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Sample records for headpiece c-terminal domain

  1. MscCG from Corynebacterium glutamicum: functional significance of the C-terminal domain.

    PubMed

    Becker, Michael; Krämer, Reinhard

    2015-10-01

    Corynebacterium glutamicum is used in microbial biotechnology for the production of amino acids, e.g., glutamate and lysine. Excretion of glutamate into the surrounding medium under production conditions is mediated by MscCG, an MscS-type mechanosensitive channel. In difference to most other MscS-type channel proteins, MscCG carries, in addition to the N-terminal pore domain, a long C-terminal domain that amounts to about half of the size of the protein and harbors an additional transmembrane segment. Here we study the impact of the C-terminal domain on both functions of MscCG as mechanosensitive channel and as glutamate exporter. Sequential truncations of the C-terminal domain were applied, as well as deletion of particular subdomains, replacement of these segments by other amino acid sequences, and sequence randomization. Several parameters of cell physiology and bioenergetics of the obtained mutants related to both glutamate excretion and response to osmotic stress were quantified. All three subdomains of the C-terminal domain, i.e., the periplasmic loop, the fourth transmembrane segment, and the cytoplasmic loop, proved to be of core significance for MscCG function, in particular for glutamate excretion. PMID:26033538

  2. A summary of staphylococcal C-terminal SH3b_5 cell wall binding domains.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcal peptidoglycan hydrolases are a potential new source of antimicrobials. A large subset of these proteins contain a C-terminal SH3b_5 cell wall binding domain that has been shown for some to be essential for accurate cell wall recognition and subsequent staphylolytic activity, propert...

  3. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    SciTech Connect

    Singh, Pratibha; Savithri, H.S.

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  4. Defining the Intrinsically Disordered C-Terminal Domain of SSB Reveals DNA-Mediated Compaction.

    PubMed

    Green, Matthew; Hatter, Louise; Brookes, Emre; Soultanas, Panos; Scott, David J

    2016-01-29

    The bacterial single-stranded DNA (ssDNA) binding protein SSB is a strictly conserved and essential protein involved in diverse functions of DNA metabolism, including replication and repair. SSB comprises a well-characterized tetrameric core of N-terminal oligonucleotide binding OB folds that bind ssDNA and four intrinsically disordered C-terminal domains of unknown structure that interact with partner proteins. The generally accepted, albeit speculative, mechanistic model in the field postulates that binding of ssDNA to the OB core induces the flexible, undefined C-terminal arms to expand outwards encouraging functional interactions with partner proteins. In this structural study, we show that the opposite is true. Combined small-angle scattering with X-rays and neutrons coupled to coarse-grained modeling reveal that the intrinsically disordered C-terminal arms are relatively collapsed around the tetrameric OB core and collapse further upon ssDNA binding. This implies a mechanism of action, in which the disordered C-terminal domain collapse traps the ssDNA and pulls functional partners onto the ssDNA. PMID:26707201

  5. Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

    PubMed Central

    Lykkemark, Simon; Mandrup, Ole Aalund; Friis, Niels Anton; Kristensen, Peter

    2014-01-01

    Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression. PMID:25426869

  6. Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant.

    PubMed

    Lykkemark, Simon; Mandrup, Ole Aalund; Friis, Niels Anton; Kristensen, Peter

    2014-01-01