal epithelium and cancer stem cells (CSCs). The transcription factor Ascl2 (Wnt target gene) is fate decider of intestinal cryptic stem cells and colon cancer stem cells. It is unclear how Wnt signaling is translated into Ascl2 expression and keeping the self-renewal of CRC progenitor cells. We showed that the exogenous Ascl2 in colorectal cancer (CRC) cells activated the endogenous Ascl2 expression via a direct autoactivatory loop, including Ascl2 binding to its own promoter and further transcriptional activation. Higher Ascl2 expression in human CRC cancerous tissues led to greater enrichment in Ascl2 immunoprecipitated DNA within the Ascl2 promoter in the CRC cancerous sample than the peri-cancerous mucosa. Ascl2 binding to its own promoter and inducing further transcriptional activation of the Ascl2 gene was predominant in the CD133 + CD44 + CRC population. R-spondin1/Wnt activated Ascl2 expression dose-dependently in the CD133 + CD44 + CRC population, but not in the CD133 - CD44 - CRC population, which was caused by differences in Ascl2 autoregulation under R-spondin1/Wnt activation. R-spondin1/Wnt treatment in the CD133 + CD44 + or CRC CD133 - CD44 - populations exerted a different pattern of stemness maintenance, which was defined by alterations of the mRNA levels of stemness-associated genes, the protein expression levels (Bmi1, C-myc, Oct-4 and Nanog) and tumorsphere formation. The results indicated that Ascl2 autoregulation formed a transcriptional switch that was enhanced by Wnt signaling in the CD133 + CD44 + CRC population, thus conferring their self-renewal.

Alteration of the C-terminal ligand specificity of the erbin PDZ domain by allosteric mutational effects.

PubMed

Murciano-Calles, Javier; McLaughlin, Megan E; Erijman, Ariel; Hooda, Yogesh; Chakravorty, Nishant; Martinez, Jose C; Shifman, Julia M; Sidhu, Sachdev S

2014-10-23

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity. Copyright © 2014 Elsevier Ltd. All rights reserved.

RNA binding protein Lin28B confers gastric cancer cells stemness via directly binding to NRP-1.

PubMed

Wang, Xiaocong; Hu, Huihua; Liu, Hebo

2018-05-19

This work aims to explore the roles and related mechanisms of RNA binding protein Lin28B in gastric cancer cells stemness. We found that Lin28B expression was negatively correlated with the overall survival (OS) of gastric cancer patients, and significantly increased in gastric cancer cells compared with that in gastric epithelial cells. Lin28B overexpression increased spheroid formation, expression of gastric cancer stemness-related markers, and decreased cisplatin sensitivity in gastric cancer cells. Mechanistically, Lin28B could directly bind to NRP-1 3'UTR, thus increasing NRP-1 mRNA stability and expression, and activate the downstream Wnt/β-catenin signaling. Knockdown of NRP-1 or treatment with Wnt/β-catenin antagonist could rescue the promotive effects of Lin28B on gastric cancer stemness. Thus, thes results indicate that Lin28B could facilitate gastric cancer stemness via directly binding to NRP-1 3'UTR and activating the downstream Wnt/β-catenin signaling. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Dynamics and unfolding pathway of chimeric azurin variants: insights from molecular dynamics simulation.

PubMed

Evoli, Stefania; Guzzi, Rita; Rizzuti, Bruno

2013-10-01

The spectroscopic, thermal, and functional properties of blue copper proteins can be modulated by mutations in the metal binding loop. Molecular dynamics simulation was used to compare the conformational properties of azurin and two chimeric variants, which were obtained by inserting into the azurin scaffold the copper binding loop of amicyanin and plastocyanin, respectively. Simulations at room temperature show that the proteins retain their overall structure and exhibit concerted motions among specific inner regions, as revealed by principal component analysis. Molecular dynamics at high temperature indicates that the first events in the unfolding pathway are structurally similar in the three proteins and unfolding starts from the region of the α-helix that is far from the metal binding loop. The results provide details of the denaturation process that are consistent with experimental data and in close agreement with other computational approaches, suggesting a distinct mechanism of unfolding of azurin and its chimeric variants. Moreover, differences observed in the dynamics of specific regions in the three proteins correlate with their thermal behavior, contributing to the determination of the basic factors that influence the stability.

The HILDA Complex Coordinates a Conditional Switch in the 3′-Untranslated Region of the VEGFA mRNA

PubMed Central

Yao, Peng; Potdar, Alka A.; Ray, Partho Sarothi; Eswarappa, Sandeepa M.; Flagg, Andrew C.; Willard, Belinda; Fox, Paul L.

2013-01-01

Cell regulatory circuits integrate diverse, and sometimes conflicting, environmental cues to generate appropriate, condition-dependent responses. Here, we elucidate the components and mechanisms driving a protein-directed RNA switch in the 3′UTR of vascular endothelial growth factor (VEGF)-A. We describe a novel HILDA (hypoxia-inducible hnRNP L–DRBP76–hnRNP A2/B1) complex that coordinates a three-element RNA switch, enabling VEGFA mRNA translation during combined hypoxia and inflammation. In addition to binding the CA-rich element (CARE), heterogeneous nuclear ribonucleoprotein (hnRNP) L regulates switch assembly and function. hnRNP L undergoes two previously unrecognized, condition-dependent posttranslational modifications: IFN-γ induces prolyl hydroxylation and von Hippel-Lindau (VHL)-mediated proteasomal degradation, whereas hypoxia stimulates hnRNP L phosphorylation at Tyr359, inducing binding to hnRNP A2/B1, which stabilizes the protein. Also, phospho-hnRNP L recruits DRBP76 (double-stranded RNA binding protein 76) to the 3′UTR, where it binds an adjacent AU-rich stem-loop (AUSL) element, “flipping” the RNA switch by disrupting the GAIT (interferon-gamma-activated inhibitor of translation) element, preventing GAIT complex binding, and driving robust VEGFA mRNA translation. The signal-dependent, HILDA complex coordinates the function of a trio of neighboring RNA elements, thereby regulating translation of VEGFA and potentially other mRNA targets. The VEGFA RNA switch might function to ensure appropriate angiogenesis and tissue oxygenation during conflicting signals from combined inflammation and hypoxia. We propose the VEGFA RNA switch as an archetype for signal-activated, protein-directed, multi-element RNA switches that regulate posttranscriptional gene expression in complex environments. PMID:23976881

The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation

PubMed Central

Sundar, Shankar; Baker, Tania A; Sauer, Robert T

2012-01-01

In the AAA+ HslUV protease, substrates are bound and unfolded by a ring hexamer of HslU, before translocation through an axial pore and into the HslV degradation chamber. Here, we show that the N-terminal residues of an Arc substrate initially bind in the HslU axial pore, with key contacts mediated by a pore loop that is highly conserved in all AAA+ unfoldases. Disordered loops from the six intermediate domains of the HslU hexamer project into a funnel-shaped cavity above the pore and are positioned to contact protein substrates. Mutations in these I-domain loops increase KM and decrease Vmax for degradation, increase the mobility of bound substrates, and prevent substrate stimulation of ATP hydrolysis. HslU-ΔI has negligible ATPase activity. Thus, the I domain plays an active role in coordinating substrate binding, ATP hydrolysis, and protein degradation by the HslUV proteolytic machine. PMID:22102327

RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

PubMed

Stefanovic, Branko

2013-01-01

Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

Conserved Regulation of MAP Kinase Expression by PUF RNA-Binding Proteins

PubMed Central

Lee, Myon-Hee; Hook, Brad; Pan, Guangjin; Kershner, Aaron M; Merritt, Christopher; Seydoux, Geraldine; Thomson, James A; Wickens, Marvin; Kimble, Judith

2007-01-01

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression. PMID:18166083

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

PubMed

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines.

PubMed

Kristensen, Tatjana P; Maria Cherian, Reeja; Gray, Fiona C; MacNeill, Stuart A

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

PubMed

Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

2011-12-01

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

Structure-guided design of an engineered streptavidin with reusability to purify streptavidin-binding peptide tagged proteins or biotinylated proteins.

PubMed

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4 × 10(-14) M to 4.45 × 10(-10) M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

PubMed Central

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4×10−14 M to 4.45×10−10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible. PMID:23874971

Structural insights into the stabilization of the human immunodeficiency virus type 1 capsid protein by the cyclophilin-binding domain and implications on the virus cycle.

PubMed

Cortines, Juliana R; Lima, Luís Mauricio T R; Mohana-Borges, Ronaldo; Millen, Thiago de A; Gaspar, Luciane Pinto; Lanman, Jason K; Prevelige, Peter E; Silva, Jerson L

2015-05-01

During infection, human immunodeficiency virus type 1 (HIV-1) interacts with the cellular host factor cyclophilin A (CypA) through residues 85-93 of the N-terminal domain of HIV-1's capsid protein (CA). The role of the CA:CypA interaction is still unclear. Previous studies showed that a CypA-binding loop mutant, Δ87-97, has increased ability to assemble in vitro. We used this mutant to infer whether the CypA-binding region has an overall effect on CA stability, as measured by pressure and chemical perturbation. We built a SAXS-based envelope model for the dimer of both WT and Δ87-97. A new conformational arrangement of the dimers is described, showing the structural plasticity that CA can adopt. In protein folding studies, the deletion of the loop drastically reduces CA stability, as assayed by high hydrostatic pressure and urea. We hypothesize that the deletion promotes a rearrangement of helix 4, which may enhance the heterotypic interaction between the N- and C-terminal domains of CA dimers. In addition, we propose that the cyclophilin-binding loop may modulate capsid assembly during infection, either in the cytoplasm or near the nucleus by binding to the nuclear protein Nup385. Copyright © 2014. Published by Elsevier B.V.

APOBEC3G Inhibits HIV-1 RNA Elongation by Inactivating the Viral Trans-Activation Response Element

PubMed Central

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-01-01

Deamination of cytidine residues in viral DNA (vDNA) is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient HIV-1 replication. dC to dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here we demonstrate that A3G provides an additional layer of defence against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. Finally, we show that free ssDNA termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′+5′ ends is sufficient for A3G deamination, identifying A3G as an efficient mutator, and that deamination of (−)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

PubMed

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-07-29

Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Lumenal Loop Met672–Pro707 of Copper-transporting ATPase ATP7A Binds Metals and Facilitates Copper Release from the Intramembrane Sites*

PubMed Central

Barry, Amanda N.; Otoikhian, Adenike; Bhatt, Sujata; Shinde, Ujwal; Tsivkovskii, Ruslan; Blackburn, Ninian J.; Lutsenko, Svetlana

2011-01-01

The copper-transporting ATPase ATP7A has an essential role in human physiology. ATP7A transfers the copper cofactor to metalloenzymes within the secretory pathway; inactivation of ATP7A results in an untreatable neurodegenerative disorder, Menkes disease. Presently, the mechanism of ATP7A-mediated copper release into the secretory pathway is not understood. We demonstrate that the characteristic His/Met-rich segment Met672–Pro707 (HM-loop) that connects the first two transmembrane segments of ATP7A is important for copper release. Mutations within this loop do not prevent the ability of ATP7A to form a phosphorylated intermediate during ATP hydrolysis but inhibit subsequent dephosphorylation, a step associated with copper release. The HM-loop inserted into a scaffold protein forms two structurally distinct binding sites and coordinates copper in a mixed His-Met environment with an ∼2:1 stoichiometry. Binding of either copper or silver, a Cu(I) analog, induces structural changes in the loop. Mutations of 4 Met residues to Ile or two His-His pairs to Ala-Gly decrease affinity for copper. Altogether, the data suggest a two-step process, where copper released from the transport sites binds to the first His(Met)2 site, triggering a structural change and binding to a second 2-coordinate His-His or His-Met site. We also show that copper binding within the HM-loop stabilizes Cu(I) and protects it from oxidation, which may further aid the transfer of copper from ATP7A to acceptor proteins. The mechanism of copper entry into the secretory pathway is discussed. PMID:21646353

A dihydropyridine receptor alpha1s loop region critical for skeletal muscle contraction is intrinsically unstructured and binds to a SPRY domain of the type 1 ryanodine receptor.

PubMed

Cui, Yanfang; Tae, Han-Shen; Norris, Nicole C; Karunasekara, Yamuna; Pouliquin, Pierre; Board, Philip G; Dulhunty, Angela F; Casarotto, Marco G

2009-03-01

The II-III loop of the dihydropyridine receptor (DHPR) alpha(1s) subunit is a modulator of the ryanodine receptor (RyR1) Ca(2+) release channel in vitro and is essential for skeletal muscle contraction in vivo. Despite its importance, the structure of this loop has not been reported. We have investigated its structure using a suite of NMR techniques which revealed that the DHPR II-III loop is an intrinsically unstructured protein (IUP) and as such belongs to a burgeoning structural class of functionally important proteins. The loop does not possess a stable tertiary fold: it is highly flexible, with a strong N-terminal helix followed by nascent helical/turn elements and unstructured segments. Its residual structure is loosely globular with the N and C termini in close proximity. The unstructured nature of the II-III loop may allow it to easily modify its interaction with RyR1 following a surface action potential and thus initiate rapid Ca(2+) release and contraction. The in vitro binding partner for the II-III was investigated. The II-III loop interacts with the second of three structurally distinct SPRY domains in RyR1, whose function is unknown. This interaction occurs through two preformed N-terminal alpha-helical regions and a C-terminal hydrophobic element. The A peptide corresponding to the helical N-terminal region is a common probe of RyR function and binds to the same SPRY domain as the full II-III loop. Thus the second SPRY domain is an in vitro binding site for the II-III loop. The possible in vivo role of this region is discussed.

Autoregulatory mechanisms controlling the microprocessor.

PubMed

Triboulet, Robinson; Gregory, Richard I

2011-01-01

The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature ∼22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part of a newly identified regulatory mechanism controlling Microprocessor activity.

Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits

PubMed Central

2016-01-01

Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the “stiffness site” affects filament mechanical properties. Incorporating a magnesium ion in the “polymerization site” does not seem to require any large-scale change to an actin subunit’s conformation. Binding of a magnesium ion in the “stiffness site” adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids. PMID:27146246

Synthetic Polymer Affinity Ligand for Bacillus thuringiensis ( Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism.

PubMed

Liu, Mingming; Huang, Rong; Weisman, Adam; Yu, Xiaoyang; Lee, Shih-Hui; Chen, Yalu; Huang, Chao; Hu, Senhua; Chen, Xiuhua; Tan, Wenfeng; Liu, Fan; Chen, Hao; Shea, Kenneth J

2018-05-24

We report a novel strategy for creating abiotic Bacillus thuringiensis ( Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R 1 proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes 280 FRGSAQGIEGS 290 , 368 RRPFNIGINNQQ 379 and 436 FRSGFSNSSVSIIR 449 located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, 368 RRPFNIGINNQQ 379 . This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP- Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of 865 NITIHITDTNNK 876 , a specific recognition site in insect receptor Bt-R 1 , to 368 RRPFNIGINNQQ 379 . The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via 368 RRPFNIGINNQQ 379 . This epitope region was then utilized as a "target" or "bait" for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.

Binding of high mobility group A proteins to the mammalian genome occurs as a function of AT-content

PubMed Central

Schübeler, Dirk

2017-01-01

Genomic location can inform on potential function and recruitment signals for chromatin-associated proteins. High mobility group (Hmg) proteins are of similar size as histones with Hmga1 and Hmga2 being particularly abundant in replicating normal tissues and in cancerous cells. While several roles for Hmga proteins have been proposed we lack a comprehensive description of their genomic location as a function of chromatin, DNA sequence and functional domains. Here we report such a characterization in mouse embryonic stem cells in which we introduce biotin-tagged constructs of wild-type and DNA-binding domain mutants. Comparative analysis of the genome-wide distribution of Hmga proteins reveals pervasive binding, a feature that critically depends on a functional DNA-binding domain and which is shared by both Hmga proteins. Assessment of the underlying queues instructive for this binding modality identifies AT richness, defined as high frequency of A or T bases, as the major criterion for local binding. Additionally, we show that other chromatin states such as those linked to cis-regulatory regions have little impact on Hmga binding both in stem and differentiated cells. As a consequence, Hmga proteins are preferentially found at AT-rich regions such as constitutively heterochromatic regions but are absent from enhancers and promoters arguing for a limited role in regulating individual genes. In line with this model, we show that genetic deletion of Hmga proteins in stem cells causes limited transcriptional effects and that binding is conserved in neuronal progenitors. Overall our comparative study describing the in vivo binding modality of Hmga1 and Hmga2 identifies the proteins’ preference for AT-rich DNA genome-wide and argues against a suggested function of Hmga at regulatory regions. Instead we discover pervasive binding with enrichment at regions of higher AT content irrespective of local variation in chromatin modifications. PMID:29267285

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

PubMed Central

Yahashiri, Atsushi; Jorgenson, Matthew A.; Weiss, David S.

2015-01-01

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division. PMID:26305949

Extended molecular dynamics of a c-kit promoter quadruplex

PubMed Central

Islam, Barira; Stadlbauer, Petr; Krepl, Miroslav; Koca, Jaroslav; Neidle, Stephen; Haider, Shozeb; Sponer, Jiri

2015-01-01

The 22-mer c-kit promoter sequence folds into a parallel-stranded quadruplex with a unique structure, which has been elucidated by crystallographic and NMR methods and shows a high degree of structural conservation. We have carried out a series of extended (up to 10 μs long, ∼50 μs in total) molecular dynamics simulations to explore conformational stability and loop dynamics of this quadruplex. Unfolding no-salt simulations are consistent with a multi-pathway model of quadruplex folding and identify the single-nucleotide propeller loops as the most fragile part of the quadruplex. Thus, formation of propeller loops represents a peculiar atomistic aspect of quadruplex folding. Unbiased simulations reveal μs-scale transitions in the loops, which emphasizes the need for extended simulations in studies of quadruplex loops. We identify ion binding in the loops which may contribute to quadruplex stability. The long lateral-propeller loop is internally very stable but extensively fluctuates as a rigid entity. It creates a size-adaptable cleft between the loop and the stem, which can facilitate ligand binding. The stability gain by forming the internal network of GA base pairs and stacks of this loop may be dictating which of the many possible quadruplex topologies is observed in the ground state by this promoter quadruplex. PMID:26245347

Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake.

PubMed

Yildiz, Ozkan; Kalthoff, Christoph; Raunser, Stefan; Kühlbrandt, Werner

2007-01-24

A binary complex of the ammonia channel Amt1 from Methanococcus jannaschii and its cognate P(II) signalling protein GlnK1 has been produced and characterized. Complex formation is prevented specifically by the effector molecules Mg-ATP and 2-ketoglutarate. Single-particle electron microscopy of the complex shows that GlnK1 binds on the cytoplasmic side of Amt1. Three high-resolution X-ray structures of GlnK1 indicate that the functionally important T-loop has an extended, flexible conformation in the absence of Mg-ATP, but assumes a compact, tightly folded conformation upon Mg-ATP binding, which in turn creates a 2-ketoglutarate-binding site. We propose a regulatory mechanism by which nitrogen uptake is controlled by the binding of both effector molecules to GlnK1. At normal effector levels, a 2-ketoglutarate molecule binding at the apex of the compact T-loop would prevent complex formation, ensuring uninhibited ammonia uptake. At low levels of Mg-ATP, the extended loops would seal the ammonia channels in the complex. Binding of both effector molecules to P(II) signalling proteins may thus represent an effective feedback mechanism for regulating ammonium uptake through the membrane.

Mechanisms of Lin28-Mediated miRNA and mRNA Regulation—A Structural and Functional Perspective

PubMed Central

Mayr, Florian; Heinemann, Udo

2013-01-01

Lin28 is an essential RNA-binding protein that is ubiquitously expressed in embryonic stem cells. Its physiological function has been linked to the regulation of differentiation, development, and oncogenesis as well as glucose metabolism. Lin28 mediates these pleiotropic functions by inhibiting let-7 miRNA biogenesis and by modulating the translation of target mRNAs. Both activities strongly depend on Lin28’s RNA-binding domains (RBDs), an N-terminal cold-shock domain (CSD) and a C-terminal Zn-knuckle domain (ZKD). Recent biochemical and structural studies revealed the mechanisms of how Lin28 controls let-7 biogenesis. Lin28 binds to the terminal loop of pri- and pre-let-7 miRNA and represses their processing by Drosha and Dicer. Several biochemical and structural studies showed that the specificity of this interaction is mainly mediated by the ZKD with a conserved GGAGA or GGAGA-like motif. Further RNA crosslinking and immunoprecipitation coupled to high-throughput sequencing (CLIP-seq) studies confirmed this binding motif and uncovered a large number of new mRNA binding sites. Here we review exciting recent progress in our understanding of how Lin28 binds structurally diverse RNAs and fulfills its pleiotropic functions. PMID:23939427

Conformation of receptor-bound visual arrestin.

PubMed

Kim, Miyeon; Vishnivetskiy, Sergey A; Van Eps, Ned; Alexander, Nathan S; Cleghorn, Whitney M; Zhan, Xuanzhi; Hanson, Susan M; Morizumi, Takefumi; Ernst, Oliver P; Meiler, Jens; Gurevich, Vsevolod V; Hubbell, Wayne L

2012-11-06

Arrestin-1 (visual arrestin) binds to light-activated phosphorylated rhodopsin (P-Rh*) to terminate G-protein signaling. To map conformational changes upon binding to the receptor, pairs of spin labels were introduced in arrestin-1 and double electron-electron resonance was used to monitor interspin distance changes upon P-Rh* binding. The results indicate that the relative position of the N and C domains remains largely unchanged, contrary to expectations of a "clam-shell" model. A loop implicated in P-Rh* binding that connects β-strands V and VI (the "finger loop," residues 67-79) moves toward the expected location of P-Rh* in the complex, but does not assume a fully extended conformation. A striking and unexpected movement of a loop containing residue 139 away from the adjacent finger loop is observed, which appears to facilitate P-Rh* binding. This change is accompanied by smaller movements of distal loops containing residues 157 and 344 at the tips of the N and C domains, which correspond to "plastic" regions of arrestin-1 that have distinct conformations in monomers of the crystal tetramer. Remarkably, the loops containing residues 139, 157, and 344 appear to have high flexibility in both free arrestin-1 and the P-Rh*complex.

Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.

PubMed

Stinson, Julie; Inoue, Toshiaki; Yates, Paula; Clancy, Anne; Norton, John D; Sharrocks, Andrew D

2003-08-15

DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.

Internal loop/bulge and hairpin loop of the iron-responsive element of ferritin mRNA contribute to maximal iron regulatory protein 2 binding and translational regulation in the iso-iron-responsive element/iso-iron regulatory protein family.

PubMed

Ke, Y; Sierzputowska-Gracz, H; Gdaniec, Z; Theil, E C

2000-05-23

Iron-responsive elements (IREs), a natural group of mRNA-specific sequences, bind iron regulatory proteins (IRPs) differentially and fold into hairpins [with a hexaloop (HL) CAGUGX] with helical distortions: an internal loop/bulge (IL/B) (UGC/C) or C-bulge. C-bulge iso-IREs bind IRP2 more poorly, as oligomers (n = 28-30), and have a weaker signal response in vivo. Two trans-loop GC base pairs occur in the ferritin IRE (IL/B and HL) but only one in C-bulge iso-IREs (HL); metal ions and protons perturb the IL/B [Gdaniec et al. (1998) Biochemistry 37, 1505-1512]. IRE function (translation) and physical properties (T(m) and accessibility to nucleases) are now compared for IL/B and C-bulge IREs and for HL mutants. Conversion of the IL/B into a C-bulge by a single deletion in the IL/B or by substituting the HL CG base pair with UA both derepressed ferritin synthesis 4-fold in rabbit reticulocyte lysates (IRP1 + IRP2), confirming differences in IRP2 binding observed for the oligomers. Since the engineered C-bulge IRE was more helical near the IL/B [Cu(phen)(2) resistant] and more stable (T(m) increased) and the HL mutant was less helical near the IL/B (ribonuclease T1 sensitive) and less stable (T(m) decreased), both CG trans-loop base pairs contribute to maximum IRP2 binding and translational regulation. The (1)H NMR spectrum of the Mg-IRE complex revealed, in contrast to the localized IL/B effects of Co(III) hexaammine observed previously, perturbation of the IL/B plus HL and interloop helix. The lower stability and greater helix distortion in the ferritin IL/B-IRE compared to the C-bulge iso-IREs create a combinatorial set of RNA/protein interactions that control protein synthesis rates with a range of signal sensitivities.

Relative stability of the open and closed conformations of the active site loop of streptavidin

NASA Astrophysics Data System (ADS)

Ignacio J., General; Meirovitch, Hagai

2011-01-01

The eight-residue surface loop, 45-52 (Ser, Ala, Val, Gly, Asn, Ala, Glu, Ser), of the homotetrameric protein streptavidin has a "closed" conformation in the streptavidin-biotin complex, where the corresponding binding affinity is one of the strongest found in nature (ΔG ˜ -18 kcal/mol). However, in most of the crystal structures of apo (unbound) streptavidin, the loop conformation is "open" and typically exhibits partial disorder and high B-factors. Thus, it is plausible to assume that the loop structure is changed from open to closed upon binding of biotin, and the corresponding difference in free energy, ΔF = Fopen - Fclosed in the unbound protein, should therefore be considered in the total absolute free energy of binding. ΔF (which has generally been neglected) is calculated here using our "hypothetical scanning molecular-dynamics" (HSMD) method. We use a protein model in which only the atoms closest to the loop are considered (the "template") and they are fixed in the x-ray coordinates of the free protein; the x-ray conformation of the closed loop is attached to the same (unbound) template and both systems are capped with the same sphere of TIP3P water. Using the force field of the assisted model building with energy refinement (AMBER), we carry out two separate MD simulations (at temperature T = 300 K), starting from the open and closed conformations, where only the atoms of the loop and water are allowed to move (the template-water and template-loop interactions are considered). The absolute Fopen and Fclosed (of loop + water) are calculated from these trajectories, where the loop and water contributions are obtained by HSMD and a thermodynamic integration (TI) process, respectively. The combined HSMD-TI procedure leads to total (loop + water) ΔF = -27.1 ± 2.0 kcal/mol, where the entropy TΔS constitutes 34% of ΔF, meaning that the effect of S is significant and should not be ignored. Also, ΔS is positive, in accord with the high flexibility of the open loop observed in crystal structures, while the energy ΔE is unexpectedly negative, thus also adding to the stability of the open loop. The loop and the 250 capped water molecules are the largest system studied thus far, which constitutes a test for the efficiency of HSMD-TI; this efficiency and technical issues related to the implementation of the method are also discussed. Finally, the result for ΔF is a prediction that will be considered in the calculation of the absolute free energy of binding of biotin to streptavidin, which constitutes our next project.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

PubMed

Shi, Wei; Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K; Straube, Jasmin; Boyle, Glen M; Al-Ejeh, Fares; Nag, Purba; Jeffery, Jessie; Harris, Janelle L; Bain, Amanda L; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J; Finnie, John; Skaar, Jeffrey R; Walkley, Carl R; Pandita, Tej K; Rowicka, Maga; Ginalski, Krzysztof; Lane, Steven W; Khanna, Kum Kum

2017-05-04

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. © 2017 by The American Society of Hematology.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress

PubMed Central

Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K.; Straube, Jasmin; Boyle, Glen M.; Al-Ejeh, Fares; Jeffery, Jessie; Harris, Janelle L.; Bain, Amanda L.; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J.; Finnie, John; Skaar, Jeffrey R.; Walkley, Carl R.; Pandita, Tej K.; Rowicka, Maga; Ginalski, Krzysztof

2017-01-01

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. PMID:28270450

Solution structure of conserved AGNN tetraloops: insights into Rnt1p RNA processing

PubMed Central

Lebars, Isabelle; Lamontagne, Bruno; Yoshizawa, Satoko; Abou Elela, Sherif; Fourmy, Dominique

2001-01-01

Rnt1p, the yeast orthologue of RNase III, cleaves rRNAs, snRNAs and snoRNAs at a stem capped with conserved AGNN tetraloop. Here we show that 9 bp long stems ending with AGAA or AGUC tetraloops bind to Rnt1p and direct specific but sequence-independent RNA cleavage when provided with stems longer than 13 bp. The solution structures of these two tetraloops reveal a common fold for the terminal loop stabilized by non-canonical A–A or A–C pairs and extensive base stacking. The conserved nucleotides are stacked at the 5′ side of the loop, exposing their Watson–Crick and Hoogsteen faces for recognition by Rnt1p. These results indicate that yeast RNase III recognizes the fold of a conserved single-stranded tetraloop to direct specific dsRNA cleavage. PMID:11743001

Deciphering the structural framework of glycine receptor anchoring by gephyrin

PubMed Central

Kim, Eun Young; Schrader, Nils; Smolinsky, Birthe; Bedet, Cécile; Vannier, Christian; Schwarz, Günter; Schindelin, Hermann

2006-01-01

Glycine is the major inhibitory neurotransmitter in the spinal cord and brain stem. Gephyrin is required to achieve a high concentration of glycine receptors (GlyRs) in the postsynaptic membrane, which is crucial for efficient glycinergic signal transduction. The interaction between gephyrin and the GlyR involves the E-domain of gephyrin and a cytoplasmic loop located between transmembrane segments three and four of the GlyR β subunit. Here, we present crystal structures of the gephyrin E-domain with and without the GlyR β-loop at 2.4 and 2.7 Å resolutions, respectively. The GlyR β-loop is bound in a symmetric ‘key and lock' fashion to each E-domain monomer in a pocket adjacent to the dimer interface. Structure-guided mutagenesis followed by in vitro binding and in vivo colocalization assays demonstrate that a hydrophobic interaction formed by Phe 330 of gephyrin and Phe 398 and Ile 400 of the GlyR β-loop is crucial for binding. PMID:16511563

Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGF-beta signaling.

PubMed

Wu, Jia Wei; Krawitz, Ariel R; Chai, Jijie; Li, Wenyu; Zhang, Fangjiu; Luo, Kunxin; Shi, Yigong

2002-11-01

The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.

Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction

PubMed Central

Becker, Matthias M. M.; Lapouge, Karine; Segnitz, Bernd; Wild, Klemens; Sinning, Irmgard

2017-01-01

Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway. PMID:27899666

STARD4 Membrane Interactions and Sterol Binding

PubMed Central

2016-01-01

The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/β helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. Among the soluble START proteins, STARD4 is expressed in most tissues and has previously been shown to transfer sterol, but the molecular mechanisms of membrane interaction and sterol binding remain unclear. In this work, we use biochemical techniques to characterize regions of STARD4 and determine their role in membrane interaction and sterol binding. Our results show that STARD4 interacts with anionic membranes through a surface-exposed basic patch and that introducing a mutation (L124D) into the Omega-1 (Ω1) loop, which covers the sterol binding pocket, attenuates sterol transfer activity. To gain insight into the attenuating mechanism of the L124D mutation, we conducted structural and biophysical studies of wild-type and L124D STARD4. These studies show that the L124D mutation reduces the conformational flexibility of the protein, resulting in a diminished level of membrane interaction and sterol transfer. These studies also reveal that the C-terminal α-helix, and not the Ω1 loop, partitions into the membrane bilayer. On the basis of these observations, we propose a model of STARD4 membrane interaction and sterol binding and release that requires dynamic movement of both the Ω1 loop and membrane insertion of the C-terminal α-helix. PMID:26168008

Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors.

PubMed

Prokop, Susanne; Perry, Nicole A; Vishnivetskiy, Sergey A; Toth, Andras D; Inoue, Asuka; Milligan, Graeme; Iverson, Tina M; Hunyady, Laszlo; Gurevich, Vsevolod V

2017-08-01

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M 2 muscarinic receptor, so that agonist activation of the M 2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M 2 , whereas its interactions with other receptors, including the β 2 -adrenergic receptor and the D 1 and D 2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the β 2 -adrenergic and D 2 dopamine receptors, while reducing its interaction with the D 1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes. Copyright © 2017. Published by Elsevier Inc.

C2 Domain of Protein Kinase Cα: Elucidation of the Membrane Docking Surface by Site-Directed Fluorescence and Spin Labeling†

PubMed Central

Kohout, Susy C.; Corbalán-García, Senena; Gómez-Fernández, Juan C.; Falke, Joseph J.

2013-01-01

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca2+-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase Cα (PKCα) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca2+ binding loops and an anion binding site on β-strands 3–4, such that the β-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca2+ binding loops and the β-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKCα C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca2+ activation or membrane docking trigger measurable fluorescence changes. Ca2+ binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca2+ binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Φ, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca2+ binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca2+ first and third Ca2+ binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on β-strands 3–4 lies near the headgroups. The data support a model in which the β-strands are tilted toward the parallel orientation relative to the membrane surface. PMID:12564928

G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

PubMed

Lynch, Jennifer R; Wang, Jenny Yingzi

2016-05-11

G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

PubMed Central

Lynch, Jennifer R.; Wang, Jenny Yingzi

2016-01-01

G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies. PMID:27187360

Amino acid signature enables proteins to recognize modified tRNA.

PubMed

Spears, Jessica L; Xiao, Xingqing; Hall, Carol K; Agris, Paul F

2014-02-25

Human tRNA(Lys3)UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNA(Lys3)UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNA(Lys3)UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N(6)-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNA(Lys3)UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA's fully modified anticodon stem and loop domain, hASL(Lys3)UUU. Peptides of this sequence specifically recognized and bound modified htRNA(Lys3)UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions.

Structural characterization of the H-NS protein from Xylella fastidiosa and its interaction with DNA.

PubMed

Rosselli-Murai, Luciana K; Sforça, Maurício L; Sassonia, Rogério C; Azzoni, Adriano R; Murai, Marcelo J; de Souza, Anete P; Zeri, Ana C

2012-10-01

The nucleoid-associated protein H-NS is a major component of the bacterial nucleoid involved in DNA compaction and transcription regulation. The NMR solution structure of the Xylella fastidiosa H-NS C-terminal domain (residues 56-134) is presented here and consists of two beta-strands and two alpha helices, with one loop connecting the two beta-strands and a second loop connecting the second beta strand and the first helix. The amide (1)H and (15)N chemical shift signals for a sample of XfH-NS(56-134) were monitored in the course of a titration series with a 14-bp DNA duplex. Most of the residues involved in contacts to DNA are located around the first and second loops and in the first helix at a positively charged side of the protein surface. The overall structure of the Xylella H-NS C-terminal domain differ significantly from Escherichia coli and Salmonella enterica H-NS proteins, even though the DNA binding motif in loop 2 adopt similar conformation, as well as β-strand 2 and loop 1. Interestingly, we have also found that the DNA binding site is expanded to include helix 1, which is not seen in the other structures. Copyright © 2012 Elsevier Inc. All rights reserved.

The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription.

PubMed Central

Ke, N; Gao, X; Keeney, J B; Boeke, J D; Voytas, D F

1999-01-01

Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated. PMID:10411136

Interplay of protein and DNA structure revealed in simulations of the lac operon.

PubMed

Czapla, Luke; Grosner, Michael A; Swigon, David; Olson, Wilma K

2013-01-01

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

Anchoring protein crystals to mounting loops with hydrogel using inkjet technology.

PubMed

Shinoda, Akira; Tanaka, Yoshikazu; Yao, Min; Tanaka, Isao

2014-11-01

X-ray crystallography is an important technique for structure-based drug discovery, mainly because it is the only technique that can reveal whether a ligand binds to the target protein as well as where and how it binds. However, ligand screening by X-ray crystallography involves a crystal-soaking experiment, which is usually performed manually. Thus, the throughput is not satisfactory for screening large numbers of candidate ligands. In this study, a technique to anchor protein crystals to mounting loops by using gel and inkjet technology has been developed; the method allows soaking of the mounted crystals in ligand-containing solution. This new technique may assist in the design of a fully automated drug-screening pipeline.

The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.

PubMed

Roth, Braden M; Ishimaru, Daniella; Hennig, Mirko

2013-09-13

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

The Core Microprocessor Component DiGeorge Syndrome Critical Region 8 (DGCR8) Is a Nonspecific RNA-binding Protein*

PubMed Central

Roth, Braden M.; Ishimaru, Daniella; Hennig, Mirko

2013-01-01

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins. PMID:23893406

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

PubMed Central

Kristensen, Tatjana P.; Maria Cherian, Reeja; Gray, Fiona C.; MacNeill, Stuart A.

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies. PMID:24723920

Reactivation of mutant p53: Constraints on mechanism highlighted by principal component analysis of the DNA binding domain.

PubMed

Ouaray, Zahra; ElSawy, Karim M; Lane, David P; Essex, Jonathan W; Verma, Chandra

2016-10-01

Most p53 mutations associated with cancer are located in its DNA binding domain (DBD). Many structures (X-ray and NMR) of this domain are available in the protein data bank (PDB) and a vast conformational heterogeneity characterizes the various free and complexed states. The major difference between the apo and the holo-complexed states appears to lie in the L1 loop. In particular, the conformations of this loop appear to depend intimately on the sequence of DNA to which it binds. This conclusion builds upon recent observations that implicate the tetramerization and the C-terminal domains (respectively TD and Cter) in DNA binding specificity. Detailed PCA analysis of the most recent collection of DBD structures from the PDB have been carried out. In contrast to recommendations that small molecules/drugs stabilize the flexible L1 loop to rescue mutant p53, our study highlights a need to retain the flexibility of the p53 DNA binding surface (DBS). It is the adaptability of this region that enables p53 to engage in the diverse interactions responsible for its functionality. Proteins 2016; 84:1443-1461. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Osmotic mechanism of the loop extrusion process

NASA Astrophysics Data System (ADS)

Yamamoto, Tetsuya; Schiessel, Helmut

2017-09-01

The loop extrusion theory assumes that protein factors, such as cohesin rings, act as molecular motors that extrude chromatin loops. However, recent single molecule experiments have shown that cohesin does not show motor activity. To predict the physical mechanism involved in loop extrusion, we here theoretically analyze the dynamics of cohesin rings on a loop, where a cohesin loader is in the middle and unloaders at the ends. Cohesin monomers bind to the loader rather frequently and cohesin dimers bind to this site only occasionally. Our theory predicts that a cohesin dimer extrudes loops by the osmotic pressure of cohesin monomers on the chromatin fiber between the two connected rings. With this mechanism, the frequency of the interactions between chromatin segments depends on the loading and unloading rates of dimers at the corresponding sites.

Structure of adenovirus bound to cellular receptor car

DOEpatents

Freimuth, Paul I.

2004-05-18

Disclosed is a mutant adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have significantly weakened binding affinity for CARD1 relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type. In the method, residues of the adenovirus fiber protein knob domain which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus to generate a mutant adenovirus. Whether or not the mutant produced exhibits altered adenovirus-CAR binding properties is then determined.

Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein.

PubMed Central

Felsenstein, K M; Goff, S P

1992-01-01

The gag-pol polyprotein of the murine and feline leukemia viruses is expressed by translational readthrough of a UAG terminator codon at the 3' end of the gag gene. To explore the cis-acting sequence requirements for the readthrough event in vivo, we generated a library of mutants of the Moloney murine leukemia virus with point mutations near the terminator codon and tested the mutant viral DNAs for the ability to direct synthesis of the gag-pol fusion protein and formation of infectious virus. The analysis showed that sequences 3' to the terminator are necessary and sufficient for the process. The results do not support a role for one proposed stem-loop structure that includes the terminator but are consistent with the involvement of another stem-loop 3' to the terminator. One mutant, containing two compensatory changes in this stem structure, was temperature sensitive for replication and for formation of the gag-pol protein. The results suggest that RNA sequence and structure are critical determinants of translational readthrough in vivo. Images PMID:1404606

Structural Basis for Antifreeze Activity of Ice-binding Protein from Arctic Yeast*

PubMed Central

Lee, Jun Hyuck; Park, Ae Kyung; Do, Hackwon; Park, Kyoung Sun; Moh, Sang Hyun; Chi, Young Min; Kim, Hak Jun

2012-01-01

Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96–115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region (243PFVPAPEVV251). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn185 provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins. PMID:22303017

CD and NMR conformational studies of a peptide encompassing the Mid Loop interface of Ship2-Sam.

PubMed

Mercurio, Flavia A; Scognamiglio, Pasqualina L; Di Natale, Concetta; Marasco, Daniela; Pellecchia, Maurizio; Leone, Marilisa

2014-11-01

The lipid phosphatase Ship2 is a protein that intervenes in several diseases such as diabetes, cancer, neurodegeneration, and atherosclerosis. It is made up of a catalytic domain and several protein docking modules such as a C-terminal Sam (Sterile alpha motif) domain. The Sam domain of Ship2 (Ship2-Sam) binds to the Sam domains of the EphA2 receptor (EphA2-Sam) and the PI3K effector protein Arap3 (Arap3-Sam). These heterotypic Sam-Sam interactions occur through formation of dimers presenting the canonical "Mid Loop/End Helix" binding mode. The central region of Ship2-Sam, spanning the C-terminal end of α2, the α3 and α4 helices together with the α2α3 and α3α4 interhelical loops, forms the Mid Loop surface that is needed to bind partners Sam domains. A peptide encompassing most of the Ship2-Sam Mid Loop interface (Shiptide) capable of binding to both EphA2-Sam and Arap3-Sam, was previously identified. Here we investigated the conformational features of this peptide, through solution CD and NMR studies in different conditions. These studies reveal that the peptide is highly flexible in aqueous buffer, while it adopts a helical conformation in presence of 2,2,2-trifluoroethanol. The discovered structural insights and in particular the identification of a helical motif, may lead to the design of more constrained and possibly cell permeable Shiptide analogs that could work as efficient antagonists of Ship2-Sam heterotypic interactions and embrace therapeutic applications. © 2014 Wiley Periodicals, Inc.

Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry

PubMed Central

Kühn-Hölsken, Eva; Lenz, Christof; Dickmanns, Achim; Hsiao, He-Hsuan; Richter, Florian M.; Kastner, Berthold; Ficner, Ralf; Urlaub, Henning

2010-01-01

Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m3G cap and snurportin 1. We were able to define previously unknown sites of protein–protein and protein–RNA interactions on the molecular level within U1 snRNP. We show that snurportin 1 interacts with its central m3G-cap-binding domain with Sm proteins and with its extreme C-terminus with stem-loop III of U1 snRNA. The crosslinking data support the idea of a larger interaction area between snurportin 1 and U snRNPs and the contact sites identified prove useful for modeling the spatial arrangement of snurportin 1 domains when bound to U1 snRNP. Moreover, this suggests a functional nuclear import complex that assembles around the m3G cap and the Sm proteins only when the Sm proteins are bound and arranged in the proper orientation to the cognate Sm site in U snRNA. PMID:20421206

Insights into the RNA quadruplex binding specificity of DDX21.

PubMed

McRae, Ewan K S; Davidson, David E; Dupas, Steven J; McKenna, Sean A

2018-06-12

Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 specifically binds to TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2'OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2'OH of loop nucleotides. Copyright © 2018. Published by Elsevier B.V.

Extended Impact of Pin1 Catalytic Loop Phosphorylation Revealed by S71E Phosphomimetic.

PubMed

Mahoney, Brendan J; Zhang, Meiling; Zintsmaster, John S; Peng, Jeffrey W

2018-03-02

Pin1 is a two-domain human protein that catalyzes the cis-trans isomerization of phospho-Ser/Thr-Pro (pS/T-P) motifs in numerous cell-cycle regulatory proteins. These pS/T-P motifs bind to Pin1's peptidyl-prolyl isomerase (PPIase) domain in a catalytic pocket, between an extended catalytic loop and the PPIase domain core. Previous studies showed that post-translational phosphorylation of S71 in the catalytic loop decreases substrate binding affinity and isomerase activity. To define the origins for these effects, we investigated a phosphomimetic Pin1 mutant, S71E-Pin1, using solution NMR. We find that S71E perturbs not only its host loop but also the nearby PPIase core. The perturbations identify a local network of hydrogen bonds and salt bridges that is more extended than previously thought, and includes interactions between the catalytic loop and the α2/α3 turn in the PPIase core. Explicit-solvent molecular dynamics simulations and phylogenetic analysis suggest that these interactions act as conserved "latches" between the loop and PPIase core that enhance binding of phosphorylated substrates, as they are absent in PPIases lacking pS/T-P specificity. Our results suggest that S71 is a hub residue within an electrostatic network primed for phosphorylation, and may illustrate a common mechanism of phosphorylation-mediated allostery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Conformation of receptor-bound visual arrestin

PubMed Central

Kim, Miyeon; Vishnivetskiy, Sergey A.; Van Eps, Ned; Alexander, Nathan S.; Cleghorn, Whitney M.; Zhan, Xuanzhi; Hanson, Susan M.; Morizumi, Takefumi; Ernst, Oliver P.; Meiler, Jens; Gurevich, Vsevolod V.; Hubbell, Wayne L.

2012-01-01

Arrestin-1 (visual arrestin) binds to light-activated phosphorylated rhodopsin (P-Rh*) to terminate G-protein signaling. To map conformational changes upon binding to the receptor, pairs of spin labels were introduced in arrestin-1 and double electron–electron resonance was used to monitor interspin distance changes upon P-Rh* binding. The results indicate that the relative position of the N and C domains remains largely unchanged, contrary to expectations of a “clam-shell” model. A loop implicated in P-Rh* binding that connects β-strands V and VI (the “finger loop,” residues 67–79) moves toward the expected location of P-Rh* in the complex, but does not assume a fully extended conformation. A striking and unexpected movement of a loop containing residue 139 away from the adjacent finger loop is observed, which appears to facilitate P-Rh* binding. This change is accompanied by smaller movements of distal loops containing residues 157 and 344 at the tips of the N and C domains, which correspond to “plastic” regions of arrestin-1 that have distinct conformations in monomers of the crystal tetramer. Remarkably, the loops containing residues 139, 157, and 344 appear to have high flexibility in both free arrestin-1 and the P-Rh*complex. PMID:23091036

Homology modeling study toward identifying structural properties in the HA2 B-loop that would influence the HA1 receptor-binding site.

PubMed

Cueno, Marni E; Imai, Kenichi; Shimizu, Kazufumi; Ochiai, Kuniyasu

2013-07-01

Influenza hemagglutinin (HA) consists of a fibrous globular stem (HA2) inserted into the viral membrane supporting a globular head (HA1). HA1 receptor-binding has been hypothesized to be structurally correlated to the HA2 B-loop, however, this was never fully understood. Here, we elucidated the structural relationship between the HA2 B-loop and the HA1 receptor-binding site (RBS). Throughout this study, we analyzed 2486 H1N1 HA homology models obtained from human, swine and avian strains during 1976-2012. Quality of all homology models were verified before further analyses. We established that amino acid residue 882 is putatively strain-conserved and differs in the human (K882), swine (H882) and avian (N882) strains. Moreover, we observed that the amino acid at residue 882 and, similarly, its orientation has the potential to influence the HA1 RBS diameter measurements which we hypothesize may consequentially affect influenza H1N1 viral infectivity, immune escape, transmissibility, and evolution. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of ligand binding on the dynamics of rice nonspecific lipid transfer protein 1: a model from molecular simulations.

PubMed

Lai, Yen-Ting; Cheng, Chao-Sheng; Liu, Yu-Nan; Liu, Yaw-Jen; Lyu, Ping-Chiang

2008-09-01

Plant nonspecific lipid transfer proteins (nsLTPs) are small, basic proteins constituted mainly of alpha-helices and stabilized by four conserved disulfide bridges. They are characterized by the presence of a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers in vitro. In this study, molecular dynamics (MD) simulations were performed at room temperature to investigate the effects of lipid binding on the dynamic properties of rice nsLTP1. Rice nsLTP1, either in the free form or complexed with one or two lipids was subjected to MD simulations. The C-terminal loop was very flexible both before and after lipid binding, as revealed by calculating the root-mean-square fluctuation. After lipid binding, the flexibility of some residues that were not in direct contact with lipid molecules increased significantly, indicating an increase of entropy in the region distal from the binding site. Essential dynamics analysis revealed clear differences in motion between unliganded and liganded rice nsLTP1s. In the free form of rice nsLTP1, loop1 exhibited the largest directional motion. This specific essential motion mode diminished after binding one or two lipid molecules. To verify the origin of the essential motion observed in the free form of rice nsLTP1, we performed multiple sequence alignments to probe the intrinsic motion encoded in the primary sequence. We found that the amino acid sequence of loop1 is highly conserved among plant nsLTP1s, thus revealing its functional importance during evolution. Furthermore, the sequence of loop1 is composed mainly of amino acids with short side chains. In this study, we show that MD simulations, together with essential dynamics analysis, can be used to determine structural and dynamic differences of rice nsLTP1 upon lipid binding. 2008 Wiley-Liss, Inc.

LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase-dependent proteolysis of the stem-cell protein SOX2.

PubMed

Zhang, Chunxiao; Hoang, Nam; Leng, Feng; Saxena, Lovely; Lee, Logan; Alejo, Salvador; Qi, Dandan; Khal, Anthony; Sun, Hong; Lu, Fei; Zhang, Hui

2018-03-09

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guan, Jian; Bywaters, Stephanie M.; Brendle, Sarah A.

2015-09-15

Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope.more » Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays. - Highlights: • We present HPV16-Fab complexes from neutralizing mAbs: H16.1A, H16.14J, and H263.A2. • The structure-function analysis revealed predominantly monovalent binding of each mAb. • Capsid–Fab interactions involved multiple loops from symmetry related L1 proteins. • Besides the known FG and HI loops, epitope mapping also identified DE, EF, and BC loops. • Neutralizing assays complement the structures to show multiple neutralization mechanisms.« less

The Solution Structure, Binding Properties, and Dynamics of the Bacterial Siderophore-binding Protein FepB*

PubMed Central

Chu, Byron C. H.; Otten, Renee; Krewulak, Karla D.; Mulder, Frans A. A.; Vogel, Hans J.

2014-01-01

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs. PMID:25173704

An α-subunit loop structure is required for GM2 activator protein binding by β-hexosaminidase A

PubMed Central

Zarghooni, Maryam; Bukovac, Scott; Tropak, Michael; Callahan, John; Mahuran, Don

2010-01-01

The α- and/or β-subunits of human β-hexosaminidase A (αβ) and B (ββ) are ~60% identical. In vivo only β-hexosaminidase A can utilize GM2 ganglioside as a substrate, but requires the GM2 activator protein to bind GM2 ganglioside and then interact with the enzyme, placing the terminal GalNAc residue in the active site of the α-subunit. A model for this interaction suggests that two loop structures, present only in the α-subunit, may be critical to this binding. Three amino acids in one of these loops are not encoded in the HEXB gene, while four from the other are removed posttranslationally from the pro-β-subunit. Natural substrate assays with forms of hexosaminidase A containing mutant α-subunits demonstrate that only the site that is removed from the β-subunit during its maturation is critical for the interaction. Our data suggest an unexpected biological role for such proteolytic processing events. PMID:15485660

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism

PubMed Central

Reilman, Ewoud; Mars, Ruben A. T.; van Dijl, Jan Maarten; Denham, Emma L.

2014-01-01

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. PMID:25217586

Determination of Surface-Exposed, Functional Domains of Gonococcal Transferrin-Binding Protein A

PubMed Central

Yost-Daljev, Mary Kate; Cornelissen, Cynthia Nau

2004-01-01

The gonococcal transferrin receptor is composed of two distinct proteins, TbpA and TbpB. TbpA is a member of the TonB-dependent family of integral outer membrane transporters, while TbpB is lipid modified and thought to be peripherally surface exposed. We previously proposed a hypothetical topology model for gonococcal TbpA that was based upon computer predictions and similarity with other TonB-dependent transporters for which crystal structures have been determined. In the present study, the hemagglutinin epitope was inserted into TbpA to probe the surface topology of this protein and secondarily to test the functional impacts of site-specific mutagenesis. Twelve epitope insertion mutants were constructed, five of which allowed us to confirm the surface exposure of loops 2, 3, 5, 7, and 10. In contrast to the predictions set forth by the hypothetical model, insertion into the plug region resulted in an epitope that was surface accessible, while epitope insertions into two putative loops (9 and 11) were not surface accessible. Insertions into putative loop 3 and β strand 9 abolished transferrin binding and utilization, and the plug insertion mutant exhibited decreased transferrin-binding affinity concomitant with an inability to utilize it. Insertion into putative β strand 16 generated a mutant that was able to bind transferrin normally but that was unable to mediate utilization. Mutants with insertions into putative loops 2, 9, and 11 maintained wild-type binding affinity but could utilize only transferrin in the presence of TbpB. This is the first demonstration of the ability of TbpB to compensate for a mutation in TbpA. PMID:14977987

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N 6-threonylcarbamoyladenosine TcdA Function.

PubMed

Fernández, Francisco J; Gómez, Sara; Navas-Yuste, Sergio; López-Estepa, Miguel; Vega, M Cristina

2017-06-21

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N 6 -threonylcarbamoyladenosine (t 6 A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t 6 A (ct 6 A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 °C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Replacing Arginine 33 for Alanine in the Hemophore HasA from Pseudomonas aeruginosa Causes Closure of the H32 Loop in the Apo-Protein

PubMed Central

Kumar, Ritesh; Qi, Yifei; Matsumura, Hirotoshi; Lovell, Scott; Yao, Huili; Battaile, Kevin P.; Im, Wonpil; Moënne-Loccoz, Pierre; Rivera, Mario

2017-01-01

Previous characterization of hemophores from Serratia marcescens (HasAs), Pseudomonas aeruginosa (HasAp) and Yersinia pestis (HasAyp) showed that hemin binds between two loops, where it is axially coordinated by H32 and Y75. The Y75 loop is structurally conserved in all three hemophores and harbors conserved ligand Y75. The other loop contains H32 in HasAs and HasAp, but a noncoordinating Q32 in HasAyp. The H32 loop in apo-HasAs and apo-HasAp is in an open conformation, which places H32 about 30 Å from the hemin-binding site. Hence, hemin binding onto the Y75 loop of HasAs or HasAp triggers a large relocation of the H32 loop from an open- to a closed-loop conformation and enables coordination of the hemin-iron by H32. In comparison, the Q32 loop in apo-HasAyp is in the closed conformation and hemin binding occurs with minimal reorganization and without coordinative interactions with the Q32 loop. Studies in crystallo and in solution have established that the open H32 loop in apo-HasAp and apo-HasAs is well structured and minimally affected by conformational dynamics. In this study we address the intriguing issue of the stability of the H32 loop in apo-HasAp and how hemin binding triggers its relocation. We address this question with a combination of NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations and find that R33 is critical to the stability of the open H32 loop. Replacing R33 with A causes the H32 loop in R33A apo-HasAp to adopt a conformation similar to that of holo-HasAp. Finally, stopped-flow absorption and resonance Raman analyses of hemin binding to apo-R33A HasAp indicates that the closed H32 loop slows down the insertion of the heme inside the binding pocket, presumably as it obstructs access to the hydrophobic platform on the Y75 loop, but accelerate the completion of the heme iron coordination. PMID:27074415

Structural analysis of the receptor binding domain of botulinum neurotoxin serotype D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Buchko, Garry W.; Qin, Lin

2010-10-28

Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65 Å resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences aremore » located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10 Å relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.« less

Structural Analysis of the Receptor Binding Domain of Botulinum Neurotoxin Serotype D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y Zhang; G Buchko; L Qin

2011-12-31

Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65{angstrom} resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are locatedmore » at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10{angstrom} relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.« less

A Novel Association between Two Trypanosome-Specific Factors and the Conserved L5-5S rRNA Complex

PubMed Central

Ciganda, Martin; Prohaska, Kimberly; Hellman, Kristina; Williams, Noreen

2012-01-01

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are involved in and essential for ribosome biogenesis. The proteins interact with the 5S rRNA with nearly identical binding characteristics. We have shown that this interaction is achieved mainly through the LoopA region of the RNA, but P34 and P37 also protect the L5 binding site located on LoopC. We now provide evidence to show that these factors form a novel pre-ribosomal particle through interactions with both 5S rRNA and the L5 ribosomal protein. Further in silico and in vitro analysis of T. brucei L5 indicates a lower affinity for 5S rRNA than expected, based on other eukaryotic L5 proteins. We hypothesize that P34 and P37 complement L5 and bridge the interaction with 5S rRNA, stabilizing it and aiding in the early steps of ribosome biogenesis. PMID:22859981

A photoaffinity scan maps regions of the p85 SH2 domain involved in phosphoprotein binding.

PubMed

Williams, K P; Shoelson, S E

1993-03-15

Src homology 2 (SH2) domains are modular phosphotyrosine binding pockets found within a wide variety of cytoplasmic signaling molecules. Here we develop a new approach to analyzing protein-protein interfaces termed photoaffinity scanning, and apply the method to map regions of the phosphatidylinositol 3-kinase p85 SH2 domain that participate in phospho-protein binding. Each residue except phosphotyrosine (pY) within a tightly binding, IRS-1-derived phosphopeptide (GNGDpYMPMSPKS) was substituted with the photoactive amino acid, benzoylphenylalanine (Bpa). Whereas most substitutions had little effect on binding affinity, Bpa substitution of either Met (+1 and +3 with respect to pY) reduced affinity 50-100-fold to confirm their importance in the pYMXM recognition motif. In three cases photolysis of SH2 domain/Bpa phosphopeptide complexes led to cross-linking of > 50% of the SH2 domain; cross-link positions were identified by microsequence, amino acid composition, and electrospray mass spectrometric analyses. Bpa-1 cross-links within alpha-helix I, whereas Bpa+1 and Bpa+4 cross-link the SH2 domain within the flexible loop C-terminal to alpha-helix II. Moreover, cross-linking at any position prevents SH2 domain cleavage at a trypsin-sensitive site within the flexible loop between beta-strands 1 and 2. Therefore, at least three distinct SH2 regions in addition to the beta-sheet participate in phosphoprotein binding; the loop cross-linked by phosphopeptide residues C-terminal to pY appears to confer specificity to the phosphoprotein/SH2 domain interaction.

Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5

PubMed Central

Azoitei, M.L.; Ban, Y.A.; Kalyuzhny, O.; Guenaga, J.; Schroeter, A.; Porter, J.; Wyatt, R.; Schief, W.R.

2015-01-01

Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope-scaffolds are a new class of antigens engineered by transplanting viral epitopes of pre-defined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope-scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody-binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope-scaffolds that display the known epitope of HIV-1 neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope-scaffold that bound 2F5 with sub-nanomolar affinity (KD = 400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope-scaffold represents a successful example of rational protein backbone engineering and protein-protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity. PMID:25043744

Analysis of Physicochemical and Structural Properties Determining HIV-1 Coreceptor Usage

PubMed Central

Bozek, Katarzyna; Lengauer, Thomas; Sierra, Saleta; Kaiser, Rolf; Domingues, Francisco S.

2013-01-01

The relationship of HIV tropism with disease progression and the recent development of CCR5-blocking drugs underscore the importance of monitoring virus coreceptor usage. As an alternative to costly phenotypic assays, computational methods aim at predicting virus tropism based on the sequence and structure of the V3 loop of the virus gp120 protein. Here we present a numerical descriptor of the V3 loop encoding its physicochemical and structural properties. The descriptor allows for structure-based prediction of HIV tropism and identification of properties of the V3 loop that are crucial for coreceptor usage. Use of the proposed descriptor for prediction results in a statistically significant improvement over the prediction based solely on V3 sequence with 3 percentage points improvement in AUC and 7 percentage points in sensitivity at the specificity of the 11/25 rule (95%). We additionally assessed the predictive power of the new method on clinically derived ‘bulk’ sequence data and obtained a statistically significant improvement in AUC of 3 percentage points over sequence-based prediction. Furthermore, we demonstrated the capacity of our method to predict therapy outcome by applying it to 53 samples from patients undergoing Maraviroc therapy. The analysis of structural features of the loop informative of tropism indicates the importance of two loop regions and their physicochemical properties. The regions are located on opposite strands of the loop stem and the respective features are predominantly charge-, hydrophobicity- and structure-related. These regions are in close proximity in the bound conformation of the loop potentially forming a site determinant for the coreceptor binding. The method is available via server under http://structure.bioinf.mpi-inf.mpg.de/. PMID:23555214

Resolved single-molecule detection of individual species within a mixture of anti-biotin antibodies using an engineered monomeric nanopore.

PubMed

Fahie, Monifa; Chisholm, Christina; Chen, Min

2015-02-24

Oligomeric protein nanopores with rigid structures have been engineered for the purpose of sensing a wide range of analytes including small molecules and biological species such as proteins and DNA. We chose a monomeric β-barrel porin, OmpG, as the platform from which to derive the nanopore sensor. OmpG is decorated with seven flexible loops that move dynamically to create a distinct gating pattern when ionic current passes through the pore. Biotin was chemically tethered to the most flexible one of these loops. The gating characteristic of the loop's movement in and out of the porin was substantially altered by analyte protein binding. The gating characteristics of the pore with bound targets were remarkably sensitive to molecular identity, even providing the ability to distinguish between homologues within an antibody mixture. A total of five gating parameters were analyzed for each analyte to create a unique fingerprint for each biotin-binding protein. Our exploitation of gating noise as a molecular identifier may allow more sophisticated sensor design, while OmpG's monomeric structure greatly simplifies nanopore production.

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

PubMed

Dai, Xinghong; Li, Zhihai; Lai, Mason; Shu, Sara; Du, Yushen; Zhou, Z Hong; Sun, Ren

2017-01-05

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

First Principles Predictions of the Structure and Function of G-Protein-Coupled Receptors: Validation for Bovine Rhodopsin

PubMed Central

Trabanino, Rene J.; Hall, Spencer E.; Vaidehi, Nagarajan; Floriano, Wely B.; Kam, Victor W. T.; Goddard, William A.

2004-01-01

G-protein-coupled receptors (GPCRs) are involved in cell communication processes and with mediating such senses as vision, smell, taste, and pain. They constitute a prominent superfamily of drug targets, but an atomic-level structure is available for only one GPCR, bovine rhodopsin, making it difficult to use structure-based methods to design receptor-specific drugs. We have developed the MembStruk first principles computational method for predicting the three-dimensional structure of GPCRs. In this article we validate the MembStruk procedure by comparing its predictions with the high-resolution crystal structure of bovine rhodopsin. The crystal structure of bovine rhodopsin has the second extracellular (EC-II) loop closed over the transmembrane regions by making a disulfide linkage between Cys-110 and Cys-187, but we speculate that opening this loop may play a role in the activation process of the receptor through the cysteine linkage with helix 3. Consequently we predicted two structures for bovine rhodopsin from the primary sequence (with no input from the crystal structure)—one with the EC-II loop closed as in the crystal structure, and the other with the EC-II loop open. The MembStruk-predicted structure of bovine rhodopsin with the closed EC-II loop deviates from the crystal by 2.84 Å coordinate root mean-square (CRMS) in the transmembrane region main-chain atoms. The predicted three-dimensional structures for other GPCRs can be validated only by predicting binding sites and energies for various ligands. For such predictions we developed the HierDock first principles computational method. We validate HierDock by predicting the binding site of 11-cis-retinal in the crystal structure of bovine rhodopsin. Scanning the whole protein without using any prior knowledge of the binding site, we find that the best scoring conformation in rhodopsin is 1.1 Å CRMS from the crystal structure for the ligand atoms. This predicted conformation has the carbonyl O only 2.82 Å from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 0.62 Å CRMS from the crystal structure. We also used HierDock to predict the binding site of 11-cis-retinal in the MembStruk-predicted structure of bovine rhodopsin (closed loop). Scanning the whole protein structure leads to a structure in which the carbonyl O is only 2.85 Å from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 2.92 Å CRMS from the crystal structure. The good agreement of the ab initio-predicted protein structures and ligand binding site with experiment validates the use of the MembStruk and HierDock first principles' methods. Since these methods are generic and applicable to any GPCR, they should be useful in predicting the structures of other GPCRs and the binding site of ligands to these proteins. PMID:15041637

Predicted stem-loop structures and variation in nucleotide sequence of 3' noncoding regions among animal calicivirus genomes.

PubMed

Seal, B S; Neill, J D; Ridpath, J F

1994-07-01

Caliciviruses are nonenveloped with a polyadenylated genome of approximately 7.6 kb and a single capsid protein. The "RNA Fold" computer program was used to analyze 3'-terminal noncoding sequences of five feline calicivirus (FCV), rabbit hemorrhagic disease virus (RHDV), and two San Miguel sea lion virus (SMSV) isolates. The FCV 3'-terminal sequences are 40-46 nucleotides in length and 72-91% similar. The FCV sequences were predicted to contain two possible duplex structures and one stem-loop structure with free energies of -2.1 to -18.2 kcal/mole. The RHDV genomic 3'-terminal RNA sequences are 54 nucleotides in length and share 49% sequence similarity to homologous regions of the FCV genome. The RHDV sequence was predicted to form two duplex structures in the 3'-terminal noncoding region with a single stem-loop structure, resembling that of FCV. In contrast, the SMSV 1 and 4 genomic 3'-terminal noncoding sequences were 185 and 182 nucleotides in length, respectively. Ten possible duplex structures were predicted with an average structural free energy of -35 kcal/mole. Sequence similarity between the two SMSV isolates was 75%. Furthermore, extensive cloverleaflike structures are predicted in the 3' noncoding region of the SMSV genome, in contrast to the predicted single stem-loop structures of FCV or RHDV.

Epidermal Growth Factor Receptor mediated cellular and subcellular targeted delivery of Iron oxide core-Titanium dioxide shell nanoparticles

NASA Astrophysics Data System (ADS)

Yuan, Ye

TiO2 nanomaterials can carry a multitude of therapeutic and diagnostic agents and the semiconductor properties of TiO2 allow for the production of cytotoxic reactive oxygen species following photoactivation. However, the delivery of these nanomaterials to specific cancer cells and specific subcellular organelles within these cells can have a substantial impact on the efficacy and safety of TiO2 nanoparticle therapeutics. Targeting cell surface receptors that are overexpressed by cancer cells is one strategy to improve the specificity of nanoparticle delivery. Therefore we decided to target the Epidermal Growth Factor Receptor (EGFR) because ligand- binding induces rapid receptor endocytosis and ligand-bound EGFR can translocate to the nucleus of cancer cells. To create NPs that can bind EGFR, we identified a peptide derived from the B-loop of Epidermal Growth Factor (EGF) that has been shown to bind and activate EGFR and conjugated it to the surface of Fe3O4 core-TiO2 shell NPs to produce B-loop NCs. We then devised a pulldown assay to show that B-loop NCs, but not bare NPs or NCs carrying a scrambled B-loop peptide, can bind and extract EGFR from HeLa cell protein extracts. Interestingly, B-loop NCs can also pulldown importin-beta, a protein that can transport EGFR to the nucleus. Furthermore, we used flow cytometry and fluorescently labeled NPs to show that B-loop peptides can significantly improve the internalization of NPs by EGFR-expressing HeLa cells. We determined that B-loop NCs can bind EGFR on the membrane of HeLa cells and that these NCs can be transported to the nucleus, by using a combination of confocal microscopy and X-ray Fluorescence Microscopy (XFM) to indirectly and directly track the subcellular distribution of NCs. Finally, we demonstrate how the Bionanoprobe, a novel high-resolution XFM apparatus that can scan whole-mounted, frozen-hydrated cells at multiple angles can be used to verify the subcellular distribution of B-loop NCs.

Rational and Modular Design of Potent Ligands Targeting the RNA that Causes Myotonic Dystrophy 2

PubMed Central

Lee, Melissa M.; Pushechnikov, Alexei; Disney, Matthew D.

2009-01-01

Most ligands targeting RNA are identified through screening a therapeutic target for binding members of a ligand library. A potential alternative way to construct RNA binders is through rational design using information about the RNA motifs ligands prefer to bind. Herein, we describe such an approach to design modularly assembled ligands targeting the RNA that causes myotonic dystrophy type 2 (DM2), a currently untreatable disease. A previous study identified that 6′-N-5-hexynoate kanamycin A (1) prefers to bind 2×2 nucleotide, pyrimidine-rich RNA internal loops. Multiple copies of such loops were found in the RNA hairpin that causes DM2. The 1 ligand was then modularly displayed on a peptoid scaffold with varied number and spacing to target several internal loops simultaneously. Modularly assembled ligands were tested for binding to a series of RNAs and for inhibiting the formation of the toxic DM2 RNA-muscleblind protein (MBNL-1) interaction. The most potent ligand displays three 1 modules, each separated by four spacing submonomers, and inhibits the formation of the RNA-protein complex with an IC50 of 25 nM. This ligand is higher affinity and more specific for binding DM2 RNA than MBNL-1. It binds the DM2 RNA at least 20-times more tightly than related RNAs and 15-fold more tightly than MBNL-1. A related control peptoid displaying 6′-N-5-hexynoate neamine (2) is >100-fold less potent at inhibiting the RNA-protein interaction and binds to DM2 RNA >125-fold more weakly. Uptake studies into a mouse myoblast cell line also show that the most potent ligand is cell permeable. PMID:19348464

Evolution of the Max and Mlx networks in animals.

PubMed

McFerrin, Lisa G; Atchley, William R

2011-01-01

Transcription factors (TFs) are essential for the regulation of gene expression and often form emergent complexes to perform vital roles in cellular processes. In this paper, we focus on the parallel Max and Mlx networks of TFs because of their critical involvement in cell cycle regulation, proliferation, growth, metabolism, and apoptosis. A basic-helix-loop-helix-zipper (bHLHZ) domain mediates the competitive protein dimerization and DNA binding among Max and Mlx network members to form a complex system of cell regulation. To understand the importance of these network interactions, we identified the bHLHZ domain of Max and Mlx network proteins across the animal kingdom and carried out several multivariate statistical analyses. The presence and conservation of Max and Mlx network proteins in animal lineages stemming from the divergence of Metazoa indicate that these networks have ancient and essential functions. Phylogenetic analysis of the bHLHZ domain identified clear relationships among protein families with distinct points of radiation and divergence. Multivariate discriminant analysis further isolated specific amino acid changes within the bHLHZ domain that classify proteins, families, and network configurations. These analyses on Max and Mlx network members provide a model for characterizing the evolution of TFs involved in essential networks.

Exploring TAR–RNA aptamer loop–loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance

PubMed Central

Lebars, Isabelle; Legrand, Pierre; Aimé, Ahissan; Pinaud, Noël; Fribourg, Sébastien; Di Primo, Carmelo

2008-01-01

In HIV-1, trans-activation of transcription of the viral genome is regulated by an imperfect hairpin, the trans-activating responsive (TAR) RNA element, located at the 5′ untranslated end of all viral transcripts. TAR acts as a binding site for viral and cellular proteins. In an attempt to identify RNA ligands that would interfere with the virus life-cycle by interacting with TAR, an in vitro selection was previously carried out. RNA hairpins that formed kissing-loop dimers with TAR were selected [Ducongé F. and Toulmé JJ (1999) RNA, 5:1605–1614]. We describe here the crystal structure of TAR bound to a high-affinity RNA aptamer. The two hairpins form a kissing complex and interact through six Watson–Crick base pairs. The complex adopts an overall conformation with an inter-helix angle of 28.1°, thus contrasting with previously reported solution and modelling studies. Structural analysis reveals that inter-backbone hydrogen bonds between ribose 2′ hydroxyl and phosphate oxygens at the stem-loop junctions can be formed. Thermal denaturation and surface plasmon resonance experiments with chemically modified 2′-O-methyl incorporated into both hairpins at key positions, clearly demonstrate the involvement of this intermolecular network of hydrogen bonds in complex stability. PMID:18996893

Conformation-selective inhibitors reveal differences in the activation and phosphate-binding loops of the tyrosine kinases Abl and Src

PubMed Central

Hari, Sanjay B.; Perera, B. Gayani K.; Ranjitkar, Pratistha; Seeliger, Markus A.; Maly, Dustin J.

2013-01-01

Over the last decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely-related tyrosine kinases, like Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abl's activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abl's activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related kinases. PMID:24106839

Studies of Xenopus laevis mitochondrial DNA: D-loop mapping and characterization of DNA-binding proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cairns, S.S.

1987-01-01

In X. laevis oocytes, mitochondrial DNA accumulates to 10/sup 5/ times the somatic cell complement, and is characterized by a high frequency of a triple-stranded displacement hoop structure at the origin of replication. To map the termini of the single strands, it was necessary to correct the nucleotide sequence of the D-loop region. The revised sequence of 2458 nucleotides contains 54 discrepancies in comparison to a previously published sequence. Radiolabeling of the nascent strands of the D-loop structure either at the 5' end or at the 3' end identifies a major species with a length of 1670 nucleotides. Cleavage ofmore » the 5' labeled strands reveals two families of ends located near several matches to an element, designated CSB-1, that is conserved in this location in several vertebrate genomes. Cleavage of 3' labeled strands produced one fragment. The unique 3' end maps to about 15 nucleotides preceding the tRNA/sup Pro/ gene. A search for proteins which may bind to mtDNA in this region to regulate nucleic acid synthesis has identified three activities in lysates of X. laevis mitochondria. The DNA-binding proteins were assayed by monitoring their ability to retard the migration of labeled double- or single-stranded DNA fragments in polyacrylamide gels. The DNA binding preference was determined by competition with an excess of either ds- or ssDNA.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonicalmore » PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo

Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain amore » canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

Mechanism of the formation of the RecA-ssDNA nucleoprotein filament structure: a coarse-grained approach.

PubMed

Mukherjee, Goutam; Pal, Arumay; Levy, Yaakov

2017-11-21

In prokaryotes, the RecA protein catalyzes the repair and strand exchange of double-stranded DNA. RecA binds to single-stranded DNA (ssDNA) and forms a presynaptic complex in which the protein polymerizes around the ssDNA to form a right-handed helical nucleoprotein filament structure. In the present work, the mechanism for the formation of the RecA-ssDNA filament structure is modeled using coarse-grained molecular dynamics simulations. Information from the X-ray structure was used to model the protein itself but not its interactions; the interactions between the protein and the ssDNA were modeled solely by electrostatic, aromatic, and repulsive energies. For the present study, the monomeric, dimeric, and trimeric units of RecA and 4, 8, and 11 NT-long ssDNA, respectively, were studied. Our results indicate that monomeric RecA is not sufficient for nucleoprotein filament formation; rather, dimeric RecA is the elementary binding unit, with higher multimeric units of RecA facilitating filament formation. Our results reveal that loop region flexibility at the primary binding site of RecA is essential for it to bind the incoming ssDNA, that the aromatic residues present in the loop region play an important role in ssDNA binding, and that ATP may play a role in guiding the ssDNA by changing the electrostatic potential of the RecA protein.

Diffusional encounter of barnase and barstar.

PubMed

Spaar, Alexander; Dammer, Christian; Gabdoulline, Razif R; Wade, Rebecca C; Helms, Volkhard

2006-03-15

We present an analysis of trajectories from Brownian dynamics simulations of diffusional protein-protein encounter for the well-studied system of barnase and barstar. This analysis reveals details about the optimal association pathways, the regions of the encounter complex, possible differences of the pathways for dissociation and association, the coupling of translational and rotation motion, and the effect of mutations on the trajectories. We found that a small free-energy barrier divides the energetically most favorable region into a region of the encounter complex above the barnase binding interface and a region around a second energy minimum near the RNA binding loop. When entering the region of the encounter complex from the region near the RNA binding loop, barstar has to change its orientation to increase the electrostatic attraction between the proteins. By concentrating the analysis on the successful binding trajectories, we found that the region of the second minimum is not essential for the binding of barstar to barnase. Nevertheless, this region may be helpful to steer barstar into the region of the encounter complex. When applying the same analysis to several barnase mutants, we found that single mutations may drastically change the free-energy landscape and may significantly alter the population of the two minima. Therefore, certain protein-protein pairs may require careful adaptation of the positions of encounter and transition states when interpreting mutation effects on kinetic rates of association and/or dissociation.

A peptide sequence on carcinoembryonic antigen binds to a 80kD protein on Kupffer cells.

PubMed

Thomas, P; Petrick, A T; Toth, C A; Fox, E S; Elting, J J; Steele, G

1992-10-30

Clearance of carcinoembryonic antigen (CEA) from the circulation is by binding to Kupffer cells in the liver. We have shown that CEA binding to Kupffer cells occurs via a peptide sequence YPELPK representing amino acids 107-112 of the CEA sequence. This peptide sequence is located in the region between the N-terminal and the first immunoglobulin like loop domain. Using native CEA and peptides containing this sequence complexed with a heterobifunctional crosslinking agent and ligand blotting with biotinylated CEA and NCA we have shown binding to an 80kD protein on the Kupffer cell surface. This binding protein may be important in the development of hepatic metastases.

Mitochondrial Genome of the Stonefly Kamimuria wangi (Plecoptera: Perlidae) and Phylogenetic Position of Plecoptera Based on Mitogenomes

PubMed Central

Yu-Han, Qian; Hai-Yan, Wu; Xiao-Yu, Ji; Wei-Wei, Yu; Yu-Zhou, Du

2014-01-01

This study determined the mitochondrial genome sequence of the stonefly, Kamimuria wangi. In order to investigate the relatedness of stonefly to other members of Neoptera, a phylogenetic analysis was undertaken based on 13 protein-coding genes of mitochondrial genomes in 13 representative insects. The mitochondrial genome of the stonefly is a circular molecule consisting of 16,179 nucleotides and contains the 37 genes typically found in other insects. A 10-bp poly-T stretch was observed in the A+T-rich region of the K. wangi mitochondrial genome. Downstream of the poly-T stretch, two regions were located with potential ability to form stem-loop structures; these were designated stem-loop 1 (positions 15848–15651) and stem-loop 2 (15965–15998). The arrangement of genes and nucleotide composition of the K. wangi mitogenome are similar to those in Pteronarcys princeps, suggesting a conserved genome evolution within the Plecoptera. Phylogenetic analysis using maximum likelihood and Bayesian inference of 13 protein-coding genes supported a novel relationship between the Plecoptera and Ephemeroptera. The results contradict the existence of a monophyletic Plectoptera and Plecoptera as sister taxa to Embiidina, and thus requires further analyses with additional mitogenome sampling at the base of the Neoptera. PMID:24466028

Mitochondrial genome of the stonefly Kamimuria wangi (Plecoptera: Perlidae) and phylogenetic position of plecoptera based on mitogenomes.

PubMed

Yu-Han, Qian; Hai-Yan, Wu; Xiao-Yu, Ji; Wei-Wei, Yu; Yu-Zhou, Du

2014-01-01

This study determined the mitochondrial genome sequence of the stonefly, Kamimuria wangi. In order to investigate the relatedness of stonefly to other members of Neoptera, a phylogenetic analysis was undertaken based on 13 protein-coding genes of mitochondrial genomes in 13 representative insects. The mitochondrial genome of the stonefly is a circular molecule consisting of 16,179 nucleotides and contains the 37 genes typically found in other insects. A 10-bp poly-T stretch was observed in the A+T-rich region of the K. wangi mitochondrial genome. Downstream of the poly-T stretch, two regions were located with potential ability to form stem-loop structures; these were designated stem-loop 1 (positions 15848-15651) and stem-loop 2 (15965-15998). The arrangement of genes and nucleotide composition of the K. wangi mitogenome are similar to those in Pteronarcys princeps, suggesting a conserved genome evolution within the Plecoptera. Phylogenetic analysis using maximum likelihood and Bayesian inference of 13 protein-coding genes supported a novel relationship between the Plecoptera and Ephemeroptera. The results contradict the existence of a monophyletic Plectoptera and Plecoptera as sister taxa to Embiidina, and thus requires further analyses with additional mitogenome sampling at the base of the Neoptera.

The interaction between the yeast telomerase RNA and the Est1 protein requires three structural elements.

PubMed

Lubin, Johnathan W; Tucey, Timothy M; Lundblad, Victoria

2012-09-01

In the budding yeast Saccharomyces cerevisiae, the telomerase enzyme is composed of a 1.3-kb TLC1 RNA that forms a complex with Est2 (the catalytic subunit) and two regulatory proteins, Est1 and Est3. Previous work has identified a conserved 5-nt bulge, present in a long helical arm of TLC1, which mediates binding of Est1 to TLC1. However, increased expression of Est1 can bypass the consequences of removal of this RNA bulge, indicating that there are additional binding site(s) for Est1 on TLC1. We report here that a conserved single-stranded internal loop immediately adjacent to the bulge is also required for the Est1-RNA interaction; furthermore, a TLC1 variant that lacks this internal loop but retains the bulge cannot be suppressed by Est1 overexpression, arguing that the internal loop may be a more critical element for Est1 binding. An additional structural feature consisting of a single-stranded region at the base of the helix containing the bulge and internal loop also contributes to recognition of TLC1 by Est1, potentially by providing flexibility to this helical arm. Association of Est1 with each of these TLC1 motifs was assessed using a highly sensitive biochemical assay that simultaneously monitors the relative levels of the Est1 and Est2 proteins in the telomerase complex. The identification of three elements of TLC1 that are required for Est1 association provides a detailed view of this particular protein-RNA interaction.

Membrane Association of the PTEN Tumor Suppressor: Electrostatic Interaction with Phos-phatidylserine-Containing Bilayers and Regulatory Role of the C-Terminal Tail

PubMed Central

Shenoy, Siddharth S.; Nanda, Hirsh; Lösche, Mathias

2012-01-01

The phosphatidylinositolphosphate phosphatase PTEN is the second most frequently mutated protein in human tumors. Its membrane association, allosteric activation and membrane dissociation are poorly understood. We recently reported PTEN binding affinities to membranes of different compositions and a preliminary investigation of the protein-membrane complex with neutron reflectometry (NR). Here we use NR to validate molecular dynamics (MD) simulations of the protein and study conformational differences of the protein in solution and on anionic membranes. NR shows that full-length PTEN binds to such membranes roughly in the conformation and orientation suggested by the crystal structure of a truncated PTEN protein, in contrast with a recently presented model which suggested that membrane binding depends critically on the SUMOylation of the CBR3 loop of PTEN’s C2 domain. Our MD simulations confirm that PTEN is peripherally bound to the bilayer surface and show slight differences of the protein structure in solution and in the membrane-bound state, where the protein body flattens against the bilayer surface. PTEN’s C2 domain binds phosphatidylserine (PS) tightly through its CBR3 loop, and its phosphatase domain also forms electrostatic interactions with PS. NR and MD results show consistently that PTEN’s unstructured, anionic C-terminal tail is repelled from the bilayer surface. In contrast, this tail is tightly tugged against the C2 domain in solution, partially obstructing the membrane-binding interface of the protein. Arresting the C-terminal tail in this conformation by phosphorylation may provide a control mechanism for PTEN’s membrane binding and activity. PMID:23073177

Membrane association of the PTEN tumor suppressor: electrostatic interaction with phosphatidylserine-containing bilayers and regulatory role of the C-terminal tail.

PubMed

Shenoy, Siddharth S; Nanda, Hirsh; Lösche, Mathias

2012-12-01

The phosphatidylinositolphosphate phosphatase PTEN is the second most frequently mutated protein in human tumors. Its membrane association, allosteric activation and membrane dissociation are poorly understood. We recently reported PTEN binding affinities to membranes of different compositions (Shenoy et al., 2012, PLoS ONE 7, e32591) and a preliminary investigation of the protein-membrane complex with neutron reflectometry (NR). Here we use NR to validate molecular dynamics (MD) simulations of the protein and study conformational differences of the protein in solution and on anionic membranes. NR shows that full-length PTEN binds to such membranes roughly in the conformation and orientation suggested by the crystal structure of a truncated PTEN protein, in contrast with a recently presented model which suggested that membrane binding depends critically on the SUMOylation of the CBR3 loop of PTEN's C2 domain. Our MD simulations confirm that PTEN is peripherally bound to the bilayer surface and show slight differences of the protein structure in solution and in the membrane-bound state, where the protein body flattens against the bilayer surface. PTEN's C2 domain binds phosphatidylserine (PS) tightly through its CBR3 loop, and its phosphatase domain also forms electrostatic interactions with PS. NR and MD results show consistently that PTEN's unstructured, anionic C-terminal tail is repelled from the bilayer surface. In contrast, this tail is tightly tugged against the C2 domain in solution, partially obstructing the membrane-binding interface of the protein. Arresting the C-terminal tail in this conformation by phosphorylation may provide a control mechanism for PTEN's membrane binding and activity. Copyright © 2012 Elsevier Inc. All rights reserved.

AtLa1 protein initiates IRES-dependent translation of WUSCHEL mRNA and regulates the stem cell homeostasis of Arabidopsis in response to environmental hazards.

PubMed

Cui, Yuchao; Rao, Shaofei; Chang, Beibei; Wang, Xiaoshuang; Zhang, Kaidian; Hou, Xueliang; Zhu, Xueyi; Wu, Haijun; Tian, Zhaoxia; Zhao, Zhong; Yang, Chengwei; Huang, Tao

2015-10-01

Plant stem cells are hypersensitive to environmental hazards throughout their life cycle, but the mechanism by which plants safeguard stem cell homeostasis in response to environmental hazards is largely unknown. The homeodomain transcription factor WUSCHEL (WUS) protein maintains the stem cell pool in the shoot apical meristem of Arabidopsis. Here, we demonstrate that the translation of WUS mRNA is directed by an internal ribosomal entry site (IRES) located in the 5'-untranslated region. The AtLa1 protein, an RNA-binding factor, binds to the 5'-untranslated region and initiates the IRES-dependent translation of WUS mRNA. Knockdown of AtLa1 expression represses the WUS IRES-dependent translation and leads to the arrest of growth and development. The AtLa1 protein is mainly located in the nucleoplasm. However, environmental hazards promote the nuclear-to-cytoplasmic translocation of the AtLa1 protein, which further enhances the IRES-dependent translation of WUS mRNA. Genetic evidence indicates that the WUS protein increases the tolerance of the shoot apical meristem to environmental hazards. Based on these results, we conclude that the stem cell niche in Arabidopsis copes with environmental hazards by enhancing the IRES-dependent translation of WUS mRNA under the control of the AtLa1 protein. © 2015 John Wiley & Sons Ltd.

Phage selection of peptide "microantibodies".

PubMed

Fujiwara, Daisuke; Fujii, Ikuo

2013-01-01

A bioactive peptide capable of inhibiting protein-protein interactions has the potential to be a molecular tool for biological studies and a therapeutic by disrupting aberrant interactions involved in diseases. We have developed combinatorial libraries of peptides with helix-loop-helix structure, from which the isolated peptides have the constrained structure to reduce entropy costs in binding, resulting in high binding affinities for target molecules. Previously, we designed a de novo peptide of helix-loop-helix structure that we termed a "microantibody." Using the microantibody as a library scaffold, we have constructed a phage-display library to successfully isolate molecular-targeting peptides against a cytokine receptor (granulocyte colony-stimulating factor receptor), a protein kinase (Aurora-A), and a ganglioside (GM1). Protocols in this article describe a general procedure for the library construction and the library screening.

Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'UTR secondary structure specifically inhibits heat shock protein mRNA translation.

PubMed Central

Hess, M A; Duncan, R F

1996-01-01

Preferential translation of Drosophila heat shock protein 70 (Hsp70) mRNA requires only the 5'-untranslated region (5'-UTR). The sequence of this region suggests that it has relatively little secondary structure, which may facilitate efficient protein synthesis initiation. To determine whether minimal 5'-UTR secondary structure is required for preferential translation during heat shock, the effect of introducing stem-loops into the Hsp70 mRNA 5'-UTR was measured. Stem-loops of -11 kcal/mol abolished translation during heat shock, but did not reduce translation in non-heat shocked cells. A -22 kcal/mol stem-loop was required to comparably inhibit translation during growth at normal temperatures. To investigate whether specific sequence elements are also required for efficient preferential translation, deletion and mutation analyses were conducted in a truncated Hsp70 5'-UTR containing only the cap-proximal and AUG-proximal segments. Linker-scanner mutations in the cap-proximal segment (+1 to +37) did not impair translation. Re-ordering the segments reduced mRNA translational efficiency by 50%. Deleting the AUG-proximal segment severely inhibited translation. A 5-extension of the full-length leader specifically impaired heat shock translation. These results indicate that heat shock reduces the capacity to unwind 5-UTR secondary structure, allowing only mRNAs with minimal 5'-UTR secondary structure to be efficiently translated. A function for specific sequences is also suggested. PMID:8710519

Fast de novo discovery of low-energy protein loop conformations.

PubMed

Wong, Samuel W K; Liu, Jun S; Kou, S C

2017-08-01

In the prediction of protein structure from amino acid sequence, loops are challenging regions for computational methods. Since loops are often located on the protein surface, they can have significant roles in determining protein functions and binding properties. Loop prediction without the aid of a structural template requires extensive conformational sampling and energy minimization, which are computationally difficult. In this article we present a new de novo loop sampling method, the Parallely filtered Energy Targeted All-atom Loop Sampler (PETALS) to rapidly locate low energy conformations. PETALS explores both backbone and side-chain positions of the loop region simultaneously according to the energy function selected by the user, and constructs a nonredundant ensemble of low energy loop conformations using filtering criteria. The method is illustrated with the DFIRE potential and DiSGro energy function for loops, and shown to be highly effective at discovering conformations with near-native (or better) energy. Using the same energy function as the DiSGro algorithm, PETALS samples conformations with both lower RMSDs and lower energies. PETALS is also useful for assessing the accuracy of different energy functions. PETALS runs rapidly, requiring an average time cost of 10 minutes for a length 12 loop on a single 3.2 GHz processor core, comparable to the fastest existing de novo methods for generating an ensemble of conformations. Proteins 2017; 85:1402-1412. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280).

PubMed

Li, Ming; Li, Chuanyu; Weingarten, Paul; Bunzow, James R; Grandy, David K; Zhou, Qun Yong

2002-03-01

Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.

A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

DOE PAGES

Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; ...

2016-05-03

Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain amore » canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

Alternative Splicing of MBD2 Supports Self-Renewal in Human Pluripotent Stem Cells

PubMed Central

Lu, Yu; Loh, Yuin-Han; Li, Hu; Cesana, Marcella; Ficarro, Scott B.; Parikh, Jignesh R.; Salomonis, Nathan; Toh, Cheng-Xu Delon; Andreadis, Stelios T.; Luckey, C. John; Collins, James J.; Daley, George Q.; Marto, Jarrod A.

2014-01-01

Summary Alternative RNA splicing (AS) regulates proteome diversity, including isoform-specific expression of several pluripotency genes. Here, we integrated global gene expression and proteomic analyses and identified a molecular signature suggesting a central role for AS in maintaining human pluripotent stem cell (hPSC) self-renewal. We demonstrate the splicing factor SFRS2 is an OCT4 target gene required for pluripotency. SFRS2 regulates AS of the methyl-CpG-binding protein MBD2, whose isoforms play opposing roles in maintenance of, and reprogramming to, pluripotency. While both MDB2a and MBD2c are enriched at the OCT4 and NANOG promoters, MBD2a preferentially interacts with repressive NuRD chromatin remodeling factors and promotes hPSC differentiation, whereas overexpression of MBD2c enhances reprogramming of fibroblasts to pluripotency. The miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and MDB2a. These data suggest that OCT4, SFRS2, and MBD2 participate in a positive feedback loop, regulating proteome diversity complexity in support of hPSC self-renewal and reprogramming. PMID:24813856

Calcium Sensing by Recoverin: Effect of Protein Conformation on Ion Affinity.

PubMed

Timr, Štěpán; Kadlec, Jan; Srb, Pavel; Ollila, O H Samuli; Jungwirth, Pavel

2018-04-05

The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors

PubMed Central

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios

2017-01-01

Abstract Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. PMID:28973457

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

PubMed

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

2017-09-29

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

PubMed

Miao, Yinglong; McCammon, J Andrew

2018-03-20

Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

Hes repressors are essential regulators of hematopoietic stem cell development downstream of Notch signaling

PubMed Central

Guiu, Jordi; Shimizu, Ritsuko; D’Altri, Teresa; Fraser, Stuart T.; Hatakeyama, Jun; Bresnick, Emery H.; Kageyama, Ryoichiro; Dzierzak, Elaine; Yamamoto, Masayuki; Espinosa, Lluis

2013-01-01

Previous studies have identified Notch as a key regulator of hematopoietic stem cell (HSC) development, but the underlying downstream mechanisms remain unknown. The Notch target Hes1 is widely expressed in the aortic endothelium and hematopoietic clusters, though Hes1-deficient mice show no overt hematopoietic abnormalities. We now demonstrate that Hes is required for the development of HSC in the mouse embryo, a function previously undetected as the result of functional compensation by de novo expression of Hes5 in the aorta/gonad/mesonephros (AGM) region of Hes1 mutants. Analysis of embryos deficient for Hes1 and Hes5 reveals an intact arterial program with overproduction of nonfunctional hematopoietic precursors and total absence of HSC activity. These alterations were associated with increased expression of the hematopoietic regulators Runx1, c-myb, and the previously identified Notch target Gata2. By analyzing the Gata2 locus, we have identified functional RBPJ-binding sites, which mutation results in loss of Gata2 reporter expression in transgenic embryos, and functional Hes-binding sites, which mutation leads to specific Gata2 up-regulation in the hematopoietic precursors. Together, our findings show that Notch activation in the AGM triggers Gata2 and Hes1 transcription, and next HES-1 protein represses Gata2, creating an incoherent feed-forward loop required to restrict Gata2 expression in the emerging HSCs. PMID:23267012

Structural Basis for Ligand Regulation of the Fatty Acid-binding Protein 5, Peroxisome Proliferator-activated Receptor β/δ (FABP5-PPARβ/δ) Signaling Pathway*

PubMed Central

Armstrong, Eric H.; Goswami, Devrishi; Griffin, Patrick R.; Noy, Noa; Ortlund, Eric A.

2014-01-01

Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain “activating” fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARβ/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing β2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling. PMID:24692551

Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

PubMed Central

McClellan, Michael J.; Wood, C. David; Ojeniyi, Opeoluwa; Cooper, Tim J.; Kanhere, Aditi; Arvey, Aaron; Webb, Helen M.; Palermo, Richard D.; Harth-Hertle, Marie L.; Kempkes, Bettina; Jenner, Richard G.; West, Michelle J.

2013-01-01

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors. PMID:24068937

pyr RNA binding to the Bacillus caldolyticus PyrR attenuation protein. Characterization and regulation by uridine and guanosine nucleotides

PubMed Central

Jørgensen, Casper Møller; Fields, Christopher J.; Chander, Preethi; Watt, Desmond; Burgner, John W.; Smith, Janet L.; Switzer, Robert L.

2011-01-01

Summary The PyrR protein regulates expression of pyrimidine biosynthetic (pyr) genes in many bacteria. PyrR binds to specific sites in the 5’ leader RNA of target operons and favors attenuation of transcription. Filter binding and gel mobility assays were used to characterize the binding of PyrR from Bacillus caldolyticus to RNA sequences (binding loops) from the three attenuation regions of the B. caldolyticus pyr operon. Binding of PyrR to the three binding loops and modulation of RNA binding by nucleotides was similar for all three RNAs. Apparent dissociation constants at 0° C ranged from 0.13 to 0.87 nM in the absence of effectors; dissociation constants were decreased by 3 to 12 fold by uridine nucleotides and increased by 40 to 200 fold by guanosine nucleotides. The binding data suggest that pyr operon expression is regulated by the ratio of intracellular uridine nucleotides to guanosine nucleotides; the effects of nucleoside addition to the growth medium on aspartate transcarbamylase (pyrB) levels in B. subtilis cells in vivo supported this conclusion. Analytical ultracentrifugation established that RNA binds to dimeric PyrR, even though the tetrameric form of unbound PyrR predominates in solution at the concentrations studied. PMID:18190533

AMP-Activated Protein Kinase β-Subunit Requires Internal Motion for Optimal Carbohydrate Binding

PubMed Central

Bieri, Michael; Mobbs, Jesse I.; Koay, Ann; Louey, Gavin; Mok, Yee-Foong; Hatters, Danny M.; Park, Jong-Tae; Park, Kwan-Hwa; Neumann, Dietbert; Stapleton, David; Gooley, Paul R.

2012-01-01

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β1 and β2). Muscle-specific β2-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β1-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β2-CBM is concurrent with an increase in flexibility in β2-CBM, which may account for the affinity differences between the two isoforms. In contrast to β1-CBM, unbound β2-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β2-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β2-CBM mutant. Insertion of a threonine into the background of β1-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms. PMID:22339867

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Qiaozhen; Krug, Robert M.; Tao, Yizhi Jane

Influenza A viruses pose a serious threat to world public health, particularly the currently circulating avian H5N1 viruses. The influenza viral nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complexes, and has a critical role in viral RNA replication. Here we report a 3.2 Angstrom crystal structure of this nucleoprotein, the overall shape of which resembles a crescent with a head and a body domain, with a protein fold different compared with that of the rhabdovirus nucleoprotein. Oligomerization of the influenza virus nucleoprotein is mediated by a flexible tail loop that is inserted inside a neighboring molecule. Thismore » flexibility in the tail loop enables the nucleoprotein to form loose polymers as well as rigid helices, both of which are important for nucleoprotein functions. Single residue mutations in the tail loop result in the complete loss of nucleoprotein oligomerization. An RNA-binding groove, which is found between the head and body domains at the exterior of the nucleoprotein oligomer, is lined with highly conserved basic residues widely distributed in the primary sequence. The nucleoprotein structure shows that only one of two proposed nuclear localization signals are accessible, and suggests that the body domain of nucleoprotein contains the binding site for the viral polymerase. Our results identify the tail loop binding pocket as a potential target for antiviral development.« less

VP40 of the Ebola Virus as a Target for EboV Therapy: Comprehensive Conformational and Inhibitor Binding Landscape from Accelerated Molecular Dynamics.

PubMed

Balmith, Marissa; Soliman, Mahmoud E S

2017-03-01

The first account of the dynamic features of the loop region of VP40 of the Ebola virus was studied using accelerated molecular dynamics simulations and reported herein. Among the proteins of the Ebola virus, the matrix protein (VP40) plays a significant role in the virus lifecycle thereby making it a promising therapeutic target. Of interest is the newly elucidated N-terminal domain loop region of VP40 comprising residues K127, T129, and N130 which when mutated to alanine have demonstrated an unrecognized role for N-terminal domain-plasma membrane interaction for efficient VP40-plasma membrane localization, oligomerization, matrix assembly, and egress. The molecular understanding of the conformational features of VP40 in complex with a known inhibitor still remains elusive. Using accelerated molecular dynamics approaches, we conducted a comparative study on VP40 apo and bound systems to understand the conformational features of VP40 at the molecular level and to determine the effect of inhibitor binding with the aid of a number of post-dynamic analytical tools. Significant features were seen in the presence of an inhibitor as per molecular mechanics/generalized born surface area binding free energy calculations. Results revealed that inhibitor binding to VP40 reduces the flexibility and mobility of the protein as supported by root mean square fluctuation and root mean square deviation calculations. The study revealed a characteristic "twisting" motion and coiling of the loop region of VP40 accompanied by conformational changes in the dimer interface upon inhibitor binding. We believe that results presented in this study will ultimately provide useful insight into the binding landscape of VP40 which could assist researchers in the discovery of potent Ebola virus inhibitors for anti-Ebola therapies.

Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5.

PubMed

Azoitei, M L; Ban, Y A; Kalyuzhny, O; Guenaga, J; Schroeter, A; Porter, J; Wyatt, R; Schief, William R

2014-10-01

Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope-scaffolds are a new class of antigens engineered by transplanting viral epitopes of predefined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope-scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody-binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope-scaffolds that display the known epitope of human immunodeficiency virus 1 (HIV-1) neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope-scaffold that bound 2F5 with subnanomolar affinity (K(D) = 400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope-scaffold represents a successful example of rational protein backbone engineering and protein-protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity. © 2014 Wiley Periodicals, Inc.

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein.

PubMed

Belfetmi, Anissa; Zargarian, Loussiné; Tisné, Carine; Sleiman, Dona; Morellet, Nelly; Lescop, Ewen; Maskri, Ouerdia; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2016-04-01

The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59. © 2016 Belfetmi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Enzymatic properties of a GH19 chitinase isolated from rice lacking a major loop structure involved in chitin binding.

PubMed

Tanaka, Jun; Fukamizo, Tamo; Ohnuma, Takayuki

2017-05-01

The catalytic domains of family GH19 chitinases have been found to consist of a conserved, α-helical core-region and different numbers (1-6) of loop structures, located at both ends of the substrate-binding groove and which extend over the glycon- and aglycon-binding sites. We expressed, purified and enzymatically characterized a GH19 chitinase from rice, Oryza sativa L. cv. Nipponbare (OsChia2a), lacking a major loop structure (loop III) connected to the functionally important β-stranded region. The new enzyme thus contained the five remaining loop structures (loops I, II, IV, V and C-term). The OsChia2a recombinant protein catalyzed hydrolysis of chitin oligosaccharides, (GlcNAc)n (n = 3-6), with inversion of anomeric configuration, indicating that OsChia2a correctly folded without loop III. From thermal unfolding experiments and calorimetric titrations using the inactive OsChia2a mutant (OsChia2a-E68Q), in which the catalytic residue Glu68 was mutated to glutamine, we found that the binding affinities towards (GlcNAc)n (n = 2-6) were almost proportional to the degree of polymerization of (GlcNAc)n, but were much lower than those obtained for a moss GH19 chitinase having only loop III [Ohnuma T, Sørlie M, Fukuda T, Kawamoto N, Taira T, Fukamizo T. 2011. Chitin oligosaccharide binding to a family GH19 chitinase from the moss, Bryum coronatum. FEBS J. 278:3991-4001]. Nevertheless, OsChia2a exhibited significant antifungal activity. It appears that loop III connected to the β-stranded region is important for (GlcNAc)n binding, but is not essential for antifungal activity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Wei; Leal, Walter S.

Pheromone-binding proteins (PBPs) are involved in the uptake of pheromones from pores on the antennae, transport through an aqueous environment surrounding the olfactory receptor neurons, and fast delivery to pheromone receptors. We tested the hypothesis that a C-terminal segment and a flexible loop are involved in the release of pheromones to membrane-bound receptors. We expressed in Escherichia coli 11 mutants of the PBP from the silkworm moth, BmorPBP, taking into consideration structural differences between the forms with high and low binding affinity. The N-terminus was truncated and His-69, His-70 and His-95 at the base of a flexible loop, and amore » cluster of acidic residues at the C-terminus were mutated. Binding assays and circular dichroism analyses support a mechanism involving protonation of acidic residues Asp-132 and Glu-141 at the C-terminus and histidines, His-70 and His-95, in the base of a loop covering the binding pocket. The former leads to the formation of a new {alpha}-helix, which competes with pheromone for the binding pocket, whereas positive charge repulsion of the histidines opens the opposite side of the binding pocket.« less

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

PubMed

Reilman, Ewoud; Mars, Ruben A T; van Dijl, Jan Maarten; Denham, Emma L

2014-10-01

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Superresolution imaging of transcription units on newt lampbrush chromosomes

PubMed Central

Kaufmann, Rainer; Cremer, Christoph; Gall, Joseph G.

2013-01-01

We have examined transcription loops on lampbrush chromosomes of the newt Notophthalmus by superresolution microscopy. Because of the favorable, essentially two-dimensional morphology of these loops, an average optical resolution in the x-y plane of about 50 nm was achieved. We analyzed the distribution of the multifunctional RNA-binding protein CELF1 on specific loops. CELF1 distribution is consistent with a model in which individual transcripts are tightly folded and hence closely packed against the loop axis. PMID:22892678

Multiprotein DNA Looping

NASA Astrophysics Data System (ADS)

Vilar, Jose M. G.; Saiz, Leonor

2006-06-01

DNA looping plays a fundamental role in a wide variety of biological processes, providing the backbone for long range interactions on DNA. Here we develop the first model for DNA looping by an arbitrarily large number of proteins and solve it analytically in the case of identical binding. We uncover a switchlike transition between looped and unlooped phases and identify the key parameters that control this transition. Our results establish the basis for the quantitative understanding of fundamental cellular processes like DNA recombination, gene silencing, and telomere maintenance.

Structures of Adnectin/Protein Complexes Reveal an Expanded Binding Footprint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramamurthy, Vidhyashankar; Krystek, Jr., Stanley R.; Bush, Alexander

2014-10-02

Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin ({sup 10}Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three {sup 10}Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibodymore » combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the {beta} strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these {beta} strand interactions, indicating that these nonloop residues can expand the available binding footprint.« less

Structures of FolT in substrate-bound and substrate-released conformations reveal a gating mechanism for ECF transporters

NASA Astrophysics Data System (ADS)

Zhao, Qin; Wang, Chengcheng; Wang, Chengyuan; Guo, Hui; Bao, Zhihao; Zhang, Minhua; Zhang, Peng

2015-07-01

Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling. Here, we report the 3.2-Å resolution crystal structure of the EcfS protein of a folate ECF transporter from Enterococcus faecalis-EfFolT, a close homologue of FolT from Lactobacillus brevis-LbFolT. Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity. Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

Dynamic Consequences of Mutation of Tryptophan 215 in Thrombin.

PubMed

Peacock, Riley B; Davis, Jessie R; Markwick, Phineus R L; Komives, Elizabeth A

2018-05-08

Thrombin normally cleaves fibrinogen to promote coagulation; however, binding of thrombomodulin to thrombin switches the specificity of thrombin toward protein C, triggering the anticoagulation pathway. The W215A thrombin mutant was reported to have decreased activity toward fibrinogen without significant loss of activity toward protein C. To understand how mutation of Trp215 may alter thrombin specificity, hydrogen-deuterium exchange experiments (HDXMS), accelerated molecular dynamics (AMD) simulations, and activity assays were carried out to compare the dynamics of Trp215 mutants with those of wild type (WT) thrombin. Variation in NaCl concentration had no detectable effect on the sodium-binding (220s CT ) loop, but appeared to affect other surface loops. Trp215 mutants showed significant increases in amide exchange in the 170s CT loop consistent with a loss of H-bonding in this loop identified by the AMD simulations. The W215A thrombin showed increased amide exchange in the 220s CT loop and in the N-terminus of the heavy chain. The AMD simulations showed that a transient conformation of the W215A thrombin has a distorted catalytic triad. HDXMS experiments revealed that mutation of Phe227, which engages in a π-stacking interaction with Trp215, also caused significantly increased amide exchange in the 170s CT loop. Activity assays showed that only the F227V mutant had wild type catalytic activity, whereas all other mutants showed markedly lower activity. Taken together, the results explain the reduced pro-coagulant activity of the W215A mutant and demonstrate the allosteric connection between Trp215, the sodium-binding loop, and the active site.

An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

NASA Astrophysics Data System (ADS)

Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D'Amelio, Nicola; Gervasio, Francesco Luigi

2016-04-01

Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

Structure-function analysis of the auxilin J-domain reveals an extended Hsc70 interaction interface.

PubMed

Jiang, Jianwen; Taylor, Alexander B; Prasad, Kondury; Ishikawa-Brush, Yumiko; Hart, P John; Lafer, Eileen M; Sousa, Rui

2003-05-20

J-domains are widespread protein interaction modules involved in recruiting and stimulating the activity of Hsp70 family chaperones. We have determined the crystal structure of the J-domain of auxilin, a protein which is involved in uncoating clathrin-coated vesicles. Comparison to the known structures of J-domains from four other proteins reveals that the auxilin J-domain is the most divergent of all J-domain structures described to date. In addition to the canonical J-domain features described previously, the auxilin J-domain contains an extra N-terminal helix and a long loop inserted between helices I and II. The latter loop extends the positively charged surface which forms the Hsc70 binding site, and is shown by directed mutagenesis and surface plasmon resonance to contain side chains important for binding to Hsc70.

Structure of an APC3–APC16 Complex: Insights into Assembly of the Anaphase-Promoting Complex/Cyclosome

DOE PAGES

Yamaguchi, Masaya; Yu, Shanshan; Qiao, Renping; ...

2014-12-06

The anaphase-promoting complex/cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the “Platform” centers around a cullin-RING-like E3 ligase catalytic core; the “Arc Lamp” is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-2-fold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3's TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, viamore » their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7 and contains a > 200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3's loop and IR tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer and, together with biochemistry and prior data, explain how APC16 recruits APC7 to APC3, show how APC3's C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J.

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2{sub 1} with four molecules per asymmetric unit. The 2.3 {angstrom} resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guaninemore » binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.« less

The loop structure and the RNA helicase p72/DDX17 influence the processing efficiency of the mice miR-132

PubMed Central

Remenyi, Judit; Bajan, Sarah; Fuller-Pace, Frances V.; Arthur, J. Simon C.; Hutvagner, Gyorgy

2016-01-01

miRNAs are small RNAs that are key regulators of gene expression in eukaryotic organisms. The processing of miRNAs is regulated by structural characteristics of the RNA and is also tightly controlled by auxiliary protein factors. Among them, RNA binding proteins play crucial roles to facilitate or inhibit miRNA maturation and can be controlled in a cell, tissue and species-specific manners or in response to environmental stimuli. In this study we dissect the molecular mechanism that promotes the overexpression of miR-132 in mice over its related, co-transcribed and co-regulated miRNA, miR-212. We have shown that the loop structure of miR-132 is a key determinant for its efficient processing in cells. We have also identified a range of RNA binding proteins that recognize the loop of miR-132 and influence both miR-132 and miR-212 processing. The DEAD box helicase p72/DDX17 was identified as a factor that facilitates the specific processing of miR-132. PMID:26947125

Optimization of a reusable, DNA pseudoknot-based electrochemical sensor for sequence-specific DNA detection in blood serum.

PubMed

Cash, Kevin J; Heeger, Alan J; Plaxco, Kevin W; Xiao, Yi

2009-01-15

We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem's loop forms part of the second stem, is modified with a methylene blue redox tag at its 3' terminus and covalently attached to a gold electrode via the 5' terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density ( approximately 1.8 x 10(13) molecules/cm(2) apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3' stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3' loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum.

Unique structural features of the AIPL1–FKBP domain that support prenyl lipid binding and underlie protein malfunction in blindness

PubMed Central

Yadav, Ravi P.; Gakhar, Lokesh; Yu, Liping

2017-01-01

FKBP-domain proteins (FKBPs) are pivotal modulators of cellular signaling, protein folding, and gene transcription. Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a distinctive member of the FKBP superfamily in terms of its biochemical properties, and it plays an important biological role as a chaperone of phosphodiesterase 6 (PDE6), an effector enzyme of the visual transduction cascade. Malfunction of mutant AIPL1 proteins triggers a severe form of Leber congenital amaurosis and leads to blindness. The mechanism underlying the chaperone activity of AIPL1 is largely unknown, but involves the binding of isoprenyl groups on PDE6 to the FKBP domain of AIPL1. We solved the crystal structures of the AIPL1–FKBP domain and its pathogenic mutant V71F, both in the apo form and in complex with isoprenyl moieties. These structures reveal a module for lipid binding that is unparalleled within the FKBP superfamily. The prenyl binding is enabled by a unique “loop-out” conformation of the β4-α1 loop and a conformational “flip-out” switch of the key W72 residue. A second major conformation of apo AIPL1–FKBP was identified by NMR studies. This conformation, wherein W72 flips into the ligand-binding pocket and renders the protein incapable of prenyl binding, is supported by molecular dynamics simulations and appears to underlie the pathogenicity of the V71F mutant. Our findings offer critical insights into the mechanisms that underlie AIPL1 function in health and disease, and highlight the structural and functional diversity of the FKBPs. PMID:28739921

The PUF binding landscape in metazoan germ cells

PubMed Central

Prasad, Aman; Porter, Douglas F.; Kroll-Conner, Peggy L.; Mohanty, Ipsita; Ryan, Anne R.; Crittenden, Sarah L.; Wickens, Marvin; Kimble, Judith

2016-01-01

PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, including FBF, is a global view of their binding sites in their native context (i.e., their “binding landscape”). To understand the interactions underlying FBF function, we used iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to determine binding landscapes of C. elegans FBF-1 and FBF-2 in the germline tissue of intact animals. Multiple iCLIP peak-calling methods were compared to maximize identification of both established FBF binding sites and positive control target mRNAs in our iCLIP data. We discovered that FBF-1 and FBF-2 bind to RNAs through canonical as well as alternate motifs. We also analyzed crosslinking-induced mutations to map binding sites precisely and to identify key nucleotides that may be critical for FBF–RNA interactions. FBF-1 and FBF-2 can bind sites in the 5′UTR, coding region, or 3′UTR, but have a strong bias for the 3′ end of transcripts. FBF-1 and FBF-2 have strongly overlapping target profiles, including mRNAs and noncoding RNAs. From a statistically robust list of 1404 common FBF targets, 847 were previously unknown, 154 were related to cell cycle regulation, three were lincRNAs, and 335 were shared with the human PUF protein PUM2. PMID:27165521

HCV RNA traffic and association with NS5A in living cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fiches, Guillaume N.; Eyre, Nicholas S.; Aloia, Amanda L.

The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3′-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA.more » Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly. - Highlights: • HCV can tolerate can bacteriophage MS2 stem-loop insertions within the 3′ UTR. • MS2 stem-loop containing HCV genomes allow for real-time imaging of HCV RNA. • HCV RNA is both static and motile and associates with NS5A and lipid droplets.« less

Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

PubMed

Grunseich, Christopher; Wang, Isabel X; Watts, Jason A; Burdick, Joshua T; Guber, Robert D; Zhu, Zhengwei; Bruzel, Alan; Lanman, Tyler; Chen, Kelian; Schindler, Alice B; Edwards, Nancy; Ray-Chaudhury, Abhik; Yao, Jianhua; Lehky, Tanya; Piszczek, Grzegorz; Crain, Barbara; Fischbeck, Kenneth H; Cheung, Vivian G

2018-02-01

R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanistic insights into phosphoprotein-binding FHA domains.

PubMed

Liang, Xiangyang; Van Doren, Steven R

2008-08-01

[Structure: see text]. FHA domains are protein modules that switch signals in diverse biological pathways by monitoring the phosphorylation of threonine residues of target proteins. As part of the effort to gain insight into cellular avoidance of cancer, FHA domains involved in the cellular response to DNA damage have been especially well-characterized. The complete protein where the FHA domain resides and the interaction partners determine the nature of the signaling. Thus, a key biochemical question is how do FHA domains pick out their partners from among thousands of alternatives in the cell? This Account discusses the structure, affinity, and specificity of FHA domains and the formation of their functional structure. Although FHA domains share sequence identity at only five loop residues, they all fold into a beta-sandwich of two beta-sheets. The conserved arginine and serine of the recognition loops recognize the phosphorylation of the threonine targeted. Side chains emanating from loops that join beta-strand 4 with 5, 6 with 7, or 10 with 11 make specific contacts with amino acids of the ligand that tailor sequence preferences. Many FHA domains choose a partner in extended conformation, somewhat according to the residue three after the phosphothreonine in sequence (pT + 3 position). One group of FHA domains chooses a short carboxylate-containing side chain at pT + 3. Another group chooses a long, branched aliphatic side chain. A third group prefers other hydrophobic or uncharged polar side chains at pT + 3. However, another FHA domain instead chooses on the basis of pT - 2, pT - 3, and pT + 1 positions. An FHA domain from a marker of human cancer instead chooses a much longer protein fragment that adds a beta-strand to its beta-sheet and that presents hydrophobic residues from a novel helix to the usual recognition surface. This novel recognition site and more remote sites for the binding of other types of protein partners were predicted for the entire family of FHA domains by a bioinformatics approach. The phosphopeptide-dependent dynamics of an FHA domain, SH2 domain, and PTB domain suggest a common theme: rigid, preformed binding surfaces support van der Waals contacts that provide favorable binding enthalpy. Despite the lack of pronounced conformational changes in FHA domains linked to binding events, more subtle adjustments may be possible. In the one FHA domain tested, phosphothreonine peptide binding is accompanied by increased flexibility just outside the binding site and increased rigidity across the beta-sandwich. The folding of the same FHA domain progresses through near-native intermediates that stabilize the recognition loops in the center of the phosphoprotein-binding surface; this may promote rigidity in the interface and affinity for targets phosphorylated on threonine.

DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1.

PubMed

Wong, Jing Ting; Akhbar, Farzanah; Ng, Amanda Yunn Ee; Tay, Mandy Li-Ian; Loi, Gladys Jing En; Pek, Jun Wei

2017-10-02

Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. Here we report that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, our study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila.Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. Here, the authors show that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis.

Surface-water Interface Induces Conformational Changes Critical for Protein Adsorption: Implications for Monolayer Formation of EAS Hydrophobin

PubMed Central

Ley, Kamron; Christofferson, Andrew; Penna, Matthew; Winkler, Dave; Maclaughlin, Shane; Yarovsky, Irene

2015-01-01

The class I hydrophobin EAS is part of a family of small, amphiphilic fungal proteins best known for their ability to self-assemble into stable monolayers that modify the hydrophobicity of a surface to facilitate further microbial growth. These proteins have attracted increasing attention for industrial and biomedical applications, with the aim of designing surfaces that have the potential to maintain their clean state by resisting non-specific protein binding. To gain a better understanding of this process, we have employed all-atom molecular dynamics to study initial stages of the spontaneous adsorption of monomeric EAS hydrophobin on fully hydroxylated silica, a commonly used industrial and biomedical substrate. Particular interest has been paid to the Cys3-Cys4 loop, which has been shown to exhibit disruptive behavior in solution, and the Cys7-Cys8 loop, which is believed to be involved in the aggregation of EAS hydrophobin at interfaces. Specific and water mediated interactions with the surface were also analyzed. We have identified two possible binding motifs, one which allows unfolding of the Cys7-Cys8 loop due to the surfactant-like behavior of the Cys3-Cys4 loop, and another which has limited unfolding due to the Cys3-Cys4 loop remaining disordered in solution. We have also identified intermittent interactions with water which mediate the protein adsorption to the surface, as well as longer lasting interactions which control the diffusion of water around the adsorption site. These results have shown that EAS behaves in a similar way at the air-water and surface-water interfaces, and have also highlighted the need for hydrophilic ligand functionalization of the silica surface in order to prevent the adsorption of EAS hydrophobin. PMID:26636091

Surface-water Interface Induces Conformational Changes Critical for Protein Adsorption: Implications for Monolayer Formation of EAS Hydrophobin.

PubMed

Ley, Kamron; Christofferson, Andrew; Penna, Matthew; Winkler, Dave; Maclaughlin, Shane; Yarovsky, Irene

2015-01-01

The class I hydrophobin EAS is part of a family of small, amphiphilic fungal proteins best known for their ability to self-assemble into stable monolayers that modify the hydrophobicity of a surface to facilitate further microbial growth. These proteins have attracted increasing attention for industrial and biomedical applications, with the aim of designing surfaces that have the potential to maintain their clean state by resisting non-specific protein binding. To gain a better understanding of this process, we have employed all-atom molecular dynamics to study initial stages of the spontaneous adsorption of monomeric EAS hydrophobin on fully hydroxylated silica, a commonly used industrial and biomedical substrate. Particular interest has been paid to the Cys3-Cys4 loop, which has been shown to exhibit disruptive behavior in solution, and the Cys7-Cys8 loop, which is believed to be involved in the aggregation of EAS hydrophobin at interfaces. Specific and water mediated interactions with the surface were also analyzed. We have identified two possible binding motifs, one which allows unfolding of the Cys7-Cys8 loop due to the surfactant-like behavior of the Cys3-Cys4 loop, and another which has limited unfolding due to the Cys3-Cys4 loop remaining disordered in solution. We have also identified intermittent interactions with water which mediate the protein adsorption to the surface, as well as longer lasting interactions which control the diffusion of water around the adsorption site. These results have shown that EAS behaves in a similar way at the air-water and surface-water interfaces, and have also highlighted the need for hydrophilic ligand functionalization of the silica surface in order to prevent the adsorption of EAS hydrophobin.

Endo-β-D-1,4-mannanase from Chrysonilia sitophila displays a novel loop arrangement for substrate selectivity.

PubMed

Gonçalves, Ana Maria D; Silva, Catarina S; Madeira, Tânia I; Coelho, Ricardo; de Sanctis, Daniele; San Romão, Maria Vitória; Bento, Isabel

2012-11-01

The crystal structure of wild-type endo-β-D-1,4-mannanase (EC 3.2.1.78) from the ascomycete Chrysonilia sitophila (CsMan5) has been solved at 1.40 Å resolution. The enzyme isolated directly from the source shows mixed activity as both an endo-glucanase and an endo-mannanase. CsMan5 adopts the (β/α)(8)-barrel fold that is well conserved within the GH5 family and has highest sequence and structural homology to the GH5 endo-mannanases. Superimposition with proteins of this family shows a unique structural arrangement of three surface loops of CsMan5 that stretch over the active centre, promoting an altered topography of the binding cleft. The most relevant feature results from the repositioning of a long loop at the extremity of the binding cleft, resulting in a shortened glycone-binding region with two subsites. The other two extended loops flanking the binding groove produce a narrower cleft compared with the wide architecture observed in GH5 homologues. Two aglycone subsites (+1 and +2) are identified and a nonconserved tryptophan (Trp271) at the +1 subsite may offer steric hindrance. Taken together, these findings suggest that the discrimination of mannan substrates is achieved through modified loop length and structure.

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

PubMed

Hayakawa-Yano, Yoshika; Suyama, Satoshi; Nogami, Masahiro; Yugami, Masato; Koya, Ikuko; Furukawa, Takako; Zhou, Li; Abe, Manabu; Sakimura, Kenji; Takebayashi, Hirohide; Nakanishi, Atsushi; Okano, Hideyuki; Yano, Masato

2017-09-15

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states. © 2017 Hayakawa-Yano et al.; Published by Cold Spring Harbor Laboratory Press.

Crystal Structure of MpPR-1i, a SCP/TAPS protein from Moniliophthora perniciosa, the fungus that causes Witches’ Broom Disease of Cacao

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baroni, Renata M.; Luo, Zhipu; Darwiche, Rabih

The pathogenic fungi Moniliophthora perniciosa causes Witches’ Broom Disease (WBD) of cacao. The structure of MpPR-1i, a protein expressed by M. perniciosa when it infects cacao, are presented. This is the first reported de novo structure determined by single-wavelength anomalous dispersion phasing upon soaking with selenourea. Each monomer has flexible loop regions linking the core alpha-beta-alpha sandwich topology that comprise ~50% of the structure, making it difficult to generate an accurate homology model of the protein. MpPR-1i is monomeric in solution but is packed as a high ~70% solvent content, crystallographic heptamer. The greatest conformational flexibility between monomers is foundmore » in loops exposed to the solvent channel that connect the two longest strands. MpPR-1i lacks the conserved CAP tetrad and is incapable of binding divalent cations. MpPR-1i has the ability to bind lipids, which may have roles in its infection of cacao. These lipids likely bind in the palmitate binding cavity as observed in tablysin-15, since MpPR-1i binds palmitate with comparable affinity as tablysin-15. Further studies are required to clarify the possible roles and underlying mechanisms of neutral lipid binding, as well as their effects on the pathogenesis of M. perniciosa so as to develop new interventions for WBD.« less

The helix-loop-helix protein id1 controls stem cell proliferation during regenerative neurogenesis in the adult zebrafish telencephalon.

PubMed

Rodriguez Viales, Rebecca; Diotel, Nicolas; Ferg, Marco; Armant, Olivier; Eich, Julia; Alunni, Alessandro; März, Martin; Bally-Cuif, Laure; Rastegar, Sepand; Strähle, Uwe

2015-03-01

The teleost brain has the remarkable ability to generate new neurons and to repair injuries during adult life stages. Maintaining life-long neurogenesis requires careful management of neural stem cell pools. In a genome-wide expression screen for transcription regulators, the id1 gene, encoding a negative regulator of E-proteins, was found to be upregulated in response to injury. id1 expression was mapped to quiescent type I neural stem cells in the adult telencephalic stem cell niche. Gain and loss of id1 function in vivo demonstrated that Id1 promotes stem cell quiescence. The increased id1 expression observed in neural stem cells in response to injury appeared independent of inflammatory signals, suggesting multiple antagonistic pathways in the regulation of reactive neurogenesis. Together, we propose that Id1 acts to maintain the neural stem cell pool by counteracting neurogenesis-promoting signals. © 2014 AlphaMed Press.

Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin

PubMed Central

Sommer, Martha E.; Hofmann, Klaus Peter; Heck, Martin

2012-01-01

G-protein-coupled receptors are universally regulated by arrestin binding. Here we show that rod arrestin induces uptake of the agonist all-trans-retinol in only half the population of phosphorylated opsin in the native membrane. Agonist uptake blocks subsequent entry of the inverse agonist 11-cis-retinal (that is, regeneration of rhodopsin), but regeneration is not blocked in the other half of aporeceptors. Environmentally sensitive fluorophores attached to arrestin reported that conformational changes in loopV−VI (N-domain) are coupled to the entry of agonist, while loopXVIII−XIX (C-domain) engages the aporeceptor even before agonist is added. The data are most consistent with a model in which each domain of arrestin engages its own aporeceptor, and the different binding preferences of the domains lead to asymmetric ligand binding by the aporeceptors. Such a mechanism would protect the rod cell in bright light by concurrently sequestering toxic all-trans-retinol and allowing regeneration with 11-cis-retinal. PMID:22871814

Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin.

PubMed

Sommer, Martha E; Hofmann, Klaus Peter; Heck, Martin

2012-01-01

G-protein-coupled receptors are universally regulated by arrestin binding. Here we show that rod arrestin induces uptake of the agonist all-trans-retinal [corrected] in only half the population of phosphorylated opsin in the native membrane. Agonist uptake blocks subsequent entry of the inverse agonist 11-cis-retinal (that is, regeneration of rhodopsin), but regeneration is not blocked in the other half of aporeceptors. Environmentally sensitive fluorophores attached to arrestin reported that conformational changes in loop(V-VI) (N-domain) are coupled to the entry of agonist, while loop(XVIII-XIX) (C-domain) engages the aporeceptor even before agonist is added. The data are most consistent with a model in which each domain of arrestin engages its own aporeceptor, and the different binding preferences of the domains lead to asymmetric ligand binding by the aporeceptors. Such a mechanism would protect the rod cell in bright light by concurrently sequestering toxic all-trans-retinal [corrected] and allowing regeneration with 11-cis-retinal.

Crystal packing modifies ligand binding affinity: the case of aldose reductase.

PubMed

Cousido-Siah, Alexandra; Petrova, Tatiana; Hazemann, Isabelle; Mitschler, André; Ruiz, Francesc X; Howard, Eduardo; Ginell, Stephan; Atmanene, Cédric; Van Dorsselaer, Alain; Sanglier-Cianférani, Sarah; Joachimiak, Andrzej; Podjarny, Alberto

2012-11-01

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used. Copyright © 2012 Wiley Periodicals, Inc.

Influence of GTP/GDP and magnesium ion on the solvated structure of the protein FtsZ: a molecular dynamics study.

PubMed

Jamous, Carla; Basdevant, Nathalie; Ha-Duong, Tap

2014-01-01

We present here a structural analysis of ten extensive all-atom molecular dynamics simulations of the monomeric protein FtsZ in various binding states. Since the polymerization and GTPase activities of FtsZ depend on the nature of a bound nucleotide as well as on the presence of a magnesium ion, we studied the structural differences between the average conformations of the following five systems: FtsZ-Apo, FtsZ-GTP, FtsZ-GDP, FtsZ-GTP-Mg, and FtsZ-GDP-Mg. The in silico solvated average structure of FtsZ-Apo significantly differs from the crystallographic structure 1W59 of FtsZ which was crystallized in a dimeric form without nucleotide and magnesium. The simulated Apo form of the protein also clearly differs from the FtsZ structures when it is bound to its ligand, the most important discrepancies being located in the loops surrounding the nucleotide binding pocket. The three average structures of FtsZ-GTP, FtsZ-GDP, and FtsZ-GTP-Mg are overall similar, except for the loop T7 located at the opposite side of the binding pocket and whose conformation in FtsZ-GDP notably differs from the one in FtsZ-GTP and FtsZ-GTP-Mg. The presence of a magnesium ion in the binding pocket has no impact on the FtsZ conformation when it is bound to GTP. In contrast, when the protein is bound to GDP, the divalent cation causes a translation of the nucleotide outwards the pocket, inducing a significant conformational change of the loop H6-H7 and the top of helix H7.

Unbiased, scalable sampling of protein loop conformations from probabilistic priors.

PubMed

Zhang, Yajia; Hauser, Kris

2013-01-01

Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion.

Unbiased, scalable sampling of protein loop conformations from probabilistic priors

PubMed Central

2013-01-01

Background Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Results Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Conclusion Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion. PMID:24565175

Crystallographic and Molecular Dynamics Analysis of Loop Motions Unmasking the Peptidoglycan-Binding Site in Stator Protein MotB of Flagellar Motor

PubMed Central

Nahar, Musammat F.; Buckle, Ashley M.; Roujeinikova, Anna

2011-01-01

Background The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues. Methodology/Principal Findings We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the β-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan. Conclusion/Significance Our structural analysis provides a new insight into the mechanism by which MotB inserts into the peptidoglycan mesh, thus anchoring the power-generating complex to the cell wall. PMID:21533052

Role of four conserved aspartic acid residues of EF-loops in the metal ion binding and in the self-assembly of ciliate Euplotes octocarinatus centrin.

PubMed

Liu, Wen; Duan, Lian; Sun, Tijian; Yang, Binsheng

2016-12-01

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. Four mutants (D37K, D73K, D110K and D146K) were created firstly to elucidate the importance of the first aspartic acid residues (Asp37, Asp73, Asp110 and Asp146) in the beginning of the four EF-loops of EoCen. Aromatic-sensitized Tb 3+ fluorescence indicates that the aspartic acid residues are very important for the metal-binding of EoCen, except for Asp73 (in EF-loop II). Resonance light scattering (RLS) measurements for different metal ions (Ca 2+ and Tb 3+ ) binding proteins suggest that the order of four conserved aspartic acid residues for contributing to the self-assembly of EoCen is Asp37 > Asp146 > Asp110 > Asp73. Cross-linking experiment also exhibits that Asp37 and Asp146 play critical role in the self-assembly of EoCen. Asp37, in site I, which is located in the N-terminal domain, plays the most important role in the metal ion-dependent self-assembly of EoCen, and there is cooperativity between N-terminal and C-terminal domain (especially the site IV). In addition, the dependence of Tb 3+ induced self-assembly of EoCen and the mutants on various factors, including ionic strength and pH, were characterized using RLS. Finally, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) binding, ionic strength and pH control experiments indicate that in the process of EoCen self-assembly, molecular interactions are mediated by both electrostatic and hydrophobic forces, and the hydrophobic interaction has the important status.

MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA.

PubMed

Lai, Ruenn Chai; Tan, Soon Sim; Yeo, Ronne Wee Yeh; Choo, Andre Boon Hwa; Reiner, Agnes T; Su, Yan; Shen, Yang; Fu, Zhiyan; Alexander, Lezhava; Sze, Siu Kwan; Lim, Sai Kiang

2016-01-01

Mesenchymal stem cell (MSC), a widely used adult stem cell candidate for regenerative medicine, has been shown to exert some of its therapeutic effects through the secretion of extracellular vesicles (EVs). These homogenously sized EVs of 100-150 ηm exhibited many exosome-like biophysical and biochemical properties and carry both proteins and RNAs. Recently, exosome-associated proteins in this MSC EV preparation were found to segregate primarily to those EVs that bind cholera toxin B chain (CTB), a GM1 ganglioside-specific ligand, and pulse-chase experiments demonstrated that these EVs have endosomal origin and carried many of the exosome-associated markers. Here, we report that only a fraction of the MSC EV proteome was found in CTB-bound EVs. Using Annexin V (AV) and Shiga toxin B subunit (ST) with affinities for phosphatidylserine and globotriaosylceramide, respectively, AV- and a ST-binding EV were identified. CTB-, AV- and ST-binding EVs all carried actin. However, the AV-binding EVs carried low or undetectable levels of the exosome-associated proteins. Only the ST-binding EVs carried RNA and EDA-containing fibronectin. Proteins in AV-binding EVs were also different from those released by apoptotic MSCs. CTB- and AV-binding activities were localized to the plasma membrane and cytoplasm of MSCs, while ST-binding activity was localized to the nucleus. Together, this study demonstrates that cells secrete many types of EVs. Specifically, MSCs secrete at least 3 types. They can be differentially isolated based on their affinities for membrane lipid-binding ligands. As the subcellular sites of the binding activities of these ligands and cargo load are different for each EV type, they are likely to have a different biogenesis pathway and possibly different functions.

NMR structure and dynamics of the engineered fluorescein-binding lipocalin FluA reveal rigidification of beta-barrel and variable loops upon enthalpy-driven ligand binding.

PubMed

Mills, Jeffrey L; Liu, Gaohua; Skerra, Arne; Szyperski, Thomas

2009-08-11

The NMR structure of the 21 kDa lipocalin FluA, which was previously obtained by combinatorial design, elucidates a reshaped binding site specific for the dye fluorescein resulting from 21 side chain replacements with respect to the parental lipocalin, the naturally occurring bilin-binding protein (BBP). As expected, FluA exhibits the lipocalin fold of BBP, comprising eight antiparallel beta-strands forming a beta-barrel with an alpha-helix attached to its side. Comparison of the NMR structure of free FluA with the X-ray structures of BBP.biliverdin IX(gamma) and FluA.fluorescein complexes revealed significant conformational changes in the binding pocket, which is formed by four loops at the open end of the beta-barrel as well as adjoining beta-strand segments. An "induced fit" became apparent for the side chain conformations of Arg 88 and Phe 99, which contact the bound fluorescein in the complex and undergo concerted rearrangement upon ligand binding. Moreover, slower internal motional modes of the polypeptide backbone were identified by measuring transverse (15)N backbone spin relaxation times in the rotating frame for free FluA and also for the FluA.fluorescein complex. A reduction in the level of such motions was detected upon complex formation, indicating rigidification of the protein structure and loss of conformational entropy. This hypothesis was confirmed by isothermal titration calorimetry, showing that ligand binding is enthalpy-driven, thus overcompensating for the negative entropy associated with both ligand binding per se and rigidification of the protein. Our investigation of the solution structure and dynamics as well as thermodynamics of lipocalin-ligand interaction not only provides insight into the general mechanism of small molecule accommodation in the deep and narrow cavity of this abundant class of proteins but also supports the future design of corresponding binding proteins with novel specificities, so-called "anticalins".

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

Pathophysiology and Toxicokinetic Studies of Blue-Green Algae Intoxication in the Swine Model

DTIC Science & Technology

1987-08-31

bovine aer, albamin caused similar reductions !u toxicity, suggesting nonspecific protein binding. Saturation at the N-methyldehydroalanine of MCYST-A...need to conserve toxin and the goal of creating a model for naturally occurring toxicosis caused us to evaluate the oral administration of microcystin...the isolated ileal loop preparation. Pancreatic enzymes and protein-binding were examined as 2 possible causes for the low oral toxicity of MCYST-A

Detection of Naja atra Cardiotoxin Using Adenosine-Based Molecular Beacon.

PubMed

Shi, Yi-Jun; Chen, Ying-Jung; Hu, Wan-Ping; Chang, Long-Sen

2017-01-07

This study presents an adenosine (A)-based molecular beacon (MB) for selective detection of Naja atra cardiotoxin (CTX) that functions by utilizing the competitive binding between CTX and the poly(A) stem of MB to coralyne. The 5'- and 3'-end of MB were labeled with a reporter fluorophore and a non-fluorescent quencher, respectively. Coralyne induced formation of the stem-loop MB structure through A₂-coralyne-A₂ coordination, causing fluorescence signal turn-off due to fluorescence resonance energy transfer between the fluorophore and quencher. CTX3 could bind to coralyne. Moreover, CTX3 alone induced the folding of MB structure and quenching of MB fluorescence. Unlike that of snake venom α-neurotoxins, the fluorescence signal of coralyne-MB complexes produced a bell-shaped concentration-dependent curve in the presence of CTX3 and CTX isotoxins; a turn-on fluorescence signal was noted when CTX concentration was ≤80 nM, while a turn-off fluorescence signal was noted with a further increase in toxin concentrations. The fluorescence signal of coralyne-MB complexes yielded a bell-shaped curve in response to varying concentrations of N. atra crude venom but not those of Bungarus multicinctus and Protobothrops mucrosquamatus venoms. Moreover, N. nigricollis venom also functioned as N. atra venom to yield a bell-shaped concentration-dependent curve of MB fluorescence signal, again supporting that the hairpin-shaped MB could detect crude venoms containing CTXs. Taken together, our data validate that a platform composed of coralyne-induced stem-loop MB structure selectively detects CTXs.

Structural basis for ligand regulation of the fatty acid-binding protein 5, peroxisome proliferator-activated receptor β/δ (FABP5-PPARβ/δ) signaling pathway.

PubMed

Armstrong, Eric H; Goswami, Devrishi; Griffin, Patrick R; Noy, Noa; Ortlund, Eric A

2014-05-23

Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain "activating" fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARβ/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing β2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock.

PubMed

Pruneda-Paz, Jose L; Breton, Ghislain; Para, Alessia; Kay, Steve A

2009-03-13

Transcriptional feedback loops constitute the molecular circuitry of the plant circadian clock. In Arabidopsis, a core loop is established between CCA1 and TOC1. Although CCA1 directly represses TOC1, the TOC1 protein has no DNA binding domains, which suggests that it cannot directly regulate CCA1. We established a functional genomic strategy that led to the identification of CHE, a TCP transcription factor that binds specifically to the CCA1 promoter. CHE is a clock component partially redundant with LHY in the repression of CCA1. The expression of CHE is regulated by CCA1, thus adding a CCA1/CHE feedback loop to the Arabidopsis circadian network. Because CHE and TOC1 interact, and CHE binds to the CCA1 promoter, a molecular linkage between TOC1 and CCA1 gene regulation is established.

Mapping interactions between the RNA chaperone FinO and its RNA targets

PubMed Central

Arthur, David C.; Tsutakawa, Susan; Tainer, John A.; Frost, Laura S.; Glover, J. N. Mark

2011-01-01

Bacterial conjugation is regulated by two-component repression comprising the antisense RNA FinP, and its protein co-factor FinO. FinO mediates base-pairing of FinP to the 5′-untranslated region (UTR) of traJ mRNA, which leads to translational inhibition of the transcriptional activator TraJ and subsequent down regulation of conjugation genes. Yet, little is known about how FinO binds to its RNA targets or how this interaction facilitates FinP and traJ mRNA pairing. Here, we use solution methods to determine how FinO binds specifically to its minimal high affinity target, FinP stem–loop II (SLII), and its complement SLIIc from traJ mRNA. Ribonuclease footprinting reveals that FinO contacts the base of the stem and the 3′ single-stranded tails of these RNAs. The phosphorylation or oxidation of the 3′-nucleotide blocks FinO binding, suggesting FinO binds the 3′-hydroxyl of its RNA targets. The collective results allow the generation of an energy-minimized model of the FinO–SLII complex, consistent with small-angle X-ray scattering data. The repression complex model was constrained using previously reported cross-linking data and newly developed footprinting results. Together, these data lead us to propose a model of how FinO mediates FinP/traJ mRNA pairing to down regulate bacterial conjugation. PMID:21278162

Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries.

PubMed

Nuttall, S D; Krishnan, U V; Hattarki, M; De Gori, R; Irving, R A; Hudson, P J

2001-08-01

The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein. To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages. On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis. One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E. coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein. Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues. Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries.

Sequences Flanking the Gephyrin-Binding Site of GlyRβ Tune Receptor Stabilization at Synapses

PubMed Central

Grünewald, Nora; Salvatico, Charlotte; Kress, Vanessa

2018-01-01

Abstract The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 310-helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells. PMID:29464196

The role of modifications in codon discrimination by tRNA(Lys)UUU.

PubMed

Murphy, Frank V; Ramakrishnan, Venki; Malkiewicz, Andrzej; Agris, Paul F

2004-12-01

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.

Cross-linking of cell surface amyloid precursor protein leads to increased β-amyloid peptide production in hippocampal neurons: implications for Alzheimer's disease.

PubMed

Lefort, Roger; Pozueta, Julio; Shelanski, Michael

2012-08-01

The accumulation of the β-amyloid peptide (Aβ) in Alzheimer's disease (AD) is thought to play a causative role in triggering synaptic dysfunction in neurons, leading to their eventual demise through apoptosis. Aβ is produced and secreted upon sequential cleavage of the amyloid precursor protein (APP) by β-secretases and γ-secretases. However, while Aβ levels have been shown to be increased in the brains of AD patients, little is known about how the cleavage of APP and the subsequent generation of Aβ is influenced, or whether the cleavage process changes over time. It has been proposed that Aβ can bind APP and promote amyloidogenic processing of APP, further enhancing Aβ production. Proof of this idea has remained elusive because a clear mechanism has not been identified, and the promiscuous nature of Aβ binding complicates the task of demonstrating the idea. To work around these problems, we used an antibody-mediated approach to bind and cross-link cell-surface APP in cultured rat primary hippocampal neurons. Here we show that cross-linking of APP is sufficient to raise the levels of Aβ in viable neurons with a concomitant increase in the levels of the β-secretase BACE1. This appears to occur as a result of a sorting defect that stems from the caspase-3-mediated inactivation of a key sorting adaptor protein, namely GGA3, which prevents the lysosomal degradation of BACE1. Together, our data suggest the occurrence of a positive pathogenic feedback loop involving Aβ and APP in affected neurons possibly allowing Aβ to spread to nearby healthy neurons.

The Ndc80 internal loop is required for recruitment of the Ska complex to establish end-on microtubule attachment to kinetochores.

PubMed

Zhang, Gang; Kelstrup, Christian D; Hu, Xiao-Wen; Kaas Hansen, Mathilde J; Singleton, Martin R; Olsen, Jesper V; Nilsson, Jakob

2012-07-01

The Ndc80 complex establishes end-on attachment of kinetochores to microtubules, which is essential for chromosome segregation. The Ndc80 subunit is characterized by an N-terminal region that binds directly to microtubules, and a long coiled-coil region that interacts with Nuf2. A loop region in Ndc80 that generates a kink in the structure disrupts the long coiled-coil region but the exact function of this loop, has until now, not been clear. Here we show that this loop region is essential for end-on attachment of kinetochores to microtubules in human cells. Cells expressing loop mutants of Ndc80 are unable to align the chromosomes, and stable kinetochore fibers are absent. Through quantitative mass spectrometry and immunofluorescence we found that the binding of the spindle and kinetochore associated (Ska) complex depends on the loop region, explaining why end-on attachment is defective. This underscores the importance of the Ndc80 loop region in coordinating chromosome segregation through the recruitment of specific proteins to the kinetochore.

Cooperative Allosteric Ligand Binding in Calmodulin

NASA Astrophysics Data System (ADS)

Nandigrami, Prithviraj

Conformational dynamics is often essential for a protein's function. For example, proteins are able to communicate the effect of binding at one site to a distal region of the molecule through changes in its conformational dynamics. This so called allosteric coupling fine tunes the sensitivity of ligand binding to changes in concentration. A conformational change between a "closed" (apo) and an "open" (holo) conformation upon ligation often produces this coupling between binding sites. Enhanced sensitivity between the unbound and bound ensembles leads to a sharper binding curve. There are two basic conceptual frameworks that guide our visualization about ligand binding mechanisms. First, a ligand can stabilize the unstable "open" state from a dynamic ensemble of conformations within the unbound basin. This binding mechanism is called conformational selection. Second, a ligand can weakly bind to the low-affinity "closed" state followed by a conformational transition to the "open" state. In this dissertation, I focus on molecular dynamics simulations to understand microscopic origins of ligand binding cooperativity. A minimal model of allosteric binding transitions must include ligand binding/unbinding events, while capturing the transition mechanism between two distinct meta-stable free energy basins. Due in part to computational timescales limitations, work in this dissertation describes large-scale conformational transitions through a simplified, coarse-grained model based on the energy basins defined by the open and closed conformations of the protein Calmodulin (CaM). CaM is a ubiquitous calcium-binding protein consisting of two structurally similar globular domains connected by a flexible linker. The two domains of CaM, N-terminal domain (nCaM) and C-terminal domain (cCaM) consists of two helix-loop-helix motifs (the EF-hands) connected by a flexible linker. Each domain of CaM consists of two binding loops and binds 2 calcium ions each. The intact domain binds up to 4 calcium ions. The simulations use a coupled molecular dynamics/monte carlo scheme where the protein dynamics is simulated explicitly, while ligand binding/unbinding are treated implicitly. In the model, ligand binding/unbinding events coupled with a conformational change of the protein within the grand canonical ensemble. Here, ligand concentration is controlled through the chemical potential (micro). This allows us to use a simple thermodynamic model to analyze the simulated data and quantify binding cooperativity. Simulated binding titration curves are calculated through equilibrium simulations at different values of micro. First, I study domain opening transitions of isolated nCaM and cCaM in the absence of calcium. This work is motivated by results from a recent analytic variational model that predicts distinct domain opening transition mechanism for the domains of CaM. This is a surprising result because the domains have the same folded state topology. In the simulations, I find the two domains of CaM have distinct transition mechanism over a broad range of temperature, in harmony with the analytic predictions. In particular, the simulated transition mechanism of nCaM follows a two-state behavior, while domain opening in cCaM involves global unfolding and refolding of the tertiary structure. The unfolded intermediate also appears in the landscape of nCaM, but at a higher temperature than it appears in cCaM's energy landscape. This is consistent with nCaM's higher thermal stability. Under approximate physiological conditions, majority of the sampled transitions in cCaM involves unfolding and refolding during conformational change. Kinetically, the transient unfolding and refolding in cCaM significantly slows the domain opening and closing rates in cCaM. Second, I investigate the structural origins of binding affinity and allosteric cooperativity of binding 2 calcium-ions to each domain of CaM. In my work, I predict the order of binding strength of CaM's loops. I analyze simulated binding curves within the framework of the classic Monod-Wyman-Changeux (MWC) model of allostery to extract the binding free energies to the closed and open ensembles. The simulations predict that cCaM binds calcium with higher affinity and greater cooperativity than nCaM. Where it is possible to compare, these predictions are in good agreement with experimental results. The analysis of the simulations offers a rationale for why the two domains differ in cooperativity: the higher cooperativity of cCaM is due to larger difference in affinity of its binding loops. Third, I extend the work to investigate structural origins of binding cooperativity of 4 calcium-ions to intact CaM. I characterize the microscopic cooperativities of each ligation state and provide a kinetic description of the binding mechanism. Due to the heterogeneous nature of CaM's loops, as predicted in our simulations of isolated domains, I focus on investigating the influence of this heterogeneity on the kinetic flux of binding pathways as a function of concentration. The formalism developed for Network Models of protein folding kinetics, is used to evaluate the directed flux of all possible pathways between unligated and fully loaded CaM. (Abstract shortened by ProQuest.).

Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

NASA Astrophysics Data System (ADS)

Rustgi, Anil K.; Dyson, Nicholas; Bernards, Rene

1991-08-01

THE proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms1. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization2-5. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation6,7. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.

Lanthanide binding and IgG affinity construct: Potential applications in solution NMR, MRI, and luminescence microscopy

PubMed Central

Barb, Adam W; Ho, Tienhuei Grace; Flanagan-Steet, Heather; Prestegard, James H

2012-01-01

Paramagnetic lanthanide ions when bound to proteins offer great potential for structural investigations that utilize solution nuclear magnetic resonance spectroscopy, magnetic resonance imaging, or optical microscopy. However, many proteins do not have native metal ion binding sites and engineering a chimeric protein to bind an ion while retaining affinity for a protein of interest represents a significant challenge. Here we report the characterization of an immunoglobulin G-binding protein redesigned to include a lanthanide binding motif in place of a loop between two helices (Z-L2LBT). It was shown to bind Tb3+ with 130 nM affinity. Ions such as Dy3+, Yb3+, and Ce3+ produce paramagnetic effects on NMR spectra and the utility of these effects is illustrated by their use in determining a structural model of the metal-complexed Z-L2LBT protein and a preliminary characterization of the dynamic distribution of IgG Fc glycan positions. Furthermore, this designed protein is demonstrated to be a novel IgG-binding reagent for magnetic resonance imaging (Z-L2LBT:Gd3+ complex) and luminescence microscopy (Z-L2LBT: Tb3+ complex). PMID:22851279

Visualizing Active-Site Dynamics in Single Crystals of HePTP: Opening of the WPD Loop Involves Coordinated Movement of the E Loop

DOE Office of Scientific and Technical Information (OSTI.GOV)

D Critton; L Tautz; R Page

2011-12-31

Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding and catalysis is comparatively unexplored. Hematopoietic PTP (HePTP) is a member of the kinase interaction motif (KIM) PTP family. Compared to other PTPs, KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loopmore » in the transition between the closed state and the open state of HePTP, we identified a novel crystal form of HePTP that allowed the closed-state-to-open-state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an 'atypically open' conformation and, importantly, that ligands can be exchanged at the active site, which is critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel secondary site and function to coordinate the PTP, WPD, and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E-loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for the coordinated dynamics of the WPD and E loops in the catalytic cycle, which, as we show, is relevant to multiple PTP families.« less

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy.

PubMed

Leder, Verena; Lummer, Martina; Tegeler, Kathrin; Humpert, Fabian; Lewinski, Martin; Schüttpelz, Mark; Staiger, Dorothee

2014-10-10

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased Kd value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R(49) that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding. Copyright © 2014 Elsevier Inc. All rights reserved.

Attenuation of cancer-initiating cells stemness properties by abrogating S100A4 calcium binding ability in head and neck cancers.

PubMed

Cheng, Li-Hao; Hung, Kai-Feng; Huang, Tung-Fu; Hsieh, Hsin-Pei; Wang, Shu-Ying; Huang, Chih-Yang; Lo, Jeng-Fan

2016-11-29

S100A4 is a calcium-binding protein capable of promoting epithelial-mesenchymal transition. Previously, we have demonstrated that S100A4 is required to sustain the head and neck cancer-initiating cells (HN-CICs) subpopulation. In this study, to further investigate the molecular mechanism, we established the head and neck squamous cell carcinoma (HNSCC) cell lines stably expressing mutant S100A4 proteins with defective calcium-binding sites on either N-terminal (NM) or C-terminal (CM), or a deletion of the last 15 amino-acid residues (CD). We showed that the NM, CM and CD harboring sphere cells that were enriched with HN-CICs population exhibited impaired stemness and malignant properties in vitro, as well as reduced tumor growth ability in vivo. Mechanistically, we demonstrated that mutant S100A4 proteins decreased the promoter activity of Nanog, likely through inhibition of p53. Moreover, the biophysical analyses of purified recombinant mutant S100A4 proteins suggest that both NM and CM mutant S100A4 were very similar to the WT S100A4 with subtle difference on the secondary structure, and that the CD mutant protein displayed the unexpected monomeric form in the solution phase.Taken together, our results suggest that both the calcium-binding ability and the C-terminal region of S100A4 are important for HN-CICs to sustain its stemness property and malignancy, and that the mechanism could be mediated by repressing p53 and subsequently activating the Nanog expression.

The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element.

PubMed

Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

2013-07-01

AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5'-NNCCAC-3' and 5'-GCGMGN'N'-3' (M:A or C; N and N' form Watson-Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences.

The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

PubMed Central

Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

2013-01-01

AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences. PMID:23709277

Musashi RNA-Binding Proteins as Cancer Drivers and Novel Therapeutic Targets.

PubMed

Kudinov, Alexander E; Karanicolas, John; Golemis, Erica A; Boumber, Yanis

2017-05-01

Aberrant gene expression that drives human cancer can arise from epigenetic dysregulation. Although much attention has focused on altered activity of transcription factors and chromatin-modulating proteins, proteins that act posttranscriptionally can potently affect expression of oncogenic signaling proteins. The RNA-binding proteins (RBP) Musashi-1 (MSI1) and Musashi-2 (MSI2) are emerging as regulators of multiple critical biological processes relevant to cancer initiation, progression, and drug resistance. Following identification of Musashi as a regulator of progenitor cell identity in Drosophila , the human Musashi proteins were initially linked to control of maintenance of hematopoietic stem cells, then stem cell compartments for additional cell types. More recently, the Musashi proteins were found to be overexpressed and prognostic of outcome in numerous cancer types, including colorectal, lung, and pancreatic cancers; glioblastoma; and several leukemias. MSI1 and MSI2 bind and regulate the mRNA stability and translation of proteins operating in essential oncogenic signaling pathways, including NUMB/Notch, PTEN/mTOR, TGFβ/SMAD3, MYC, cMET, and others. On the basis of these activities, MSI proteins maintain cancer stem cell populations and regulate cancer invasion, metastasis, and development of more aggressive cancer phenotypes, including drug resistance. Although RBPs are viewed as difficult therapeutic targets, initial efforts to develop MSI-specific inhibitors are promising, and RNA interference-based approaches to inhibiting these proteins have had promising outcomes in preclinical studies. In the interim, understanding the function of these translational regulators may yield insight into the relationship between mRNA expression and protein expression in tumors, guiding tumor-profiling analysis. This review provides a current overview of Musashi as a cancer driver and novel therapeutic target. Clin Cancer Res; 23(9); 2143-53. ©2017 AACR . ©2017 American Association for Cancer Research.

The yeast telomerase recruitment module requires a specific RNA architecture.

PubMed

Laterreur, Nancy; Lemieux, Bruno; Neumann, Hannah; Berger-Dancause, Jean-Christophe; Lafontaine, Daniel; Wellinger, Raymund J

2018-05-18

Telomerases are ribonucleoprotein (RNP) enzymes that are related to reverse transcriptases. While they maintain genome stability, their composition varies significantly between species. Yeast telomerase RNPs contain an RNA that is comparatively large and its overall folding shows long helical segments with distal functional parts. Here we investigated the essential stem IVc module of the budding yeast telomerase RNA, called Tlc1. The distal part of stem IVc includes a conserved sequence element CS2a and structurally conserved features to which bind the Pop1/Pop6/Pop7 proteins and which together function analogously to the P3 domains of the RNase P/MRP RNPs. A more proximal bulged stem with the CS2 element is thought to associate with Est1. Previous data showed that changes in CS2a cause a loss of all of the proteins, not just the Pop-proteins, from stem IVc. The results here show that the association of Est1 with stem IVc indeed requires both the proximal bulged stem and the presence of the Tlc1 P3 domain with the associated Pop-proteins. Separating the P3-domain from the Est1 binding site by inserting only 2 base pairs into the helical stem between the two sites causes a complete loss of Est1 from the RNP and hence a telomerase-negative phenotype in vivo. Still, the distal P3 domain with the associated Pop-proteins remains intact. Moreover, the P3 domain also ensures Est2 stability on the RNP independently of the Est1 association. Therefore, the recruitment module of the Tlc1 RNA requires a very tight architectural organization for telomerase function in vivo. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET.

PubMed

Clerte, Caroline; Declerck, Nathalie; Margeat, Emmanuel

2013-02-01

The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5'-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic.

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis

PubMed Central

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K.

2012-01-01

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3′ untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior. PMID:22645327

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.

PubMed

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K

2012-06-12

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

Conformational motions regulate phosphoryl transfer in related protein tyrosine phosphatases

PubMed Central

Whittier, Sean K.; Hengge, Alvan C.; Loria, J. Patrick

2014-01-01

Many studies have implicated a role for conformational motions during the catalytic cycle, acting to optimize the binding pocket or facilitate product release, but a more intimate role in the chemical reaction has not been described. We address this by monitoring active-site loop motion in two protein tyrosine phosphatases (PTPs) using NMR spectroscopy. The PTPs, YopH and PTP1B, have very different catalytic rates, however we find in both that the active-site loop closes to its catalytically competent position at rates that mirror the phosphotyrosine cleavage kinetics. This loop contains the catalytic acid, suggesting that loop closure occurs concomitantly with the protonation of the leaving group tyrosine and explains the different kinetics of two otherwise chemically and mechanistically indistinguishable enzymes. PMID:23970698

Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247.

PubMed

Kirby, Karen A; Ong, Yee Tsuey; Hachiya, Atsuko; Laughlin, Thomas G; Chiang, Leslie A; Pan, Yun; Moran, Jennifer L; Marchand, Bruno; Singh, Kamalendra; Gallazzi, Fabio; Quinn, Thomas P; Yoshimura, Kazuhisa; Murakami, Toshio; Matsushita, Shuzo; Sarafianos, Stefan G

2015-01-01

Humanized monoclonal antibody KD-247 targets the Gly(312)-Pro(313)-Gly(314)-Arg(315) arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg(315) of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg(315) of the V3 loop is based on a network of interactions that involve Tyr(L32), Tyr(L92), and Asn(L27d) that directly interact with Arg(315), thus elucidating the molecular interactions of KD-247 with its V3 loop target. © FASEB.

Mechanisms underlying radiosensitivity : iIvestigations in xrs-5, an X-ray sensitive hamster cell line

NASA Astrophysics Data System (ADS)

Johnston, Peter James

The damage caused to cells by ionising radiation is believed to center on damage to the DNA. In particular, the induction of DNA double strand breaks (DSB) have been implicated in biological end-points such as cell killing and the formation of chromosomal aberrations. The xrs-5 cell line is a mutant Chinese hamster ovary fibroblast (CHO-K1) mutant which exhibits sensitivity to ionising radiation and a number of other DNA damaging agents. This mutation, postulated to involve the hamster homologue of the human XRCC5 gene, is believed to be involved in the repair of the DSB. In addition, there are constitutive differences between the wild type and xrs cells involving the structure and function of the nucleus and higher order chromatin structures. The aims of this thesis were to study further the xrs-5 cell line and its response to DNA damage and to investigate the possible link between chromatin structure and DSB repair. By the examination of the response of xrs-5 cells to a number of DNA damaging agents and potential modulators of this response using the cytokinesis block micronucleus assay [Fenech and Morley, 1985] a possible cell cycle defect was identified in addition to elevated levels of chromosomal damage. Xrs-5 cells appeared to be partially defective in the cell cycle checkpoints involving the passage from G2 phase to mitosis. By the use of a modified neutral filter elution procedure variations in the repair of DSB were observed between xrs-5 and CHO. Conventional neutral filter elution requires harsh lysis conditions to remove higher order chromatin structures which interfere with the elution of DNA containing DSB. By lysing cells with non-ionic detergent in the presence of 2 M NaC1, histone depleted structures which retain the higher order nuclear matrix organisation, including chromatin loops, can be produced. Elution from these structures will only occur if two or more DSB lie within a single looped domain delineated by points of attachment to the nuclear matrix. Repair experiments indicate that in CHO cells repair of DSB in loops containing multiple DSB are repaired with "slow" kinetics (t1/2 = 5 hrs) whilst DSB occurring in loops containing single DSB are repaired with "fast" kinetics (t1/2 " 10 min). Xrs- 5 cells are incapable of repairing these multiply damaged loops. This work indicates that the spatial orientation of DSB in higher order structures of chromatin are a possible factor in the repair of these lesions. By construction of a mathematical model of the process of elution from chromatin loops it was possible to postulate the size of the loops to approximate to 2.5-3 Mbp. Further evidence of a potential structural defect in the chromatin of xrs-5 cells was provided by examination of the polypeptide composition and DNA binding activity of nuclear extracts. The affinity of extracted proteins for double-stranded calf-thymus DNA was measured in nuclear extracts of xrs-5 and CHO cells. There was an alteration in the DNA binding activity of salt extractable proteins from xrs-5 as measured by a filter binding assay. By the use of SDS-PAGE and the technique of South-Western blotting, it was possible to identify the approximate molecular weights of these DNA binding proteins. Differences were found in DNA binding between proteins from CHO and xrs-5 extracts of both non-irradiated and irradiated cells. Two proteins with apparent molecular weights of 32.2 and 31.8 kDa exhibited a lower DNA binding activity in xrs-5 than proteins of similar extracts from CHO. The amount of the 32.2 kDa protein was less in the xrs-5 extracts than in CHO extracts, as measured by Coomassie blue staining. The two proteins have not yet been identified but comprise a major DNA binding activity in CHO extracts obtained by detergent-free extraction procedures. This work provides circumstantial evidence that suggests these two polypeptides may form part of the histone H1 family.

A molecular mechanism of P-loop pliability of Rho-kinase investigated by molecular dynamic simulation

NASA Astrophysics Data System (ADS)

Gohda, Keigo; Hakoshima, Toshio

2008-11-01

Rho-kinase is a leading player in the regulation of cytoskeletal events involving smooth muscle contraction and neurite growth-cone collapse and retraction, and is a promising drug target in the treatment of both vascular and neurological disorders. Recent crystal structure of Rho-kinase complexed with a small-molecule inhibitor fasudil has revealed structural details of the ATP-binding site, which represents the target site for the inhibitor, and showed that the conserved phenylalanine on the P-loop occupies the pocket, resulting in an increase of protein-ligand contacts. Thus, the P-loop pliability is considered to play an important role in inhibitor binding affinity and specificity. In this study, we carried out a molecular dynamic simulation for Rho-kinase-fasudil complexes with two different P-loop conformations, i.e., the extended and folded conformations, in order to understand the P-loop pliability and dynamics at atomic level. A PKA-fasudil complex was also used for comparison. In the MD simulation, the flip-flop movement of the P-loop conformation starting either from the extended or folded conformation was not able to be observed. However, a significant conformational change in a long loop region covering over the P-loop, and also alteration of ionic interaction-manner of fasudil with acidic residues in the ATP binding site were shown only in the Rho-kinase-fasudil complex with the extended P-loop conformation, while Rho-kinase with the folded P-loop conformation and PKA complexes did not show large fluctuations, suggesting that the Rho-kinase-fasudil complex with the extended P-loop conformation represents a meta-stable state. The information of the P-loop pliability at atomic level obtained in this study could provide valuable clues to designing potent and/or selective inhibitors for Rho-kinase.

Functional characterization of mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 lacking short domains within the large, lumen-exposed loop of the chlorophyll protein CP47 in photosystem II.

PubMed

Gleiter, H M; Haag, E; Shen, J R; Eaton-Rye, J J; Inoue, Y; Vermaas, W F; Renger, G

1994-10-11

Several autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions or a single-site mutation within the large, lumen-exposed loop (loop E) of the chlorophyll a-binding photosystem II core protein, CP47, are analyzed for their functional properties by measuring the flash-induced pattern of thermoluminescence, oxygen yield, and fluorescence quantum yield. A physiological and biochemical characterization of these mutant strains has been given in two previous reports [Eaton-Rye, J.J., & Vermaas, W.F.J. (1991) Plant Mol. Biol. 17, 1165-1177; Haag, E., Eaton-Rye, J.J., Renger, G., & Vermaas, S. F.J. (1993) Biochemistry 32, 4444-4454]. The results of the present study show that deletion of charged and conserved amino acids in a region roughly located between residues 370 and 390 decreases the binding affinity of the extrinsic PS II-O protein to photosystem II. Marked differences with PSII-O deletion mutants are observed with respect to Ca2+ requirement and the flash-induced pattern of oxygen evolution. Under conditions where a sufficient light activation is provided, the psbB mutants assayed in this study reveal normal S-state parameters and lifetimes. The results bear two basic implications: (i) the manganese involved in water oxidation can still be bound in a functionally normal or only slightly distorted manner, and (ii) the binding of the extrinsic PS II-O protein to photosystem II is impaired in mutants carrying a deletion in the domain between residues 370 and 390, but the presence of the PS II-O protein is still of functional relevance for the PS II complex, e.g., for maintenance of a high-affinity binding site for Ca2+ and/or involvement during the process of photoactivation.

A Bridging [4Fe-4S] Cluster and Nucleotide Binding Are Essential for Function of the Cfd1-Nbp35 Complex as a Scaffold in Iron-Sulfur Protein Maturation*

PubMed Central

Netz, Daili J. A.; Pierik, Antonio J.; Stümpfig, Martin; Bill, Eckhard; Sharma, Anil K.; Pallesen, Leif J.; Walden, William E.; Lill, Roland

2012-01-01

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo. PMID:22362766

Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stefely, Jonathan A.; Reidenbach, Andrew G.; Ulbrich, Arne

The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. In this paper, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flipsmore » this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Finally, our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.« less

Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis

DOE PAGES

Stefely, Jonathan A.; Reidenbach, Andrew G.; Ulbrich, Arne; ...

2014-12-11

The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. In this paper, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flipsmore » this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Finally, our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.« less

Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor*

PubMed Central

Clayton, Cecilea C.; Donthamsetti, Prashant; Lambert, Nevin A.; Javitch, Jonathan A.; Neve, Kim A.

2014-01-01

Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212–215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213–215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to β2-adaptin (the β2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and β2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified. PMID:25336643

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

NASA Astrophysics Data System (ADS)

Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

2017-01-01

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

Biogenesis of influenza a virus hemagglutinin cross-protective stem epitopes.

PubMed

Magadán, Javier G; Altman, Meghan O; Ince, William L; Hickman, Heather D; Stevens, James; Chevalier, Aaron; Baker, David; Wilson, Patrick C; Ahmed, Rafi; Bennink, Jack R; Yewdell, Jonathan W

2014-06-01

Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

DTIC Science & Technology

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

Split green fluorescent protein as a modular binding partner for protein crystallization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Hau B.; Hung, Li-Wei; Yeates, Todd O.

2013-12-01

A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was testedmore » by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization.« less

Regulation of Steroid Hormone Receptor Function By the 52-kDa FK506-Binding Protein (FKBP52)

PubMed Central

Sivils, Jeffrey C.; Storer, Cheryl L.; Galigniana, Mario D.; Cox, Marc B.

2011-01-01

The large FK506-binding protein FKBP52 has been characterized as an important positive regulator of androgen, glucocorticoid and progesterone receptor signaling pathways. FKBP52 associates with receptor-Hsp90 complexes and is proposed to have roles in both receptor hormone binding and receptor subcellular localization. Data from biochemical and cellular studies has been corroborated in whole animal models as fkbp52-deficient male and female mice display characteristics of androgen, glucocorticoid and/or progesterone insensitivity. FKBP52 receptor specificity and the specific phenotypes displayed by the fkbp52-deficient mice have firmly established FKBP52 as a promising target for the treatment of a variety of hormone-dependent diseases. Recent studies demonstrated that the FKBP52 FK1 domain and the proline-rich loop within this domain are functionally important for FKBP52 regulation of receptor function. Based on these data, efforts are currently underway to target the FKBP52 FK1 domain and the proline-rich loop with small molecule inhibitors. PMID:21511531

Crystal structure of the YGR205w protein from Saccharomyces cerevisiae: close structural resemblance to E. coli pantothenate kinase.

PubMed

Li de La Sierra-Gallay, Ines; Collinet, Bruno; Graille, Marc; Quevillon-Cheruel, Sophie; Liger, Dominique; Minard, Philippe; Blondeau, Karine; Henckes, Gilles; Aufrère, Robert; Leulliot, Nicolas; Zhou, Cong-Zhao; Sorel, Isabelle; Ferrer, Jean-Luc; Poupon, Anne; Janin, Joël; van Tilbeurgh, Herman

2004-03-01

The protein product of the YGR205w gene of Saccharomyces cerevisiae was targeted as part of our yeast structural genomics project. YGR205w codes for a small (290 amino acids) protein with unknown structure and function. The only recognizable sequence feature is the presence of a Walker A motif (P loop) indicating a possible nucleotide binding/converting function. We determined the three-dimensional crystal structure of Se-methionine substituted protein using multiple anomalous diffraction. The structure revealed a well known mononucleotide fold and strong resemblance to the structure of small metabolite phosphorylating enzymes such as pantothenate and phosphoribulo kinase. Biochemical experiments show that YGR205w binds specifically ATP and, less tightly, ADP. The structure also revealed the presence of two bound sulphate ions, occupying opposite niches in a canyon that corresponds to the active site of the protein. One sulphate is bound to the P-loop in a position that corresponds to the position of beta-phosphate in mononucleotide protein ATP complex, suggesting the protein is indeed a kinase. The nature of the phosphate accepting substrate remains to be determined. Copyright 2004 Wiley-Liss, Inc.

Characterization of secretory phospholipase A₂ with phospholipase A₁ activity in tobacco, Nicotiana tabacum (L.).

PubMed

Fujikawa, Yukichi; Fujikawa, Ritsuko; Iijima, Noriaki; Esaka, Muneharu

2012-03-01

A cDNA encoding protein with homology to plant secretory phospholipase A₂ (sPLA₂), denoted as Nt1 PLA₂, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA₂ has 12 cysteines, Ca²⁺ binding loop and catalytic site domain that are commonly conserved in plant sPLA₂s. The recombinant Nt1 PLA₂ was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA₂ could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²⁺ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA₂ mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.

Herpes Simplex Virus Glycoprotein B Associates with Target Membranes via Its Fusion Loops▿

PubMed Central

Hannah, Brian P.; Cairns, Tina M.; Bender, Florent C.; Whitbeck, J. Charles; Lou, Huan; Eisenberg, Roselyn J.; Cohen, Gary H.

2009-01-01

Herpes simplex virus (HSV) glycoproteins gB, gD, and gH/gL are necessary and sufficient for virus entry into cells. Structural features of gB are similar to those of vesicular stomatitis virus G and baculovirus gp64, and together they define the new class III group of fusion proteins. Previously, we used mutagenesis to show that three hydrophobic residues (W174, Y179, and A261) within the putative gB fusion loops are integral to gB function. Here we expanded our analysis, using site-directed mutagenesis of each residue in both gB fusion loops. Mutation of most of the nonpolar or hydrophobic amino acids (W174, F175, G176, Y179, and A261) had severe effects on gB function in cell-cell fusion and null virus complementation assays. Of the six charged amino acids, mutation of H263 or R264 also negatively affected gB function. To further analyze the mutants, we cloned the ectodomains of the W174R, Y179S, H263A, and R264A mutants into a baculovirus expression system and compared them with the wild-type (WT) form, gB730t. As shown previously, gB730t blocks virus entry into cells, suggesting that gB730t competes with virion gB for a cell receptor. All four mutant proteins retained this function, implying that fusion loop activity is separate from gB-receptor binding. However, unlike WT gB730t, the mutant proteins displayed reduced binding to cells and were either impaired or unable to bind naked, cholesterol-enriched liposomes, suggesting that it may be gB-lipid binding that is disrupted by the mutations. Furthermore, monoclonal antibodies with epitopes proximal to the fusion loops abrogated gB-liposome binding. Taken together, our data suggest that gB associates with lipid membranes via a fusion domain of key hydrophobic and hydrophilic residues and that this domain associates with lipid membranes during fusion. PMID:19369321

Piwi and potency: PIWI proteins in animal stem cells and regeneration.

PubMed

van Wolfswinkel, Josien C

2014-10-01

PIWI proteins are well known for their roles in the animal germline. They are essential for germline development and maintenance, and together with their binding partners, the piRNAs, they mediate transposon silencing. More recently, PIWI proteins have also been identified in somatic stem cells in diverse animals. The expression of PIWI proteins in these cells could be related to the ability of such cells to contribute to the germline. However, evaluation of stem cell systems across many different animal phyla suggests that PIWI proteins have an ancestral role in somatic stem cells, irrespective of their contribution to the germ cell lineage. Moreover, the data currently available reveal a possible correlation between the differentiation potential of a cell and its PIWI levels. © The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain.

PubMed

Law, S F; Zhang, Y Z; Fashena, S J; Toby, G; Estojak, J; Golemis, E A

1999-10-10

HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells. While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition. In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif. This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47. We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth. Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins. These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation. Copyright 1999 Academic Press.

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

PubMed Central

Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

2015-01-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

Structural Determination of Functional Domains in Early B-cell Factor (EBF) Family of Transcription Factors Reveals Similarities to Rel DNA-binding Proteins and a Novel Dimerization Motif*

PubMed Central

Siponen, Marina I.; Wisniewska, Magdalena; Lehtiö, Lari; Johansson, Ida; Svensson, Linda; Raszewski, Grzegorz; Nilsson, Lennart; Sigvardsson, Mikael; Berglund, Helena

2010-01-01

The early B-cell factor (EBF) transcription factors are central regulators of development in several organs and tissues. This protein family shows low sequence similarity to other protein families, which is why structural information for the functional domains of these proteins is crucial to understand their biochemical features. We have used a modular approach to determine the crystal structures of the structured domains in the EBF family. The DNA binding domain reveals a striking resemblance to the DNA binding domains of the Rel homology superfamily of transcription factors but contains a unique zinc binding structure, termed zinc knuckle. Further the EBF proteins contain an IPT/TIG domain and an atypical helix-loop-helix domain with a novel type of dimerization motif. The data presented here provide insights into unique structural features of the EBF proteins and open possibilities for detailed molecular investigations of this important transcription factor family. PMID:20592035

A change in structural integrity of c-Kit mutant D816V causes constitutive signaling.

PubMed

Raghav, Pawan Kumar; Singh, Ajay Kumar; Gangenahalli, Gurudutta

2018-03-01

Several signaling pathways, ligands, and genes that regulate proliferative and self-renewal properties of the Hematopoietic Stem Cells (HSCs) have been studied meticulously. One of the signaling pathways that play a crucial role in the process of hematopoiesis is the Stem Cell Factor (SCF) mediated c-Kit pathway. The c-Kit is a Receptor Tyrosine Kinase (RTK), which is expressed in the cells including HSCs. It undergoes dimerization upon binding with its cognate ligand SCF. As a result, phosphorylation of the Juxtamembrane (JM) domain of c-Kit takes place at Tyr568 and Tyr570 residues. These phosphorylated residues become the docking sites for protein tyrosine phosphatases (PTPs) namely SHP-1 and SHP-2, which in turn cause dephosphorylation and negative regulation of the downstream signaling responsible for the cell proliferation. Interestingly, it has been reported that the mutation of c-Kit at D816V makes it independent of SCF stimulation and SHP-1/SHP-2 inhibition, thereby, causing its constitutive activation. The present study was commenced to elucidate the structural behavior of this mutation in the JM and A-loop region of c-Kit using Molecular Dynamics (MD) simulations of the wild-type and mutant c-Kit in unphosphorylated and phosphorylated states. The energy difference computed between the wild type and mutant (D816V) c-Kit, and protein-protein docking and complex analysis revealed the impact of this single residue mutation on the integrity dynamics of c-Kit that makes it independent of SHP-1/SHP-2 negative regulation. Copyright © 2018 Elsevier B.V. All rights reserved.

Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation.

PubMed

Zhang, Hua; Song, Lei; Cong, Haolong; Tien, Po

2015-10-01

Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5' untranslated region (5'UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mechanistic events underlying odorant binding protein chemoreception.

PubMed

Golebiowski, Jérôme; Antonczak, Serge; Fiorucci, Sébastien; Cabrol-Bass, Daniel

2007-05-01

Odorant binding proteins (OBP's) are small hydrophilic proteins, belonging to the lipocalin family dedicated to bind and transport small hydrophobic ligands. Despite many works, the mechanism of ligand binding, together with the functional role of these proteins remains a topic of debate and little is known at the atomic level. The present work reports a computational study of odorants capture and release by an OBP, using both constrained and unconstrained simulations, giving a glimpse on the molecular mechanism of chemoreception. The residues at the origin of the regulation of the protein door opening are identified and a tyrosine amino-acid together with other nearby residues appear to play a crucial role in allowing this event to occur. The simulations reveal that this tyrosine and the protein's L5 loop are implicated in the ligand contact with the protein and act as an anchoring point for the ligand. The protein structural features required for the ligand entry are highly conserved among many transport proteins, suggesting that this mechanism could somewhat be extended to some members of the larger family of lipocalin. (c) 2007 Wiley-Liss, Inc.

The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor.

PubMed

Kokabu, Shoichiro; Nakatomi, Chihiro; Matsubara, Takuma; Ono, Yusuke; Addison, William N; Lowery, Jonathan W; Urata, Mariko; Hudnall, Aaron M; Hitomi, Suzuro; Nakatomi, Mitsushiro; Sato, Tsuyoshi; Osawa, Kenji; Yoda, Tetsuya; Rosen, Vicki; Jimi, Eijiro

2017-08-04

Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1

DOE PAGES

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany; ...

2017-01-31

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

Impact of low-frequency hotspot mutation R282Q on the structure of p53 DNA-binding domain as revealed by crystallography at 1.54 Å resolution

PubMed Central

Tu, Chao; Tan, Yu-Hong; Shaw, Gary; Zhou, Zheng; Bai, Yawen; Luo, Ray; Ji, Xinhua

2008-01-01

Tumor suppressor p53 is a sequence-specific DNA-binding protein and its central DNA-binding domain (DBD) harbors six hotspots (Arg175, Gly245, Arg248, Arg249, Arg273 and Arg282) for human cancers. Here, the crystal structure of a low-frequency hotspot mutant, p53DBD(R282Q), is reported at 1.54 Å resolution together with the results of molecular-dynamics simulations on the basis of the structure. In addition to eliminating a salt bridge, the R282Q mutation has a significant impact on the properties of two DNA-binding loops (L1 and L3). The L1 loop is flexible in the wild type, but it is not flexible in the mutant. The L3 loop of the wild type is not flexible, whereas it assumes two conformations in the mutant. Molecular-dynamics simulations indicated that both conformations of the L3 loop are accessible under biological conditions. It is predicted that the elimination of the salt bridge and the inversion of the flexibility of L1 and L3 are directly or indirectly responsible for deactivating the tumor suppressor p53. PMID:18453682

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

PubMed

Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

2016-08-01

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

PubMed Central

Takahashi, Yuji; Bigler, Dora; Ito, Yasuhiko; White, Judith M.

2001-01-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions. PMID:11294888

Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of beta1 integrin-associated proteins CD9, CD81, and CD98.

PubMed

Takahashi, Y; Bigler, D; Ito, Y; White, J M

2001-04-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-alpha6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-alpha6 mAb, or by mAbs against either the alphav or beta3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other beta1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg beta1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface "tetraspan web" facilitates fertilization and that it may do so by fostering ADAM-integrin interactions.

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

PubMed Central

Pallesen, Jesper; Wang, Nianshuang; Corbett, Kizzmekia S.; Wrapp, Daniel; Kirchdoerfer, Robert N.; Turner, Hannah L.; Cottrell, Christopher A.; Becker, Michelle M.; Wang, Lingshu; Shi, Wei; Kong, Wing-Pui; Andres, Erica L.; Kettenbach, Arminja N.; Denison, Mark R.; Chappell, James D.; Graham, Barney S.; Ward, Andrew B.

2017-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also determined high-resolution structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines. PMID:28807998

A Sequence in the loop domain of hepatitis C virus E2 protein identified in silico as crucial for the selective binding to human CD81

PubMed Central

Chang, Chun-Chun; Hsu, Hao-Jen; Yen, Jui-Hung; Lo, Shih-Yen

2017-01-01

Hepatitis C virus (HCV) is a species-specific pathogenic virus that infects only humans and chimpanzees. Previous studies have indicated that interactions between the HCV E2 protein and CD81 on host cells are required for HCV infection. To determine the crucial factors for species-specific interactions at the molecular level, this study employed in silico molecular docking involving molecular dynamic simulations of the binding of HCV E2 onto human and rat CD81s. In vitro experiments including surface plasmon resonance measurements and cellular binding assays were applied for simple validations of the in silico results. The in silico studies identified two binding regions on the HCV E2 loop domain, namely E2-site1 and E2-site2, as being crucial for the interactions with CD81s, with the E2-site2 as the determinant factor for human-specific binding. Free energy calculations indicated that the E2/CD81 binding process might follow a two-step model involving (i) the electrostatic interaction-driven initial binding of human-specific E2-site2, followed by (ii) changes in the E2 orientation to facilitate the hydrophobic and van der Waals interaction-driven binding of E2-site1. The sequence of the human-specific, stronger-binding E2-site2 could serve as a candidate template for the future development of HCV-inhibiting peptide drugs. PMID:28481946

Manipulation of a DNA aptamer-protein binding site through arylation of internal guanine residues.

PubMed

Van Riesen, Abigail J; Fadock, Kaila L; Deore, Prashant S; Desoky, Ahmed; Manderville, Richard A; Sowlati-Hashjin, Shahin; Wetmore, Stacey D

2018-05-23

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

Distinguishing West Nile virus infection using a recombinant envelope protein with mutations in the conserved fusion-loop.

PubMed

Chabierski, Stefan; Barzon, Luisa; Papa, Anna; Niedrig, Matthias; Bramson, Jonathan L; Richner, Justin M; Palù, Giorgio; Diamond, Michael S; Ulbert, Sebastian

2014-05-09

West Nile Virus (WNV) is an emerging mosquito-transmitted flavivirus that continues to spread and cause disease throughout several parts of the world, including Europe and the Americas. Specific diagnosis of WNV infections using current serological testing is complicated by the high degree of cross-reactivity between antibodies against other clinically relevant flaviviruses, including dengue, tick-borne encephalitis (TBEV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. Cross-reactivity is particularly problematic in areas where different flaviviruses co-circulate or in populations that have been immunized with vaccines against TBEV, JEV, or YFV. The majority of cross-reactive antibodies against the immunodominant flavivirus envelope (E) protein target a conserved epitope in the fusion loop at the distal end of domain II. We tested a loss-of-function bacterially expressed recombinant WNV E protein containing mutations in the fusion loop and an adjacent loop domain as a possible diagnostic reagent. By comparing the binding of sera from humans infected with WNV or other flaviviruses to the wild type and the mutant E proteins, we analyzed the potential of this technology to specifically detect WNV antibodies. Using this system, we could reliably determine WNV infections. Antibodies from WNV-infected individuals bound equally well to the wild type and the mutant protein. In contrast, sera from persons infected with other flaviviruses showed significantly decreased binding to the mutant protein. By calculating the mean differences between antibody signals detected using the wild type and the mutant proteins, a value could be assigned for each of the flaviviruses, which distinguished their pattern of reactivity. Recombinant mutant E proteins can be used to discriminate infections with WNV from those with other flaviviruses. The data have important implications for the development of improved, specific serological assays for the detection of WNV antibodies in regions where other flaviviruses co-circulate or in populations that are immunized with other flavivirus vaccines.

Structural insights into the T6SS effector protein Tse3 and the Tse3-Tsi3 complex from Pseudomonas aeruginosa reveal a calcium-dependent membrane-binding mechanism.

PubMed

Lu, Defen; Shang, Guijun; Zhang, Heqiao; Yu, Qian; Cong, Xiaoyan; Yuan, Jupeng; He, Fengjuan; Zhu, Chunyuan; Zhao, Yanyu; Yin, Kun; Chen, Yuanyuan; Hu, Junqiang; Zhang, Xiaodan; Yuan, Zenglin; Xu, Sujuan; Hu, Wei; Cang, Huaixing; Gu, Lichuan

2014-06-01

The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm-localized immunity protein Tsi3 to prevent potential self-intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3-Tsi3 complex. Tse3 contains an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. One loop in the N-terminal domain (Loop 12) and one helix (α9) from the C-terminal domain together anchor Tse3 and the Tse3-Tsi3 complex to membrane in a calcium-dependent manner in vitro, and this membrane-binding ability is essential for Tse3's activity. In the C-terminal domain, a Y-shaped groove present on the surface likely serves as the PG binding site. Two calcium-binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3-Tsi3 structure, three loops of Tsi3 insert into the substrate-binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3-Tsi3 complex. © 2014 John Wiley & Sons Ltd.

Crystal structure of the coat protein from the GA bacteriophage: model of the unassembled dimer.

PubMed Central

Ni, C. Z.; White, C. A.; Mitchell, R. S.; Wickersham, J.; Kodandapani, R.; Peabody, D. S.; Ely, K. R.

1996-01-01

There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L. 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R. Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263). The structures differ in the FG loops and in the first turn of the alpha A helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface. Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F. Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010). This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine. PMID:8976557

Crystal structure of the coat protein from the GA bacteriophage: model of the unassembled dimer.

PubMed

Ni, C Z; White, C A; Mitchell, R S; Wickersham, J; Kodandapani, R; Peabody, D S; Ely, K R

1996-12-01

There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L. 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R. Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263). The structures differ in the FG loops and in the first turn of the alpha A helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface. Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F. Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010). This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine.

Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP).

PubMed

Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin; Chung, Chong-Pyoung; Park, Yoon Jeong

2012-03-09

Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Several recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor γ. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken together, a novel CBP could be a useful candidate for regenerating bone and treating osteoporosis, which result from an imbalance in osteogenesis and adipogenesis differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses.

PubMed

Johnson, Britney; Li, Jing; Adhikari, Jagat; Edwards, Megan R; Zhang, Hao; Schwarz, Toni; Leung, Daisy W; Basler, Christopher F; Gross, Michael L; Amarasinghe, Gaya K

2016-08-28

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Britney; Li, Jing; Adhikari, Jagat

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulatemore » nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.« less

Rigidification of the autolysis loop enhances Na[superscript +] binding to thrombin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pozzi, Nicola; Chen, Raymond; Chen, Zhiwei

2011-09-20

Binding of Na{sup +} to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na{sup +} is weak due to large heat capacity and enthalpy changes associated with binding, and the K{sub d} = 80 mM ensures only 64% saturation of the site at the concentration of Na{sup +} in the blood (140 mM). Residues controlling Na{sup +} binding and activation have been identified. Yet, attempts to improve the interaction of Na{sup +} with thrombin and possibly increase catalyticmore » activity under physiological conditions have so far been unsuccessful. Here we report how replacement of the flexible autolysis loop of human thrombin with the homologous rigid domain of the murine enzyme results in a drastic (up to 10-fold) increase in Na{sup +} affinity and a significant improvement in the catalytic activity of the enzyme. Rigidification of the autolysis loop abolishes the heat capacity change associated with Na{sup +} binding observed in the wild-type and also increases the stability of thrombin. These findings have general relevance to protein engineering studies of clotting proteases and trypsin-like enzymes.« less

DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

PubMed

Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

1995-09-05

Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

PubMed

Iwaya, Naoko; Takasu, Hirotoshi; Goda, Natsuko; Shirakawa, Masahiro; Tanaka, Toshiki; Hamada, Daizo; Hiroaki, Hidekazu

2013-05-01

The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

Ribosome protection by antibiotic resistance ATP-binding cassette protein.

PubMed

Su, Weixin; Kumar, Veerendra; Ding, Yichen; Ero, Rya; Serra, Aida; Lee, Benjamin Sian Teck; Wong, Andrew See Weng; Shi, Jian; Sze, Siu Kwan; Yang, Liang; Gao, Yong-Gui

2018-05-15

The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins. Copyright © 2018 the Author(s). Published by PNAS.

Non-coding stem-bulge RNAs are required for cell proliferation and embryonic development in C. elegans

PubMed Central

Kowalski, Madzia P.; Baylis, Howard A.; Krude, Torsten

2015-01-01

ABSTRACT Stem bulge RNAs (sbRNAs) are a family of small non-coding stem-loop RNAs present in Caenorhabditis elegans and other nematodes, the function of which is unknown. Here, we report the first functional characterisation of nematode sbRNAs. We demonstrate that sbRNAs from a range of nematode species are able to reconstitute the initiation of chromosomal DNA replication in the presence of replication proteins in vitro, and that conserved nucleotide sequence motifs are essential for this function. By functionally inactivating sbRNAs with antisense morpholino oligonucleotides, we show that sbRNAs are required for S phase progression, early embryonic development and the viability of C. elegans in vivo. Thus, we demonstrate a new and essential role for sbRNAs during the early development of C. elegans. sbRNAs show limited nucleotide sequence similarity to vertebrate Y RNAs, which are also essential for the initiation of DNA replication. Our results therefore establish that the essential function of small non-coding stem-loop RNAs during DNA replication extends beyond vertebrates. PMID:25908866

General transfer matrix formalism to calculate DNA-protein-drug binding in gene regulation: application to OR operator of phage lambda.

PubMed

Teif, Vladimir B

2007-01-01

The transfer matrix methodology is proposed as a systematic tool for the statistical-mechanical description of DNA-protein-drug binding involved in gene regulation. We show that a genetic system of several cis-regulatory modules is calculable using this method, considering explicitly the site-overlapping, competitive, cooperative binding of regulatory proteins, their multilayer assembly and DNA looping. In the methodological section, the matrix models are solved for the basic types of short- and long-range interactions between DNA-bound proteins, drugs and nucleosomes. We apply the matrix method to gene regulation at the O(R) operator of phage lambda. The transfer matrix formalism allowed the description of the lambda-switch at a single-nucleotide resolution, taking into account the effects of a range of inter-protein distances. Our calculations confirm previously established roles of the contact CI-Cro-RNAP interactions. Concerning long-range interactions, we show that while the DNA loop between the O(R) and O(L) operators is important at the lysogenic CI concentrations, the interference between the adjacent promoters P(R) and P(RM) becomes more important at small CI concentrations. A large change in the expression pattern may arise in this regime due to anticooperative interactions between DNA-bound RNA polymerases. The applicability of the matrix method to more complex systems is discussed.

ABC transporter Cdr1p harbors charged residues in the intracellular loop and nucleotide-binding domain critical for protein trafficking and drug resistance.

PubMed

Shah, Abdul Haseeb; Banerjee, Atanu; Rawal, Manpreet Kaur; Saxena, Ajay Kumar; Mondal, Alok Kumar; Prasad, Rajendra

2015-08-01

The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of <4 Å, an ion pair between K577 of ICL1 and E315 of NBD1 was found to be critical. The substitution, swapping and changing of the length or charge of K577 or E315 by directed mutagenesis led to a misfolded, non-rescuable protein entrapped in intracellular structures. Furthermore, the equipositional ionic pair-forming residues from ICL3 and NBD2 (R1260 and E1014) did not impact protein trafficking. These results point to a new role for ICL/NBD interacting residues in PDR ABC transporters in protein folding and trafficking. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET

PubMed Central

Clerte, Caroline; Declerck, Nathalie; Margeat, Emmanuel

2013-01-01

The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5′-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic. PMID:23303779

Mechanism of foreign DNA selection in a bacterial adaptive immune system

PubMed Central

Sashital, Dipali G.; Wiedenheft, Blake; Doudna, Jennifer A.

2012-01-01

Summary In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary “non-self” DNA sequences for destruction, while avoiding binding to “self” sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for non-self target recognition and binding. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved 3-base pair motif that is required for non-self target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade. PMID:22521690

Ubiquitin Regulates Caspase Recruitment Domain-mediated Signaling by Nucleotide-binding Oligomerization Domain-containing Proteins NOD1 and NOD2*

PubMed Central

Ver Heul, Aaron M.; Fowler, C. Andrew; Ramaswamy, S.; Piper, Robert C.

2013-01-01

NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2. PMID:23300079

Proteins mediating DNA loops effectively block transcription.

PubMed

Vörös, Zsuzsanna; Yan, Yan; Kovari, Daniel T; Finzi, Laura; Dunlap, David

2017-07-01

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Exploring the Midgut Transcriptome and Brush Border Membrane Vesicle Proteome of the Rice Stem Borer, Chilo suppressalis (Walker)

PubMed Central

Peng, Chuanhua; Wang, Xiaoping; Li, Fei; Lin, Yongjun

2012-01-01

The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt) toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV). Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac. PMID:22666467

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

PubMed

Katti, Sachin; Nyenhuis, Sarah B; Her, Bin; Srivastava, Atul K; Taylor, Alexander B; Hart, P John; Cafiso, David S; Igumenova, Tatyana I

2017-06-27

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca 2+ -dependent manner. In these cases, membrane association is triggered by Ca 2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd 2+ , in lieu of Ca 2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd 2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd 2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd 2+ -complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca 2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca 2+ ion binding to the C2 domain loop regions.

Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC

PubMed Central

Chen, Qiang; Cheng, Mary Hongying; Xu, Yan; Tang, Pei

2010-01-01

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (KD) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins. PMID:20858424

Cellular distribution of calmodulin and calmodulin-binding proteins in Vicia faba L

NASA Technical Reports Server (NTRS)

Ling, V.; Assmann, S. M.

1992-01-01

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. 125I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with Mr 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (Mr 39,000, 88,000), stems (Mr 45,000, 60,000, 64,000), and roots (Mr 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.

NFκB- and AP-1-mediated DNA looping regulates matrix metalloproteinase-9 transcription in TNF-α-treated human leukemia U937 cells.

PubMed

Chen, Ying-Jung; Chang, Long-Sen

2015-10-01

The aim of this study is to explore the spatial association of critical genomic elements in the effect of TNF-α on matrix metalloproteinase-9 (MMP-9) expression in human leukemia U937 cells. TNF-α up-regulated MMP-9 protein expression and mRNA level in U937 cells, and Akt-mediated-NFκB/p65 activation and JNK-mediated c-Jun activation were proven to be involved in TNF-α-induced MMP-9 up-regulation. Promoter luciferase activity assay revealed that NFκB (nt-600) and AP-1 (nt-79) binding sites were crucial for TNF-α-induced transcription of MMP-9 gene. The results of a chromatin immunoprecipitation assay indicated that TNF-α reduced histone deacetylase-1 (HDAC-1) recruitment but increased p300 (a histone acetyltransferase) recruitment to MMP-9 promoter regions surrounding NFκB and AP-1 binding sites. Consistently, TNF-α increased enrichment of the acetylated histone H3 mark on MMP-9 promoter regions. DNA affinity purification assay revealed that p300 and HDAC1 could bind oligonucleotides containing AP-1/c-Jun and NFκB/p65 binding sites. Chromosome conformation capture assay showed that TNF-α stimulated chromosomal loops in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun. The p300-associated acetyltransferase activity was crucial for p65/c-Jun-mediated DNA looping, and inhibition of HDAC activity increased the level of DNA looping. Reduction in the level of DNA looping eliminated all TNF-α-stimulated MMP-9 up-regulation. Taken together, our data suggest that p65/c-Jun-mediated DNA looping is involved in TNF-α-induced MMP-9 up-regulation and that the recruitment of p300 or HDAC1 to NFκB and AP-1 binding sites modifies the level of DNA looping. Copyright © 2015 Elsevier B.V. All rights reserved.

Mining protein loops using a structural alphabet and statistical exceptionality

PubMed Central

2010-01-01

Background Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. Results We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 Å). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. Conclusions We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/. PMID:20132552

Mining protein loops using a structural alphabet and statistical exceptionality.

PubMed

Regad, Leslie; Martin, Juliette; Nuel, Gregory; Camproux, Anne-Claude

2010-02-04

Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 A). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/.

The Myb-domain protein ULTRAPETALA1 INTERACTING FACTOR 1 controls floral meristem activities in Arabidopsis.

PubMed

Moreau, Fanny; Thévenon, Emmanuel; Blanvillain, Robert; Lopez-Vidriero, Irene; Franco-Zorrilla, Jose Manuel; Dumas, Renaud; Parcy, François; Morel, Patrice; Trehin, Christophe; Carles, Cristel C

2016-04-01

Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression. © 2016. Published by The Company of Biologists Ltd.

Transcriptional analysis of phloem-associated cells of potato.

PubMed

Lin, Tian; Lashbrook, Coralie C; Cho, Sung Ki; Butler, Nathaniel M; Sharma, Pooja; Muppirala, Usha; Severin, Andrew J; Hannapel, David J

2015-09-03

Numerous signal molecules, including proteins and mRNAs, are transported through the architecture of plants via the vascular system. As the connection between leaves and other organs, the petiole and stem are especially important in their transport function, which is carried out by the phloem and xylem, especially by the sieve elements in the phloem system. The phloem is an important conduit for transporting photosynthate and signal molecules like metabolites, proteins, small RNAs, and full-length mRNAs. Phloem sap has been used as an unadulterated source to profile phloem proteins and RNAs, but unfortunately, pure phloem sap cannot be obtained in most plant species. Here we make use of laser capture microdissection (LCM) and RNA-seq for an in-depth transcriptional profile of phloem-associated cells of both petioles and stems of potato. To expedite our analysis, we have taken advantage of the potato genome that has recently been fully sequenced and annotated. Out of the 27 k transcripts assembled that we identified, approximately 15 k were present in phloem-associated cells of petiole and stem with greater than ten reads. Among these genes, roughly 10 k are affected by photoperiod. Several RNAs from this day length-regulated group are also abundant in phloem cells of petioles and encode for proteins involved in signaling or transcriptional control. Approximately 22 % of the transcripts in phloem cells contained at least one binding motif for Pumilio, Nova, or polypyrimidine tract-binding proteins in their downstream sequences. Highlighting the predominance of binding processes identified in the gene ontology analysis of active genes from phloem cells, 78 % of the 464 RNA-binding proteins present in the potato genome were detected in our phloem transcriptome. As a reasonable alternative when phloem sap collection is not possible, LCM can be used to isolate RNA from specific cell types, and along with RNA-seq, provides practical access to expression profiles of phloem tissue. The combination of these techniques provides a useful approach to the study of phloem and a comprehensive picture of the mechanisms associated with long-distance signaling. The data presented here provide valuable insights into potentially novel phloem-mobile mRNAs and phloem-associated RNA-binding proteins.

On the Minimization of Fluctuations in the Response Times of Autoregulatory Gene Networks

PubMed Central

Murugan, Rajamanickam; Kreiman, Gabriel

2011-01-01

The temporal dynamics of the concentrations of several proteins are tightly regulated, particularly for critical nodes in biological networks such as transcription factors. An important mechanism to control transcription factor levels is through autoregulatory feedback loops where the protein can bind its own promoter. Here we use theoretical tools and computational simulations to further our understanding of transcription-factor autoregulatory loops. We show that the stochastic dynamics of feedback and mRNA synthesis can significantly influence the speed of response of autoregulatory genetic networks toward external stimuli. The fluctuations in the response-times associated with the accumulation of the transcription factor in the presence of negative or positive autoregulation can be minimized by confining the ratio of mRNA/protein lifetimes within 1:10. This predicted range of mRNA/protein lifetime agrees with ranges observed empirically in prokaryotes and eukaryotes. The theory can quantitatively and systematically account for the influence of regulatory element binding and unbinding dynamics on the transcription-factor concentration rise-times. The simulation results are robust against changes in several system parameters of the gene expression machinery. PMID:21943410

Molecular dynamics simulations on interaction between bacterial proteins: Implication on pathogenic activities.

PubMed

Mondal, Manas; Chakrabarti, Jaydeb; Ghosh, Mahua

2018-03-01

We perform molecular dynamics simulation studies on interaction between bacterial proteins: an outer-membrane protein STY3179 and a yfdX protein STY3178 of Salmonella Typhi. STY3179 has been found to be involved in bacterial adhesion and invasion. STY3178 is recently biophysically characterized. It is a soluble protein having antibiotic binding and chaperon activity capabilities. These two proteins co-occur and are from neighboring gene in Salmonella Typhi-occurrence of homologs of both STY3178 and STY3179 are identified in many Gram-negative bacteria. We show using homology modeling, docking followed by molecular dynamics simulation that they can form a stable complex. STY3178 belongs to aqueous phase, while the beta barrel portion of STY3179 remains buried in DPPC bilayer with extra-cellular loops exposed to water. To understand the molecular basis of interaction between STY3178 and STY3179, we compute the conformational thermodynamics which indicate that these two proteins interact through polar and acidic residues belonging to their interfacial region. Conformational thermodynamics results further reveal instability of certain residues in extra-cellular loops of STY3179 upon complexation with STY3178 which is an indication for binding with host cell protein laminin. © 2017 Wiley Periodicals, Inc.

Optimization of a Reusable, DNA Pseudoknot-Based Electrochemical Sensor for Sequence-Specific DNA Detection in Blood Serum

PubMed Central

Cash, Kevin J.; Heeger, Alan J.; Plaxco, Kevin W.; Xiao, Yi

2010-01-01

We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem’s loop forms part of the second stem, is modified with a methylene blue redox tag at its 3′ terminus and covalently attached to a gold electrode via the 5′ terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density (~1.8 × 1013 molecules/cm2 apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3′ stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3′ loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum. PMID:19093760

RNA-binding properties and RNA chaperone activity of human peroxiredoxin 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Ji-Hee; Lee, Jeong-Mi; Lee, Hae Na

2012-09-07

Highlights: Black-Right-Pointing-Pointer hPrx1 has RNA-binding properties. Black-Right-Pointing-Pointer hPrx1 exhibits helix-destabilizing activity. Black-Right-Pointing-Pointer Cold stress increases hPrx1 level in the nuclear fraction. Black-Right-Pointing-Pointer hPrx1 enhances the viability of cells exposed to cold stress. -- Abstract: Human peroxiredoxin 1 (hPrx1), a member of the peroxiredoxin family, detoxifies peroxide substrates and has been implicated in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, and redox signaling. To date, Prx1 has not been implicated in RNA metabolism. Here, we investigated the ability of hPrx1 to bind RNA and act as an RNA chaperone. In vitro, hPrx1 bound to RNA and DNA, and unwoundmore » nucleic acid duplexes. hPrx1 also acted as a transcription anti-terminator in an assay using an Escherichia coli strain containing a stem-loop structure upstream of the chloramphenicol resistance gene. The overall cellular level of hPrx1 expression was not increased at low temperatures, but the nuclear level of hPrx1 was increased. In addition, hPrx1 overexpression enhanced the survival of cells exposed to cold stress, whereas hPrx1 knockdown significantly reduced cell survival under the same conditions. These findings suggest that hPrx1 may perform biological functions as a RNA-binding protein, which are distinctive from known functions of hPrx1 as a reactive oxygen species scavenger.« less

Structure of an Arrestin2-clathrin Complex Reveals a Novel Clathrin Binding Domain that Modulates Receptor Trafficking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, D.; Kern, R; Puthenveedu, M

2009-01-01

Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin {beta}-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formedmore » by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I){sub 2}GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.« less

Mesenchymal stem/stromal cells (MSCs): role as guardians of inflammation.

PubMed

Prockop, Darwin J; Oh, Joo Youn

2012-01-01

Recent observations have demonstrated that one of the functions of mesenchymal stem/stromal cells (MSCs) is to serve as guardians against excessive inflammatory responses. One mode of action of the cells is that they are activated to express the interleukin (IL)-1 receptor antagonist. A second mode of action is to create a negative feedback loop in which tumor necrosis factor-α (TNF-α) and other proinflammatory cytokines from resident macrophages activate MSCs to secrete the multifunctional anti-inflammatory protein TNF-α stimulated gene/protein 6 (TSG-6). The TSG-6 then reduces nuclear factor-κB (NF-κB) signaling in the resident macrophages and thereby modulates the cascade of proinflammatory cytokines. A third mode of action is to create a second negative feedback loop whereby lipopolysaccharide, TNF-α, nitric oxide, and perhaps other damage-associated molecular patterns (DAMPs) from injured tissues and macrophages activate MSCs to secrete prostaglandin E(2) (PGE(2)). The PGE(2) converts macrophages to the phenotype that secretes IL-10. There are also suggestions that MSCs may produce anti-inflammatory effects through additional modes of action including activation to express the antireactive oxygen species protein stanniocalcin-1.

Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein.

PubMed

Zhang, Zhen; Palzkill, Timothy

2003-11-14

The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.

Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

PubMed

Yee, Andrew; Tan, Fen-Lai; Ginsburg, David

2013-01-01

von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

PubMed Central

Lücke, S; Xu, G L; Palfi, Z; Cross, M; Bellofatto, V; Bindereif, A

1996-01-01

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome. Images PMID:8861965

Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qualley, Dominic F., E-mail: dqualley@berry.edu; Sokolove, Victoria L.; Ross, James L.

2015-03-13

Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two othermore » retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable. - Highlights: • BLV NC binds strongly to DNA and RNA. • BLV NC promotes mini-TAR annealing as well as HIV-1 NC. • Annealing kinetics suggest a low degree of similarity between BLV NC and HTLV-1 NC.« less

CHMP6 and VPS4A mediate recycling of Ras to the plasma membrane to promote growth factor signaling

PubMed Central

Zheng, Ze-Yi; Cheng, Chiang-Min; Fu, Xin-Rong; Chen, Liuh-Yow; Xu, Lizhong; Terrillon, Sonia; Wong, Stephen T.; Bar-Sagi, Dafna; Songyang, Zhou; Chang, Eric C.

2011-01-01

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III, were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding-loop and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound N-Ras, but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked EGFR recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling. PMID:22231449

Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

NASA Astrophysics Data System (ADS)

Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn; Dwyer, Mary; Benkovic, Stephen J.; Hammes, Gordon G.

2002-10-01

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s1 and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

PubMed

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

The cell pole: The site of cross talk between the DNA uptake and genetic recombination machinery

PubMed Central

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann; Graumann, Peter L.; Alonso, Juan C.

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as “guardians”, protect ssDNA from degradation and limit the RecA recombinase loading. Then, the “mediators” overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by “modulators”, catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or “resolver” cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the “rescuers” will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective. PMID:23046409

Protecting Gram-negative bacterial cell envelopes from human lysozyme: Interactions with Ivy inhibitor proteins from Escherichia coli and Pseudomonas aeruginosa.

PubMed

Liu, Zhihong; García-Díaz, Beatriz; Catacchio, Bruno; Chiancone, Emilia; Vogel, Hans J

2015-11-01

Lysozymes play an important role in host defense by degrading peptidoglycan in the cell envelopes of pathogenic bacteria. Several Gram-negative bacteria can evade this mechanism by producing periplasmic proteins that inhibit the enzymatic activity of lysozyme. The Escherichia coli inhibitor of vertebrate lysozyme, Ivyc and its Pseudomonas aeruginosa homolog, Ivyp1 have been shown to be potent inhibitors of hen egg white lysozyme (HEWL). Since human lysozyme (HL) plays an important role in the innate immune response, we have examined the binding of HL to Ivyc and Ivyp1. Our results show that Ivyp1 is a weaker inhibitor of HL than Ivyc even though they inhibit HEWL with similar potency. Calorimetry experiments confirm that Ivyp1 interacts more weakly with HL than HEWL. Analytical ultracentrifugation studies revealed that Ivyp1 in solution is a monomer and forms a 30kDa heterodimer with both HL and HEWL, while Ivyc is a homodimer that forms a tetramer with both enzymes. The interaction of Ivyp1 with HL was further characterized by NMR chemical shift perturbation experiments. In addition to the characteristic His-containing Ivy inhibitory loop that binds into the active site of lysozyme, an extended loop (P2) between the final two beta-strands also participates in forming protein-protein interactions. The P2 loop is not conserved in Ivyc and it constitutes a flexible region in Ivyp1 that becomes more rigid in the complex with HL. We conclude that differences in the electrostatic interactions at the binding interface between Ivy inhibitors and distinct lysozymes determine the strength of this interaction. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides. Copyright © 2015 Elsevier B.V. All rights reserved.

Recombineering reveals a diverse collection of ribosomal proteins L4 and L22 that confer resistance to macrolide antibiotics

PubMed Central

Diner, Elie J.; Hayes, Christopher S.

2009-01-01

Summary Mutations in ribosomal proteins L4 and L22 confer resistance to erythromycin and other macrolide antibiotics in a variety of bacteria. L4 and L22 have elongated loops whose tips converge in the peptide exit tunnel near the macrolide binding site, and resistance mutations typically affect residues within these loops. Here, we use bacteriophage λ Red-mediated recombination, or “recombineering”, to uncover new L4 and L22 alleles that confer macrolide resistance in Escherichia coli. We randomized residues at the tips of the L4 and L22 loops using recombineered oligonucleotide libraries, and selected the mutagenized cells for erythromycin-resistant mutants. These experiments led to the identification of 341 different resistance mutations encoding 278 unique L4 and L22 proteins – the overwhelming majority of which are novel. Many resistance mutations were complex, involving multiple missense mutations, in-frame deletions, and insertions. Transfer of L4 and L22 mutations into wild-type cells by phage P1-mediated transduction demonstrated that each allele was sufficient to confer macrolide resistance. Although L4 and L22 mutants are typically resistant to most macrolides, selections carried out on different antibiotics revealed macrolide-specific resistance mutations. L22 Lys90Trp is one such allele, which confers resistance to erythromycin, but not tylosin or spiramycin. Purified L22 Lys90Trp ribosomes show reduced erythromycin binding, but have the same affinity for tylosin as wild-type ribosomes. Moreover, DMS methylation protection assays demonstrated that L22 Lys90Trp ribosomes bind tylosin more readily than erythromycin in vivo. This work underscores the exceptional functional plasticity of the L4 and L22 proteins, and highlights the utility of Red-mediated recombination in targeted genetic selections. PMID:19150357

Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging

PubMed Central

Stewart, H.; Bingham, R.J.; White, S. J.; Dykeman, E. C.; Zothner, C.; Tuplin, A. K.; Stockley, P. G.; Twarock, R.; Harris, M.

2016-01-01

The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core-RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly. PMID:26972799

Quaking Is a Key Regulator of Endothelial Cell Differentiation, Neovascularization, and Angiogenesis

PubMed Central

Cochrane, Amy; Kelaini, Sophia; Tsifaki, Marianna; Bojdo, James; Vilà‐González, Marta; Drehmer, Daiana; Caines, Rachel; Magee, Corey; Eleftheriadou, Magdalini; Hu, Yanhua; Grieve, David; Stitt, Alan W.; Zeng, Lingfang; Xu, Qingbo

2017-01-01

Abstract The capability to derive endothelial cell (ECs) from induced pluripotent stem cells (iPSCs) holds huge therapeutic potential for cardiovascular disease. This study elucidates the precise role of the RNA‐binding protein Quaking isoform 5 (QKI‐5) during EC differentiation from both mouse and human iPSCs (hiPSCs) and dissects how RNA‐binding proteins can improve differentiation efficiency toward cell therapy for important vascular diseases. iPSCs represent an attractive cellular approach for regenerative medicine today as they can be used to generate patient‐specific therapeutic cells toward autologous cell therapy. In this study, using the model of iPSCs differentiation toward ECs, the QKI‐5 was found to be an important regulator of STAT3 stabilization and vascular endothelial growth factor receptor 2 (VEGFR2) activation during the EC differentiation process. QKI‐5 was induced during EC differentiation, resulting in stabilization of STAT3 expression and modulation of VEGFR2 transcriptional activation as well as VEGF secretion through direct binding to the 3′ UTR of STAT3. Importantly, mouse iPS‐ECs overexpressing QKI‐5 significantly improved angiogenesis and neovascularization and blood flow recovery in experimental hind limb ischemia. Notably, hiPSCs overexpressing QKI‐5, induced angiogenesis on Matrigel plug assays in vivo only 7 days after subcutaneous injection in SCID mice. These results highlight a clear functional benefit of QKI‐5 in neovascularization, blood flow recovery, and angiogenesis. Thus, they provide support to the growing consensus that elucidation of the molecular mechanisms underlying EC differentiation will ultimately advance stem cell regenerative therapy and eventually make the treatment of cardiovascular disease a reality. The RNA binding protein QKI‐5 is induced during EC differentiation from iPSCs. RNA binding protein QKI‐5 was induced during EC differentiation in parallel with the EC marker CD144. Immunofluorescence staining showing that QKI‐5 is localized in the nucleus and stained in parallel with CD144 in differentiated ECs (scale bar = 50 µm). stem cells 2017 Stem Cells 2017;35:952–966 PMID:28207177

Domain- and nucleotide-specific Rev response element regulation of feline immunodeficiency virus production

PubMed Central

Na, Hong; Huisman, Willem; Ellestad, Kristofor K.; Phillips, Tom R.; Power, Christopher

2010-01-01

Computational analysis of feline immunodeficiency virus (FIV) RNA sequences indicated that common FIV strains contain a rev response element (RRE) defined by a long unbranched hairpin with 6 stem-loop sub-domains, termed stem-loop A (SLA). To examine the role of the RNA secondary structure of the RRE, mutational analyses were performed in both an infectious FIV molecular clone and a FIV CAT-RRE reporter system. These studies disclosed that the stems within SLA (SA1, 2, 3, 4, and 5) of the RRE were critical but SA6 was not essential for FIV replication and CAT expression. These studies also revealed that the secondary structure rather than an antisense protein (ASP) mediates virus expression and replication in vitro. In addition, a single synonymous mutation within the FIV-RRE, SA3/45, reduced viral reverse transcriptase activity and p24 expression after transfection but in addition also showed a marked reduction in viral expression and production following infection. PMID:20570310

Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

PubMed

Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

2013-02-15

PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

Characterization of the Interaction of Sclerostin with the Low Density Lipoprotein Receptor-related Protein (LRP) Family of Wnt Co-receptors*

PubMed Central

Holdsworth, Gill; Slocombe, Patrick; Doyle, Carl; Sweeney, Bernadette; Veverka, Vaclav; Le Riche, Kelly; Franklin, Richard J.; Compson, Joanne; Brookings, Daniel; Turner, James; Kennedy, Jeffery; Garlish, Rachael; Shi, Jiye; Newnham, Laura; McMillan, David; Muzylak, Mariusz; Carr, Mark D.; Henry, Alistair J.; Ceska, Thomas; Robinson, Martyn K.

2012-01-01

LRP5 and LRP6 are proteins predicted to contain four six-bladed β-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the β-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another β-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways. PMID:22696217

YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase.

PubMed

Harris, Kimberly A; Jones, Victoria; Bilbille, Yann; Swairjo, Manal A; Agris, Paul F

2011-09-01

The post-transcriptional nucleoside modifications of tRNA's anticodon domain form the loop structure and dynamics required for effective and accurate recognition of synonymous codons. The N(6)-threonylcarbamoyladenosine modification at position 37 (t(6)A(37)), 3'-adjacent to the anticodon, of many tRNA species in all organisms ensures the accurate recognition of ANN codons by increasing codon affinity, enhancing ribosome binding, and maintaining the reading frame. However, biosynthesis of this complex modification is only partially understood. The synthesis requires ATP, free threonine, a single carbon source for the carbamoyl, and an enzyme yet to be identified. Recently, the universal protein family Sua5/YciO/YrdC was associated with t(6)A(37) biosynthesis. To further investigate the role of YrdC in t(6)A(37) biosynthesis, the interaction of the Escherichia coli YrdC with a heptadecamer anticodon stem and loop of lysine tRNA (ASL(Lys)(UUU)) was examined. YrdC bound the unmodified ASL(Lys)(UUU) with high affinity compared with the t(6)A(37)-modified ASL(Lys)(UUU) (K(d) = 0.27 ± 0.20 μM and 1.36 ± 0.39 μM, respectively). YrdC also demonstrated specificity toward the unmodified versus modified anticodon pentamer UUUUA and toward threonine and ATP. The protein did not significantly alter the ASL architecture, nor was it able to base flip A(37), as determined by NMR, circular dichroism, and fluorescence of 2-aminopuine at position 37. Thus, current data support the hypothesis that YrdC, with many of the properties of a putative threonylcarbamoyl transferase, most likely functions as a component of a heteromultimeric protein complex for t(6)A(37) biosynthesis.

Shrimp miRNAs regulate innate immune response against white spot syndrome virus infection.

PubMed

Kaewkascholkul, Napol; Somboonviwat, Kulwadee; Asakawa, Shuichi; Hirono, Ikuo; Tassanakajon, Anchalee; Somboonwiwat, Kunlaya

2016-07-01

MicroRNAs are short noncoding RNAs of RNA interference pathways that regulate gene expression through partial complementary base-pairing to target mRNAs. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon, were identified using next generation sequencing. Forty-six miRNA homologs were identified from WSSV-infected shrimp hemocyte. Stem-loop real-time RT-PCR analysis showed that 11 out of 16 selected miRNAs were differentially expressed upon WSSV infection. Of those, pmo-miR-315 and pmo-miR-750 were highly responsive miRNAs. miRNA target prediction revealed that the miRNAs were targeted at 5'UTR, ORF, and 3'UTR of several immune-related genes such as genes encoding antimicrobial peptides, signaling transduction proteins, heat shock proteins, oxidative stress proteins, proteinases or proteinase inhibitors, proteins in blood clotting system, apoptosis-related proteins, proteins in prophenoloxidase system, pattern recognition proteins and other immune molecules. The highly conserved miRNA homolog, pmo-bantam, was characterized for its function in shrimp. The pmo-bantam was predicted to target the 3'UTR of Kunitz-type serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was analyzed by luciferase reporter assay. Correlation of pmo-bantam and KuSPI expression was observed in lymphoid organ of WSSV-infected shrimp. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response. Copyright © 2016. Published by Elsevier Ltd.

Evidence for an RNA pseudoknot loop-helix interaction essential for efficient -1 ribosomal frameshifting.

PubMed

Liphardt, J; Napthine, S; Kontos, H; Brierley, I

1999-05-07

RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2. Copyright 1999 Academic Press.

Stepwise assembly of multiple Lin28 proteins on the terminal loop of let-7 miRNA precursors

PubMed Central

Desjardins, Alexandre; Bouvette, Jonathan; Legault, Pascale

2014-01-01

Lin28 inhibits the biogenesis of let-7 miRNAs through direct interactions with let-7 precursors. Previous studies have described seemingly inconsistent Lin28 binding sites on pre-let-7 RNAs. Here, we reconcile these data by examining the binding mechanism of Lin28 to the terminal loop of pre-let-7g (TL-let-7g) using biochemical and biophysical methods. First, we investigate Lin28 binding to TL-let-7g variants and short RNA fragments and identify three independent binding sites for Lin28 on TL-let-7g. We then determine that Lin28 assembles in a stepwise manner on TL-let-7g to form a stable 1:3 complex. We show that the cold-shock domain (CSD) of Lin28 is responsible for remodelling the terminal loop of TL-let-7g, whereas the NCp7-like domain facilitates the initial binding of Lin28 to TL-let-7g. This stable binding of multiple Lin28 molecules to the terminal loop of pre-let-7g extends to other precursors of the let-7 family, but not to other pre-miRNAs tested. We propose a model for stepwise assembly of the 1:1, 1:2 and 1:3 pre-let-7g/Lin28 complexes. Stepwise multimerization of Lin28 on pre-let-7 is required for maximum inhibition of Dicer cleavage for a least one member of the let-7 family and may be important for orchestrating the activity of the several factors that regulate let-7 biogenesis. PMID:24452802

RNA binding specificity of Ebola virus transcription factor VP30.

PubMed

Schlereth, Julia; Grünweller, Arnold; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K

2016-09-01

The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.

RosettaScripts: a scripting language interface to the Rosetta macromolecular modeling suite.

PubMed

Fleishman, Sarel J; Leaver-Fay, Andrew; Corn, Jacob E; Strauch, Eva-Maria; Khare, Sagar D; Koga, Nobuyasu; Ashworth, Justin; Murphy, Paul; Richter, Florian; Lemmon, Gordon; Meiler, Jens; Baker, David

2011-01-01

Macromolecular modeling and design are increasingly useful in basic research, biotechnology, and teaching. However, the absence of a user-friendly modeling framework that provides access to a wide range of modeling capabilities is hampering the wider adoption of computational methods by non-experts. RosettaScripts is an XML-like language for specifying modeling tasks in the Rosetta framework. RosettaScripts provides access to protocol-level functionalities, such as rigid-body docking and sequence redesign, and allows fast testing and deployment of complex protocols without need for modifying or recompiling the underlying C++ code. We illustrate these capabilities with RosettaScripts protocols for the stabilization of proteins, the generation of computationally constrained libraries for experimental selection of higher-affinity binding proteins, loop remodeling, small-molecule ligand docking, design of ligand-binding proteins, and specificity redesign in DNA-binding proteins.

CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sterjovski, Jasminka; Churchill, Melissa J.; Roche, Michael

2011-02-20

CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained bymore » the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.« less

Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models

NASA Astrophysics Data System (ADS)

Dodani, Sheel C.; Kiss, Gert; Cahn, Jackson K. B.; Su, Ye; Pande, Vijay S.; Arnold, Frances H.

2016-05-01

The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate.

Visualization of chromatin domains created by the gypsy insulator of Drosophila.

PubMed

Byrd, Keith; Corces, Victor G

2003-08-18

Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops.

Identification of a conserved 8 aa insert in the PIP5K protein in the Saccharomycetaceae family of fungi and the molecular dynamics simulations and structural analysis to investigate its potential functional role.

PubMed

Khadka, Bijendra; Gupta, Radhey S

2017-08-01

Homologs of the phosphatidylinositol-4-phosphate-5-kinase (PIP5K), which controls a multitude of essential cellular functions, contain a 8 aa insert in a conserved region that is specific for the Saccharomycetaceae family of fungi. Using structures of human PIP4K proteins as templates, structural models were generated of the Saccharomyces cerevisiae and human PIP5K proteins. In the modeled S. cerevisiae PIP5K, the 8 aa insert forms a surface exposed loop, present on the same face of the protein as the activation loop of the kinase domain. Electrostatic potential analysis indicates that the residues from 8 aa conserved loop form a highly positively charged surface patch, which through electrostatic interaction with the anionic portions of phospholipid head groups, is expected to play a role in the membrane interaction of the yeast PIP5K. To unravel this prediction, molecular dynamics (MD) simulations were carried out to examine the binding interaction of PIP5K, either containing or lacking the conserved signature insert, with two different membrane lipid bilayers. The results from MD studies provide insights concerning the mechanistic of interaction of PIP5K with lipid bilayer, and support the contention that the identified 8 aa conserved insert in fungal PIP5K plays an important role in the binding of this protein with membrane surface. Proteins 2017; 85:1454-1467. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole

NASA Astrophysics Data System (ADS)

Tsvetkov, Vladimir B.; Varizhuk, Anna M.; Pozmogova, Galina E.; Smirnov, Igor P.; Kolganova, Natalia A.; Timofeev, Edward N.

2015-11-01

In this study we describe new modified analogs of the thrombin binding aptamer (TBA) containing 5-nitroindole residues. It has been shown that all modified TBAs form an anti-parallel G-quadruplex structure and retain the ability to inhibit thrombin. The most advanced TBA variant (TBA-N8) has a substantially increased clotting time and two-fold lower IC50 value compared to the unmodified prototype. Molecular modelling studies suggest that the improved anticoagulant properties of TBA-N8 result from changes in the binding mode of the analog. A modified central loop in TBA-N8 is presumed to participate in the binding of the target protein. Studies of FAM labelled TBA and TBA-N8 showed an improved binding affinity of the modified aptamer and provided evidence of a direct interaction between the modified central loop and thrombin. Our findings have implications for the design of new aptamers with improved binding affinities.

A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole

PubMed Central

Tsvetkov, Vladimir B.; Varizhuk, Anna M.; Pozmogova, Galina E.; Smirnov, Igor P.; Kolganova, Natalia A.; Timofeev, Edward N.

2015-01-01

In this study we describe new modified analogs of the thrombin binding aptamer (TBA) containing 5-nitroindole residues. It has been shown that all modified TBAs form an anti-parallel G-quadruplex structure and retain the ability to inhibit thrombin. The most advanced TBA variant (TBA-N8) has a substantially increased clotting time and two-fold lower IC50 value compared to the unmodified prototype. Molecular modelling studies suggest that the improved anticoagulant properties of TBA-N8 result from changes in the binding mode of the analog. A modified central loop in TBA-N8 is presumed to participate in the binding of the target protein. Studies of FAM labelled TBA and TBA-N8 showed an improved binding affinity of the modified aptamer and provided evidence of a direct interaction between the modified central loop and thrombin. Our findings have implications for the design of new aptamers with improved binding affinities.

Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

NASA Technical Reports Server (NTRS)

He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

1992-01-01

The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

NASA Technical Reports Server (NTRS)

He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

1992-01-01

The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

Characterizing Solution Surface Loop Conformational Flexibility of the GM2 Activator Protein

PubMed Central

2015-01-01

GM2AP has a β-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL. PMID:25127419

Asymmetric processing of a substrate protein in sequential allosteric cycles of AAA+ nanomachines

NASA Astrophysics Data System (ADS)

Kravats, Andrea N.; Tonddast-Navaei, Sam; Bucher, Ryan J.; Stan, George

2013-09-01

Essential protein quality control includes mechanisms of substrate protein (SP) unfolding and translocation performed by powerful ring-shaped AAA+ (ATPases associated with various cellular activities) nanomachines. These SP remodeling actions are effected by mechanical forces imparted by AAA+ loops that protrude into the central channel. Sequential intra-ring allosteric motions, which underlie repetitive SP-loop interactions, have been proposed to comprise clockwise (CW), counterclockwise (CCW), or random (R) conformational transitions of individual AAA+ subunits. To probe the effect of these allosteric mechanisms on unfoldase and translocase functions, we perform Langevin dynamics simulations of a coarse-grained model of an all-alpha SP processed by the single-ring ClpY ATPase or by the double-ring p97 ATPase. We find that, in all three allosteric mechanisms, the SP undergoes conformational transitions along a common set of pathways, which reveals that the active work provided by the ClpY machine involves single loop-SP interactions. Nevertheless, the rates and yields of SP unfolding and translocation are controlled by mechanism-dependent loop-SP binding events, as illustrated by faster timescales of SP processing in CW allostery compared with CCW and R allostery. The distinct efficacy of allosteric mechanisms is due to the asymmetric collaboration of adjacent subunits, which involves CW-biased structural motions of AAA+ loops and results in CW-compatible torque applied onto the SP. Additional simulations of mutant ClpY rings, which render a subset of subunits catalytically-defective or reduce their SP binding affinity, reveal that subunit-based conformational transitions play the major role in SP remodeling. Based on these results we predict that the minimally functional AAA+ ring includes three active subunits, only two of which are adjacent.

Introduction of N-cadherin-binding motif to alginate hydrogels for controlled stem cell differentiation.

PubMed

Lee, Jae Won; An, Hyoseok; Lee, Kuen Yong

2017-07-01

Control of stem cell fate and phenotype using biomimetic synthetic extracellular matrices (ECMs) is an important tissue engineering approach. Many studies have focused on improving cell-matrix interactions. However, proper control of cell-cell interactions using synthetic ECMs could be critical for tissue engineering, especially with undifferentiated stem cells. In this study, alginate hydrogels were modified with a peptide derived from the low-density lipoprotein receptor-related protein 5 (LRP5), which is known to bind to N-cadherin, as a cell-cell interaction motif. In vitro changes in the morphology and differentiation of mouse bone marrow stromal cells (D1 stem cells) cultured in LRP5-alginate hydrogels were investigated. LRP5-alginate gels successfully induced stem cell aggregation and enhanced chondrogenic differentiation of D1 stem cells, compared to RGD-alginate gels, at low cell density. This approach to tailoring synthetic biomimetic ECMs using cell-cell interaction motifs may be critical in tissue engineering approaches using stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

The basic helix-loop-helix region of the transcriptional repressor hairy and enhancer of split 1 is preorganized to bind DNA.

PubMed

Popovic, Matija; Wienk, Hans; Coglievina, Maristella; Boelens, Rolf; Pongor, Sándor; Pintar, Alessandro

2014-04-01

Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions. Copyright © 2014 Wiley Periodicals, Inc.

The stoichiometry of the TMEM16A ion channel determined in intact plasma membranes of COS-7 cells using liquid-phase electron microscopy.

PubMed

Peckys, Diana B; Stoerger, Christof; Latta, Lorenz; Wissenbach, Ulrich; Flockerzi, Veit; de Jonge, Niels

2017-08-01

TMEM16A is a membrane protein forming a calcium-activated chloride channel. A homodimeric stoichiometry of the TMEM16 family of proteins has been reported but an important question is whether the protein resides always in a dimeric configuration in the plasma membrane or whether monomers of the protein are also present in its native state within in the intact plasma membrane. We have determined the stoichiometry of the human (h)TMEM16A within whole COS-7 cells in liquid. For the purpose of detecting TMEM16A subunits, single proteins were tagged by the streptavidin-binding peptide within extracellular loops accessible by streptavidin coated quantum dot (QD) nanoparticles. The labeled proteins were then imaged using correlative light microscopy and environmental scanning electron microscopy (ESEM) using scanning transmission electron microscopy (STEM) detection. The locations of 19,583 individual proteins were determined of which a statistical analysis using the pair correlation function revealed the presence of a dimeric conformation of the protein. The amounts of detected label pairs and single labels were compared between experiments in which the TMEM16A SBP-tag position was varied, and experiments in which tagged and non-tagged TMEM16A proteins were present. It followed that hTMEM16A resides in the plasma membrane as dimer only and is not present as monomer. This strategy may help to elucidate the stoichiometry of other membrane protein species within the context of the intact plasma membrane in future. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

PubMed Central

Buchko, Garry W.; Berg, Howard R.; Kaur, Jagdeep; Pandurangi, Raghu S.; Smith, Thomas J.; Shah, Dilip M.

2013-01-01

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β2 and β3 strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA. PMID:24324798

Structural dynamics of Casein Kinase I (CKI) from malarial parasite Plasmodium falciparum (Isolate 3D7): Insights from theoretical modelling and molecular simulations.

PubMed

Dehury, Budheswar; Behera, Santosh Kumar; Mahapatra, Namita

2017-01-01

The protein kinases (PKs), belonging to serine/threonine kinase (STKs), are important drug targets for a wide spectrum of diseases in human. Among protein kinases, the Casein Kinases (CKs) are vastly expanded in various organisms, where, the malarial parasite Plasmodium falciparum possesses a single member i.e., PfCKI, which can phosphorylate various proteins in parasite extracts in vitro condition. But, the structure-function relationship of PfCKI and dynamics of ATP binding is yet to be understood. Henceforth, an attempt was made to study the dynamics, stability, and ATP binding mechanisms of PfCKI through computational modelling, docking, molecular dynamics (MD) simulations, and MM/PBSA binding free energy estimation. Bi-lobed catalytic domain of PfCKI shares a high degree of secondary structure topology with CKI domains of rice, human, and mouse indicating co-evolution of these kinases. Molecular docking study revealed that ATP binds to the active site where the glycine-rich ATP-binding motif (G16-X-G18-X-X-G21) along with few conserved residues plays a crucial role maintaining stability of the complex. Structural superposition of PfCKI with close structural homologs depicted that the location and length of important loops are different, indicating the dynamic properties of these loops among CKIs, which is consistent with principal component analysis (PCA). PCA displayed that the overall global motion of ATP-bound form is comparatively higher than that of apo form. The present study provides insights into the structural features of PfCKI, which could contribute towards further understanding of related protein structures, dynamics of catalysis and phosphorylation mechanism in these important STKs from malarial parasite in near future. Copyright © 2016 Elsevier Inc. All rights reserved.

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors.

PubMed

Suebsuwong, Chalada; Pinkas, Daniel M; Ray, Soumya S; Bufton, Joshua C; Dai, Bing; Bullock, Alex N; Degterev, Alexei; Cuny, Gregory D

2018-02-15

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity.

PubMed

Jin, Lily L; Wybenga-Groot, Leanne E; Tong, Jiefei; Taylor, Paul; Minden, Mark D; Trudel, Suzanne; McGlade, C Jane; Moran, Michael F

2015-03-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of a Tetrameric Streptavidin Mutein with Reversible Biotin Binding Capability: Engineering a Mobile Loop as an Exit Door for Biotin

PubMed Central

O'Sullivan, Valerie J.; Barrette-Ng, Isabelle; Hommema, Eric; Hermanson, Greg T.; Schofield, Mark; Wu, Sau-Ching; Honetschlaeger, Claudia; Ng, Kenneth K.-S.; Wong, Sui-Lam

2012-01-01

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop3–4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop3–4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop7–8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop7–8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (koff of 4.28×10−4 s−1 and Kd of 1.9×10−8 M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible. PMID:22536357

A Small Stem Loop Structure Of The Ebola Virus Trailer Is Essential For Replication And Interacts With Heat Shock Protein A8

DTIC Science & Technology

2016-12-02

agarose gel electrophoresis TR-16-205 Nucleic Acids Research, 2016 3 (Seakem GTG , Sigma-Aldrich) and purified using the QI- Aquick Gel Extraction Kit... gtg +cga 1923-1938 3′ LNA2 +caa+aaa+ga+aa+gaa+gaa 3E-5E eGFP, 3E-5E plasmid containing enhanced green fluorescent protein; aiSHAPE, antisense-interfered

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

DOE PAGES

Hayashi, Yohei; Caboni, Laura; Das, Debanu; ...

2015-03-30

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutantsmore » based on the protein–DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings indicate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.« less

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

PubMed Central

Hayashi, Yohei; Caboni, Laura; Das, Debanu; Yumoto, Fumiaki; Clayton, Thomas; Deller, Marc C.; Nguyen, Phuong; Farr, Carol L.; Chiu, Hsiu-Ju; Miller, Mitchell D.; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Tomoda, Kiichiro; Conklin, Bruce R.; Wilson, Ian A.; Yamanaka, Shinya; Fletterick, Robert J.

2015-01-01

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein–DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering. PMID:25825768

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayashi, Yohei; Caboni, Laura; Das, Debanu

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutantsmore » based on the protein–DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings indicate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.« less

Computational study of Gleevec and G6G reveals molecular determinants of kinase inhibitor selectivity

DOE PAGES

Lin, Yen -Lin; Meng, Yilin; Huang, Lei; ...

2014-10-22

Gleevec is a potent inhibitor of Abl tyrosine kinase but not of the highly homologous c-Src kinase. Because the ligand binds to an inactive form of the protein in which an Asp-Phe-Gly structural motif along the activation loop adopts a so-called DFG-out conformation, it was suggested that binding specificity was controlled by a “conformational selection” mechanism. In this context, the binding affinity displayed by the kinase inhibitor G6G poses an intriguing challenge. Although it possesses a chemical core very similar to that of Gleevec, G6G is a potent inhibitor of both Abl and c-Src kinases. Both inhibitors bind to themore » DFG-out conformation of the kinases, which seems to be in contradiction with the conformational selection mechanism. To address this issue and display the hidden thermodynamic contributions affecting the binding selectivity, molecular dynamics free energy simulations with explicit solvent molecules were carried out. Relative to Gleevec, G6G forms highly favorable van der Waals dispersive interactions upon binding to the kinases via its triazine functional group, which is considerably larger than the corresponding pyridine moiety in Gleevec. Upon binding of G6G to c-Src, these interactions offset the unfavorable free energy cost of the DFG-out conformation. When binding to Abl, however, G6G experiences an unfavorable free energy penalty due to steric clashes with the phosphate-binding loop, yielding an overall binding affinity that is similar to that of Gleevec. Such steric clashes are absent when G6G binds to c-Src, due to the extended conformation of the phosphate-binding loop.« less

D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function

PubMed Central

Burtscher, Laura; Hajdu, Dorottya; Muñoz, Alberto; Gáspári, Zoltán; Read, Nick D.; Batta, Gyula; Marx, Florentine

2017-01-01

The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF. PMID:28072824

Crystal structure of the TRIM25 B30.2 (PRYSPRY) domain: a key component of antiviral signalling.

PubMed

D'Cruz, Akshay A; Kershaw, Nadia J; Chiang, Jessica J; Wang, May K; Nicola, Nicos A; Babon, Jeffrey J; Gack, Michaela U; Nicholson, Sandra E

2013-12-01

TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen β-strands arranged into two β-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp(488) and Trp(621)) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs.

Crystal structure of the TRIM25 B30.2 (PRYSPRY) domain: a key component of antiviral signalling

PubMed Central

D'Cruz, Akshay A.; Kershaw, Nadia J.; Chiang, Jessica J.; Wang, May K.; Nicola, Nicos A.; Babon, Jeffrey J.; Gack, Michaela U.; Nicholson, Sandra E.

2014-01-01

TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen β-strands arranged into two β-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp488 and Trp621) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs. PMID:24015671

Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated.

PubMed Central

Hoe, N P; Goguen, J D

1993-01-01

The lcrF gene of Yersinia pestis encodes a transcription activator responsible for inducing expression of several virulence-related proteins in response to temperature. The mechanism of this thermoregulation was investigated. An lcrF clone was found to produce much lower levels of LcrF protein at 26 than at 37 degrees C in Y. pestis, although it was transcribed at similar levels at both temperatures. High-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at 26 and 37 degrees C, suggesting that translation rate or message degradation is thermally controlled. The lcrF mRNA appears to be highly unstable and could not be reliably detected in Y. pestis. Insertion of the lcrF gene into plasmid pET4a, which produces high levels of plasmid-length RNA, aided detection of lcrF-specific message in E. coli. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37 degrees C indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented. Images PMID:7504666

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shetty, Nishant D.; Reddy, Manchi C.M.; Palaninathan, Satheesh K.

2010-10-11

PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 {angstrom} resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T-loop ofmore » one subunit and the C-loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T-loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T-loop makes direct contact with the {gamma}-phosphate of the ATP molecule replacing the Mg{sup 2+} position seen in the Methanococcus jannaschii GlnK1 structure. The C-loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C-terminal 3{sub 10} helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.« less

The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression

PubMed Central

Garay-Arroyo, Adriana; Ortiz-Moreno, Enrique; de la Paz Sánchez, María; Murphy, Angus S; García-Ponce, Berenice; Marsch-Martínez, Nayelli; de Folter, Stefan; Corvera-Poiré, Adriana; Jaimes-Miranda, Fabiola; Pacheco-Escobedo, Mario A; Dubrovsky, Joseph G; Pelaz, Soraya; Álvarez-Buylla, Elena R

2013-01-01

Elucidating molecular links between cell-fate regulatory networks and dynamic patterning modules is a key for understanding development. Auxin is important for plant patterning, particularly in roots, where it establishes positional information for cell-fate decisions. PIN genes encode plasma membrane proteins that serve as auxin efflux transporters; mutations in members of this gene family exhibit smaller roots with altered root meristems and stem-cell patterning. Direct regulators of PIN transcription have remained elusive. Here, we establish that a MADS-box gene (XAANTAL2, XAL2/AGL14) controls auxin transport via PIN transcriptional regulation during Arabidopsis root development; mutations in this gene exhibit altered stem-cell patterning, root meristem size, and root growth. XAL2 is necessary for normal shootward and rootward auxin transport, as well as for maintaining normal auxin distribution within the root. Furthermore, this MADS-domain transcription factor upregulates PIN1 and PIN4 by direct binding to regulatory regions and it is required for PIN4-dependent auxin response. In turn, XAL2 expression is regulated by auxin levels thus establishing a positive feedback loop between auxin levels and PIN regulation that is likely to be important for robust root patterning. PMID:24121311

Allostery in the ferredoxin protein motif does not involve a conformational switch.

PubMed

Nechushtai, Rachel; Lammert, Heiko; Michaeli, Dorit; Eisenberg-Domovich, Yael; Zuris, John A; Luca, Maria A; Capraro, Dominique T; Fish, Alex; Shimshon, Odelia; Roy, Melinda; Schug, Alexander; Whitford, Paul C; Livnah, Oded; Onuchic, José N; Jennings, Patricia A

2011-02-08

Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain plant-type ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the iron-sulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.

Dynamics of human protein kinase Aurora A linked to drug selectivity

DOE PAGES

Pitsawong, Warintra; Buosi, Vanessa; Otten, Renee; ...

2018-06-14

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Auroramore » A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.« less

Dynamics of human protein kinase Aurora A linked to drug selectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pitsawong, Warintra; Buosi, Vanessa; Otten, Renee

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Auroramore » A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.« less

Prenatal arsenic exposure alters REST/NRSF and microRNA regulators of embryonic neural stem cell fate in a sex-dependent manner

PubMed Central

Tyler, Christina R.; Labrecque, Matthew T.; Solomon, Elizabeth R.; Guo, Xun; Allan, Andrea M.

2016-01-01

Exposure to arsenic, a common environmental toxin found in drinking water, leads to a host of neurological pathologies. We have previously demonstrated that developmental exposure to a low level of arsenic (50 ppb) alters epigenetic processes that underlie deficits in adult hippocampal neurogenesis leading to aberrant behavior. It is unclear if arsenic impacts the programming and regulation of embryonic neurogenesis during development when exposure occurs. The master negative regulator of neural-lineage, REST/NRSF, controls the precise timing of fate specification and differentiation of neural stem cells (NSCs). Early in development (embryonic day 14), we observed increased expression of Rest, its co-repressor, CoREST, and the inhibitory RNA binding/splicing protein, Ptbp1, and altered expression of mRNA spliced isoforms of Pbx1 that are directly regulated by these factors in the male brain in response to prenatal 50 ppb arsenic exposure. These increases were concurrent with decreased expression of microRNA-9 (miR-9), miR-9*, and miR-124, all of which are REST/NRSF targets and inversely regulate Rest expression to allow for maturation of NSCs. Exposure to arsenic decreased the formation of neuroblasts in vitro from NSCs derived from male pup brains. The female response to arsenic was limited to increased expression of CoREST and Ptbp2, an RNA binding protein that allows for appropriate splicing of genes involved in the progression of neurogenesis. These changes were accompanied by increased neuroblast formation in vitro from NSCs derived from female pups. Unexposed male mice express transcriptomic factors to induce differentiation earlier in development compared to unexposed females. Thus, arsenic exposure likely delays differentiation of NSCs in males while potentially inducing precocious differentiation in females early in development. These effects are mitigated by embryonic day 18 of development. Arsenic-induced dysregulation of the regulatory loop formed by REST/NRSF, its target microRNAs, miR-9 and miR-124, and RNA splicing proteins, PTBP1 and 2, leads to aberrant programming of NSC function that is perhaps perpetuated into adulthood inducing deficits in differentiation we have previously observed. PMID:27751817

Eye patches: Protein assembly of index-gradient squid lenses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, J.; Townsend, J. P.; Dodson, T. C.

A parabolic relationship between lens radius and refractive index allows spherical lenses to avoid spherical aberration. We show that in squid, patchy colloidal physics resulted from an evolutionary radiation of globular S-crystallin proteins. Small-angle x-ray scattering experiments on lens tissue show colloidal gels of S-crystallins at all radial positions. Sparse lens materials form via low-valence linkages between disordered loops protruding from the protein surface. The loops are polydisperse and bind via a set of hydrogen bonds between disordered side chains. Peripheral lens regions with low particle valence form stable, volume-spanning gels at low density, whereas central regions with higher averagemore » valence gel at higher densities. The proteins demonstrate an evolved set of linkers for self-assembly of nanoparticles into volumetric materials.« less

SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain

PubMed Central

Kozlowski, Maya; Larose, Louise; Lee, Fai; Le, Duc Mingh; Rottapel, Robert; Siminovitch, Katherine A.

1998-01-01

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation. PMID:9528781

The Sequence-specific Peptide-binding Activity of the Protein Sulfide Isomerase AGR2 Directs Its Stable Binding to the Oncogenic Receptor EpCAM.

PubMed

Mohtar, M Aiman; Hernychova, Lenka; O'Neill, J Robert; Lawrence, Melanie L; Murray, Euan; Vojtesek, Borek; Hupp, Ted R

2018-04-01

AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Organic bioelectronics probing conformational changes in surface confined proteins

NASA Astrophysics Data System (ADS)

Macchia, Eleonora; Alberga, Domenico; Manoli, Kyriaki; Mangiatordi, Giuseppe F.; Magliulo, Maria; Palazzo, Gerardo; Giordano, Francesco; Lattanzi, Gianluca; Torsi, Luisa

2016-06-01

The study of proteins confined on a surface has attracted a great deal of attention due to its relevance in the development of bio-systems for laboratory and clinical settings. In this respect, organic bio-electronic platforms can be used as tools to achieve a deeper understanding of the processes involving protein interfaces. In this work, biotin-binding proteins have been integrated in two different organic thin-film transistor (TFT) configurations to separately address the changes occurring in the protein-ligand complex morphology and dipole moment. This has been achieved by decoupling the output current change upon binding, taken as the transducing signal, into its component figures of merit. In particular, the threshold voltage is related to the protein dipole moment, while the field-effect mobility is associated with conformational changes occurring in the proteins of the layer when ligand binding occurs. Molecular Dynamics simulations on the whole avidin tetramer in presence and absence of ligands were carried out, to evaluate how the tight interactions with the ligand affect the protein dipole moment and the conformation of the loops surrounding the binding pocket. These simulations allow assembling a rather complete picture of the studied interaction processes and support the interpretation of the experimental results.

Organic bioelectronics probing conformational changes in surface confined proteins

PubMed Central

Macchia, Eleonora; Alberga, Domenico; Manoli, Kyriaki; Mangiatordi, Giuseppe F.; Magliulo, Maria; Palazzo, Gerardo; Giordano, Francesco; Lattanzi, Gianluca; Torsi, Luisa

2016-01-01

The study of proteins confined on a surface has attracted a great deal of attention due to its relevance in the development of bio-systems for laboratory and clinical settings. In this respect, organic bio-electronic platforms can be used as tools to achieve a deeper understanding of the processes involving protein interfaces. In this work, biotin-binding proteins have been integrated in two different organic thin-film transistor (TFT) configurations to separately address the changes occurring in the protein-ligand complex morphology and dipole moment. This has been achieved by decoupling the output current change upon binding, taken as the transducing signal, into its component figures of merit. In particular, the threshold voltage is related to the protein dipole moment, while the field-effect mobility is associated with conformational changes occurring in the proteins of the layer when ligand binding occurs. Molecular Dynamics simulations on the whole avidin tetramer in presence and absence of ligands were carried out, to evaluate how the tight interactions with the ligand affect the protein dipole moment and the conformation of the loops surrounding the binding pocket. These simulations allow assembling a rather complete picture of the studied interaction processes and support the interpretation of the experimental results. PMID:27312768

Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin

Highlights: Black-Right-Pointing-Pointer CBP sequence is identified from BSP and has collagen binding activity. Black-Right-Pointing-Pointer CBP directly activates the MAPK signaling, especially ERK1/2. Black-Right-Pointing-Pointer CBP increase osteoblastic differentiation by the activation of Runx2. Black-Right-Pointing-Pointer CBP decrease adipogenic differentiation by the inhibition of PPAR{gamma}. -- Abstract: Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Severalmore » recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor {gamma}. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken together, a novel CBP could be a useful candidate for regenerating bone and treating osteoporosis, which result from an imbalance in osteogenesis and adipogenesis differentiation.« less

Regulation of Plant Microprocessor Function in Shaping microRNA Landscape.

PubMed

Dolata, Jakub; Taube, Michał; Bajczyk, Mateusz; Jarmolowski, Artur; Szweykowska-Kulinska, Zofia; Bielewicz, Dawid

2018-01-01

MicroRNAs are small molecules (∼21 nucleotides long) that are key regulators of gene expression. They originate from long stem-loop RNAs as a product of cleavage by a protein complex called Microprocessor. The core components of the plant Microprocessor are the RNase type III enzyme Dicer-Like 1 (DCL1), the zinc finger protein Serrate (SE), and the double-stranded RNA binding protein Hyponastic Leaves 1 (HYL1). Microprocessor assembly and its processing of microRNA precursors have been reported to occur in discrete nuclear bodies called Dicing bodies. The accessibility of and modifications to Microprocessor components affect microRNA levels and may have dramatic consequences in plant development. Currently, numerous lines of evidence indicate that plant Microprocessor activity is tightly regulated. The cellular localization of HYL1 is dependent on a specific KETCH1 importin, and the E3 ubiquitin ligase COP1 indirectly protects HYL1 from degradation in a light-dependent manner. Furthermore, proper localization of HYL1 in Dicing bodies is regulated by MOS2. On the other hand, the Dicing body localization of DCL1 is regulated by NOT2b, which also interacts with SE in the nucleus. Post-translational modifications are substantial factors that contribute to protein functional diversity and provide a fine-tuning system for the regulation of protein activity. The phosphorylation status of HYL1 is crucial for its activity/stability and is a result of the interplay between kinases (MPK3 and SnRK2) and phosphatases (CPL1 and PP4). Additionally, MPK3 and SnRK2 are known to phosphorylate SE. Several other proteins (e.g., TGH, CDF2, SIC, and RCF3) that interact with Microprocessor have been found to influence its RNA-binding and processing activities. In this minireview, recent findings on the various modes of Microprocessor activity regulation are discussed.

A small modified hammerhead ribozyme and its conformational characteristics determined by mutagenesis and lattice calculation.

PubMed Central

Lustig, B; Lin, N H; Smith, S M; Jernigan, R L; Jeang, K T

1995-01-01

A prototypic hammerhead ribozyme has three helices that surround an asymmetrical central core loop. We have mutagenized a hammerhead type ribozyme. In agreement with previous studies, progressive removal of stem-loop II from a three stemmed ribozyme showed that this region is not absolutely critical for catalysis. However, complete elimination of stem II and its loop did reduce, but did not eliminate, function. In a stem-loop II-deleted ribozyme, activity was best preserved when a purine, preferably a G, was present at position 10.1. This G contributed to catalysis irregardless of its role as either one part of a canonical pair with a C residue at 11.1 or a lone nucleotide with C (11.1) deleted. Computational methods using lattices generated 87 million three-dimensional chain forms for a stem-loop II-deleted RNA complex that preserved one potential G.C base pair at positions 10.1 and 11.1. This exhaustive set of chain forms included one major class of structures with G(10.1) being spatially proximal to the GUCX cleavage site of the substrate strand. Strong correlations were observed between colinear arrangement of stems I and III, constraints of base-pairing in the central core loop, and one particular placement of G(10.1) relative to the cleavage site. Our calculations of a stem-loop II-deleted ribozyme indicate that without needing to invoke any other constraints, the inherent asymmetry in the lengths of the two loop strands (3 nt in one and 7 nt in the other) that compose the core and flank G10.1-C11.1 stipulated strongly this particular G placement. This suggests that the hammerhead ribozyme maintains an asymmetry in its internal loop for a necessary structure/function reason. Images PMID:7567466

Influence of dynamic flow conditions on adsorbed plasma protein corona and surface-induced thrombus generation on antifouling brushes.

PubMed

Yu, Kai; Andruschak, Paula; Yeh, Han Hung; Grecov, Dana; Kizhakkedathu, Jayachandran N

2018-06-01

The information regarding the nature of protein corona (and its changes) and cell binding on biomaterial surface under dynamic conditions is critical to dissect the mechanism of surface-induced thrombosis. In this manuscript, we investigated the nature of protein corona and blood cell binding in heparinized recalcified human plasma, platelet rich plasma and whole blood on three highly hydrophilic antifouling polymer brushes, (poly(N, N-dimethylacrylamide) (PDMA), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) using an in vitro blood loop model at comparable arterial and venous flow, and static conditions. A fluid dynamics model was used initially to better understand the resulting flow patterns in a vertical channel containing the substrates to arrive at the placement of the substrates within the blood loop. The protein binding on the brush modified substrates was determined using ellipsometry, fluorescence microscopy and the nature of the protein corona was investigated using mass spectrometry based proteomics. The flow elevated fouling on brush coated surface from blood. The extent of plasma protein adsorption and platelet adhesion onto PDMA brush was lower than other surfaces in both static and flow conditions. The profiles of adsorbed protein corona showed strong dependence on the test conditions (static vs. flow), and the chemistry of the polymer brushes. Specially, the PDMA brush under flow conditions was more enriched with coagulation proteins, complement proteins, vitronectin and fibronectin but was less enriched with serum albumin. Apolipoprotein B-100 and complement proteins were the most abundant proteins seen on PMPC and PHPMA surfaces under both flow and static conditions, respectively. Unlike PDMA brush, the flow conditions did not affect the composition of protein corona on PMPC and PHPMA brushes. The nature of the protein corona formed in flow conditions influenced the platelet and red blood cell binding. The dependence of shear stress on platelet adhesion from platelet rich plasma and whole blood highlights the contribution of red blood cells in enhancing platelet adhesion on the surface under high shear condition. Copyright © 2018 Elsevier Ltd. All rights reserved.

Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

PubMed Central

Mitra, Sayantan; Zhu, Wanlong; Qin, Haina; Pasquale, Elena B.; Song, Jianxing

2013-01-01

The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll β-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD) simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD), which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J–K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity might be to target specific dynamic states of the Eph receptor LBDs. PMID:24086308

Epithelial-mesenchymal transition and tumor suppression are controlled by a reciprocal feedback loop between ZEB1 and Grainyhead-like-2

PubMed Central

Cieply, Benjamin; Farris, Joshua; Denvir, James; Ford, Heide; Frisch, Steven M.

2013-01-01

Epithelial-mesenchymal transition (EMT) in carcinoma cells enhances malignant progression by promoting invasion and survival. EMT is induced by microenvironmental factors including TGF-β and Wnt agonists, and by the E-box-binding transcription factors Twist, Snail and ZEB. Grainyhead-like-2 (GRHL2), a member of the mammalian Grainyhead family of wound healing regulatory transcription factors, suppresses EMT and restores sensitivity to anoikis by repressing ZEB1 expression and inhibiting TGF-β signaling. In this study, we elucidate the functional relationship between GRHL2 and ZEB1 in EMT/MET and tumor biology. At least three homeodomain proteins, Six1, LBX1, and HoxA5, transactivated the ZEB1 promoter, in the case of Six1, through direct protein-promoter interaction. GRHL2 altered the Six1-DNA complex, inhibiting this transactivation. Correspondingly, GRHL2 expression prevented tumor initiation in xenograft assays, sensitized breast cancer cells to paclitaxel and suppressed the emergence of CD44highCD24low cells (defining the cancer stem cell phenotype in the cell type studied). GRHL2 was down-regulated in recurrent mouse tumors that had evolved to an oncogene-independent, EMT-like state, supporting a role for GRHL2 down-regulation in this phenotypic transition, modeling disease recurrence. The combination of TGF-β and Wnt activation repressed GRHL2 expression by direct interaction of ZEB1 with the GRHL2 promoter, inducing EMT. Together, our observations indicate that a reciprocal feedback loop between GRHL2 and ZEB1 controls epithelial vs. mesenchymal phenotypes and EMT-driven tumor progression. PMID:23943797

Thermodynamics and NMR studies on Duck, Heron and Human HBV encapsidation signals

PubMed Central

Girard, Frederic C.; Ottink, Otmar M.; Ampt, Kirsten A.M.; Tessari, Marco; Wijmenga, Sybren S.

2007-01-01

Hepatitis B virus (HBV) replication is initiated by binding of its reverse transcriptase (P) to the apical stem-loop (AL) and primer loop (PL) of epsilon, a highly conserved RNA element at the 5′-end of the RNA pregenome. Mutation studies on duck/heron and human in vitro systems have shown similarities but also differences between their P–epsilon interaction. Here, NMR and UV thermodynamic data on AL (and PL) from these three species are presented. The stabilities of the duck and heron ALs were found to be similar, and much lower than that of human. NMR data show that this low stability stems from an 11-nt internal bulge destabilizing the stem of heron AL. In duck, although structured at low temperature, this region also forms a weak point as its imino resonances broaden to disappearance between 30 and 35°C well below the overall AL melting temperature. Surprisingly, the duck- and heron ALs were both found to be capped by a stable well-structured UGUU tetraloop. All avian ALs are expected to adhere to this because of their conserved sequence. Duck PL is stable and structured and, in view of sequence similarities, the same is expected for heron - and human PL. PMID:17430968

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin.

PubMed

Elgeti, Matthias; Kazmin, Roman; Rose, Alexander S; Szczepek, Michal; Hildebrand, Peter W; Bartl, Franz J; Scheerer, Patrick; Hofmann, Klaus Peter

2018-03-23

Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G t ) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the G t α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, G t α-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

PubMed Central

Owczarek, C M; Layton, M J; Metcalf, D; Lock, P; Willson, T A; Gough, N M; Nicola, N A

1993-01-01

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins. Images PMID:8253075

miR-21 promotes the differentiation of hair follicle-derived neural crest stem cells into Schwann cells

PubMed Central

Ni, Yuxin; Zhang, Kaizhi; Liu, Xuejuan; Yang, Tingting; Wang, Baixiang; Fu, Li; A, Lan; Zhou, Yanmin

2014-01-01

Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair follicles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA (miR)-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist (agomir-21), the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist (antagomir-21), the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3′-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regulating SOX protein expression by binding to the 3′-UTR of SOX2 mRNA. PMID:25206896

RNA-Binding Protein L1TD1 Interacts with LIN28 via RNA and is Required for Human Embryonic Stem Cell Self-Renewal and Cancer Cell Proliferation

PubMed Central

Närvä, Elisa; Rahkonen, Nelly; Emani, Maheswara Reddy; Lund, Riikka; Pursiheimo, Huha-Pekka; Nästi, Juuso; Autio, Reija; Rasool, Omid; Denessiouk, Konstantin; Lähdesmäki, Harri; Rao, Anjana; Lahesmaa, Ritta

2012-01-01

Human embryonic stem cells (hESC) have a unique capacity to self-renew and differentiate into all the cell types found in human body. Although the transcriptional regulators of pluripotency are well studied, the role of cytoplasmic regulators is still poorly characterized. Here, we report a new stem cell-specific RNA-binding protein L1TD1 (ECAT11, FLJ10884) required for hESC self-renewal and cancer cell proliferation. Depletion of L1TD1 results in immediate downregulation of OCT4 and NANOG. Furthermore, we demonstrate that OCT4, SOX2, and NANOG all bind to the promoter of L1TD1. Moreover, L1TD1 is highly expressed in seminomas, and depletion of L1TD1 in these cancer cells influences self-renewal and proliferation. We show that L1TD1 colocalizes and interacts with LIN28 via RNA and directly with RNA helicase A (RHA). LIN28 has been reported to regulate translation of OCT4 in complex with RHA. Thus, we hypothesize that L1TD1 is part of the L1TD1-RHA-LIN28 complex that could influence levels of OCT4. Our results strongly suggest that L1TD1 has an important role in the regulation of stemness. PMID:22162396

The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding to Metabolic Intermediates*

PubMed Central

Casañal, Ana; Zander, Ulrich; Muñoz, Cristina; Dupeux, Florine; Luque, Irene; Botella, Miguel Angel; Schwab, Wilfried; Valpuesta, Victoriano; Marquez, José A.

2013-01-01

Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low μm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates. PMID:24133217

Imino proton exchange and base-pair kinetics in the AMP-RNA aptamer complex.

PubMed

Nonin, S; Jiang, F; Patel, D J

1997-05-02

We report on the dynamics of base-pair opening in the ATP-binding asymmetric internal loop and flanking base-pairs of the AMP-RNA aptamer complex by monitoring the exchange characteristics of the extremely well resolved imino protons in the NMR spectrum of the complex. The kinetics of imino proton exchange as a function of basic pH or added ammonia catalyst are used to measure the apparent base-pair dissociation constants and lifetimes of Watson-Crick and mismatched base-pairs, as well as the solvent accessibility of the unpaired imino protons in the complex. The exchange characteristics of the imino protons identify the existence of four additional hydrogen bonds stabilizing the conformation of the asymmetric ATP-binding internal loop that were not detected by NOEs and coupling constants alone, but are readily accommodated in the previously reported solution structure of the AMP-RNA aptamer complex published from our laboratory. The hydrogen exchange kinetics of the non-Watson-Crick pairs in the asymmetric internal loop of the AMP-RNA aptamer complex have been characterized and yield apparent dissociation constants (alphaKd) that range from 10(-2) to 10(-7). Surprisingly, three of these alphaKd values are amongst the lowest measured for all base-pairs in the AMP-RNA aptamer complex. Comparative studies of hydrogen exchange of the imino protons in the free RNA aptamer and the AMP-RNA aptamer complex establish that complexation stabilizes not only the bases within the ATP-binding asymmetric internal loop, but also the flanking stem base-pairs (two pairs on either side) of the binding site. We also outline some preliminary results related to the exchange properties of a sugar 2'-hydroxyl proton of a guanosine residue involved in a novel hydrogen bond that has been shown to contribute to the immobilization of the bound AMP by the RNA aptamer, and whose resonance is narrow and downfield shifted in the spectrum.

Principles for computational design of binding antibodies

PubMed Central

Pszolla, M. Gabriele; Lapidoth, Gideon D.; Norn, Christoffer; Dym, Orly; Unger, Tamar; Albeck, Shira; Tyka, Michael D.; Fleishman, Sarel J.

2017-01-01

Natural proteins must both fold into a stable conformation and exert their molecular function. To date, computational design has successfully produced stable and atomically accurate proteins by using so-called “ideal” folds rich in regular secondary structures and almost devoid of loops and destabilizing elements, such as cavities. Molecular function, such as binding and catalysis, however, often demands nonideal features, including large and irregular loops and buried polar interaction networks, which have remained challenging for fold design. Through five design/experiment cycles, we learned principles for designing stable and functional antibody variable fragments (Fvs). Specifically, we (i) used sequence-design constraints derived from antibody multiple-sequence alignments, and (ii) during backbone design, maintained stabilizing interactions observed in natural antibodies between the framework and loops of complementarity-determining regions (CDRs) 1 and 2. Designed Fvs bound their ligands with midnanomolar affinities and were as stable as natural antibodies, despite having >30 mutations from mammalian antibody germlines. Furthermore, crystallographic analysis demonstrated atomic accuracy throughout the framework and in four of six CDRs in one design and atomic accuracy in the entire Fv in another. The principles we learned are general, and can be implemented to design other nonideal folds, generating stable, specific, and precise antibodies and enzymes. PMID:28973872

Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.

PubMed

Horton, Sarah J; Giotopoulos, George; Yun, Haiyang; Vohra, Shabana; Sheppard, Olivia; Bashford-Rogers, Rachael; Rashid, Mamunur; Clipson, Alexandra; Chan, Wai-In; Sasca, Daniel; Yiangou, Loukia; Osaki, Hikari; Basheer, Faisal; Gallipoli, Paolo; Burrows, Natalie; Erdem, Ayşegül; Sybirna, Anastasiya; Foerster, Sarah; Zhao, Wanfeng; Sustic, Tonci; Petrunkina Harrison, Anna; Laurenti, Elisa; Okosun, Jessica; Hodson, Daniel; Wright, Penny; Smith, Ken G; Maxwell, Patrick; Fitzgibbon, Jude; Du, Ming Q; Adams, David J; Huntly, Brian J P

2017-09-01

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.

Mechanism of nucleotide sensing in group II chaperonins.

PubMed

Pereira, Jose H; Ralston, Corie Y; Douglas, Nicholai R; Kumar, Ramya; Lopez, Tom; McAndrew, Ryan P; Knee, Kelly M; King, Jonathan A; Frydman, Judith; Adams, Paul D

2012-02-01

Group II chaperonins mediate protein folding in an ATP-dependent manner in eukaryotes and archaea. The binding of ATP and subsequent hydrolysis promotes the closure of the multi-subunit rings where protein folding occurs. The mechanism by which local changes in the nucleotide-binding site are communicated between individual subunits is unknown. The crystal structure of the archaeal chaperonin from Methanococcus maripaludis in several nucleotides bound states reveals the local conformational changes associated with ATP hydrolysis. Residue Lys-161, which is extremely conserved among group II chaperonins, forms interactions with the γ-phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ-phosphate interaction with Lys-161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160-169. We propose that Lys-161 functions as an ATP sensor and that 160-169 constitutes a nucleotide-sensing loop (NSL) that monitors the presence of the γ-phosphate. Functional analysis using NSL mutants shows a significant decrease in ATPase activity, suggesting that the NSL is involved in timing of the protein folding cycle.

Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly

PubMed Central

Chereji, Răzvan V.; Bharatula, Vasudha; Elfving, Nils; Blomberg, Jeanette; Larsson, Miriam; Morozov, Alexandre V.; Broach, James R.

2017-01-01

Abstract Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Saccharomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3′-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization. PMID:28575439

Biofunctionalization of silica-coated magnetic particles mediated by a peptide

NASA Astrophysics Data System (ADS)

Care, Andrew; Chi, Fei; Bergquist, Peter L.; Sunna, Anwar

2014-08-01

A linker peptide sequence with affinity to silica-containing materials was fused to Streptococcus protein G', an antibody-binding protein. This recombinant fusion protein, linker-protein G (LPG) was produced in E. coli and exhibited strong affinity to silica-coated magnetic particles and was able to bind to them at different pHs, indicating a true pH-independent binding. LPG was used as an anchorage point for the oriented immobilization of antibodies onto the surface of the particles. These particle-bound "LPG-Antibody complexes" mediated the binding and recovery of different cell types (e.g., human stem cells, Legionella, Cryptosporidium and Giardia), enabling their rapid and simple visualization and identification. This strategy was used also for the efficient capture of Cryptosporidium oocysts from water samples. These results demonstrate that LPG can mediate the direct biofunctionalization of silica-coated magnetic particles without the need for complex surface chemical modification.

A vicious loop of fatty acid-binding protein 4 and DNA methyltransferase 1 promotes acute myeloid leukemia and acts as a therapeutic target

PubMed Central

Yan, F; Shen, N; Pang, JX; Zhao, N; Zhang, YW; Bode, AM; Al-Kali, A; Litzow, MR; Li, B; Liu, SJ

2017-01-01

Aberrant DNA methylation mediated by deregulation of DNA methyltransferases (DNMT) is a key hallmark of acute myeloid leukemia (AML), yet efforts to target DNMT deregulation for drug development have lagged. We previously demonstrated that upregulation of fatty acid-binding protein 4 (FABP4) promotes AML aggressiveness through enhanced DNMT1-dependent DNA methylation. Here we demonstrate that FABP4 upregulation in AML cells occurs through vascular endothelial growth factor (VEGF) signaling, thus elucidating a crucial FABP4-DNMT1 regulatory feedback loop in AML biology. We show that FABP4 dysfunction by its selective inhibitor BMS309403 leads to downregulation of DNMT1, decrease of global DNA methylation and re-expression of p15INK4B tumor suppressor gene by promoter DNA hypomethylation in vitro, ex vivo and in vivo. Functionally, BMS309403 suppresses cell colony formation, induces cell differentiation, and, importantly, impairs leukemic disease progression in mouse models of leukemia. Our findings highlight AML-promoting properties of the FABP4-DNMT1 vicious loop, and identify an attractive class of therapeutic agents with a high potential for clinical use in AML patients. The results will also assist in establishing the FABP4-DNMT1 loop as a target for therapeutic discovery to enhance the index of current epigenetic therapies. PMID:28993705

Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor*

PubMed Central

Tong, Yufeng; Hota, Prasanta K.; Penachioni, Junia Y.; Hamaneh, Mehdi B.; Kim, SoonJeung; Alviani, Rebecca S.; Shen, Limin; He, Hao; Tempel, Wolfram; Tamagnone, Luca; Park, Hee-Won; Buck, Matthias

2009-01-01

Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 Å crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120GAP·H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation. PMID:19843518

The RNA-binding protein Musashi-1 is produced in the developing and adult mouse eye.

PubMed

Raji, B; Dansault, A; Leemput, J; de la Houssaye, G; Vieira, V; Kobetz, A; Arbogast, L; Masson, C; Menasche, M; Abitbol, M

2007-08-10

Musashi-1 (Msi1) is an RNA-binding protein produced in various types of stem cells including neural stem/progenitor cells and astroglial progenitor cells in the vertebrate central nervous system. Other RNA-binding proteins such as Pumilio-1, Pumilio-2, Staufen-1, and Staufen-2 have been characterized as potential markers of several types of stem or progenitor cells. We investigated the involvement of Msi1 in mouse eye development and adult mouse eye functions by analyzing the profile of Msi1 production in all ocular structures during development and adulthood. We studied Msi1 production by in situ hybridization and immunohistochemistry of ocular tissue sections and by semi-quantitative RT-PCR and western blot analysis from the embryonic stage of 12.5 days post coitum (E12.5 dpc) when the first retinal ganglion cells (RGCs) begin to appear to the adult stage when all retinal cell types are present. Msi1 mRNA was present at all studied stages of eye development. Msi1 protein was detected in the primitive neuroblastic layer (NbL), the ganglion cell layer (GCL), and in all major differentiated neurons of postnatal developing and adult retinae. During postnatal developing stages, faint diffuse Msi1 protein staining is converted to a more specific distribution once mouse retina is fully differentiated. The most striking result of our study concerns the large amounts of Msi1 protein and mRNA in several unexpected sites of adult mouse eyes including the corneal epithelium and endothelium, stromal keratocytes, progenitor cells of the limbus, equatorial lens stem cells, differentiated lens epithelial cells, and differentiating lens fibers. Msi1 was also found in the pigmented and nonpigmented cells of the ciliary processes, the melanocytes of the ciliary body, the retinal pigment epithelium, differentiated retinal neurons, and most probably in the retinal glial cells such as Müller glial cells, astrocytes, and the oligodendocytes surrounding the axons of the optic nerve. Msi1 expression was detected in the outer plexiform layer, the inner plexiform layer, and the nerve fiber layer of fully differentiated adult retina. We provide here the first demonstration that the RNA-binding protein, Msi1, is produced in mouse eyes from embryonic stages until adulthood. The relationship between the presence of Msi1 in developing ocular compartments and the possible stem/progenitor cell characteristics of these compartments remains unclear. Finally, the expression of Msi1 in several different cell types in the adult eye is extremely intriguing and should lead to further attempts to unravel the role of Msi1 in cellular and subcellular RNA metabolism and in the control of translational processes in adult eye cells particularly in adult neuronal dendrites, axons, and synapses.

Characterization of the ligand-binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N-terminal domain of the variant surface glycoprotein.

PubMed

Salmon, D; Hanocq-Quertier, J; Paturiaux-Hanocq, F; Pays, A; Tebabi, P; Nolan, D P; Michel, A; Pays, E

1997-12-15

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.

Protein NMR Studies of Substrate Binding to Human Blood Group A and B Glycosyltransferases.

PubMed

Grimm, Lena Lisbeth; Weissbach, Sophie; Flügge, Friedemann; Begemann, Nora; Palcic, Monica M; Peters, Thomas

2017-07-04

Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H, 15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H, 15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[ 13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of a Novel TGF-β-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β-Receptor-3 (TGFR-3)

PubMed Central

Diestel, Uschi; Resch, Marcus; Meinhardt, Kathrin; Weiler, Sigrid; Hellmann, Tina V.; Mueller, Thomas D.; Nickel, Joachim; Eichler, Jutta; Muller, Yves A.

2013-01-01

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3. PMID:23826237

Effects of magnesium ions on the stabilization of RNA oligomers of defined structures.

PubMed Central

Serra, Martin J; Baird, John D; Dale, Taraka; Fey, Bridget L; Retatagos, Kimberly; Westhof, Eric

2002-01-01

Optical melting was used to determine the stabilities of 11 small RNA oligomers of defined secondary structure as a function of magnesium ion concentration. The oligomers included helices composed of Watson-Crick base pairs, GA tandem base pairs, GU tandem base pairs, and loop E motifs (both eubacterial and eukaryotic). The effect of magnesium ion concentration on stability was interpreted in terms of two simple models. The first assumes an uptake of metal ion upon duplex formation. The second assumes nonspecific electrostatic attraction of metal ions to the RNA oligomer. For all oligomers, except the eubacterial loop E, the data could best be interpreted as nonspecific binding of metal ions to the RNAs. The effect of magnesium ions on the stability of the eubacterial loop E was distinct from that seen with the other oligomers in two ways. First, the extent of stabilization by magnesium ions (as measured by either change in melting temperature or free energy) was three times greater than that observed for the other helical oligomers. Second, the presence of magnesium ions produces a doubling of the enthalpy for the melting transition. These results indicate that magnesium ion stabilizes the eubacterial loop E sequence by chelating the RNA specifically. Further, these results on a rather small system shed light on the large enthalpy changes observed upon thermal unfolding of large RNAs like group I introns. It is suggested that parts of those large enthalpy changes observed in the folding of RNAs may be assigned to variations in the hydration states and types of coordinating atoms in some specifically bound magnesium ions and to an increase in the observed cooperativity of the folding transition due to the binding of those magnesium ions coupling the two stems together. Brownian dynamic simulations, carried out to visualize the metal ion binding sites, reveal rather delocalized ionic densities in all oligomers, except for the eubacterial loop E, in which precisely located ion densities were previously calculated. PMID:12003491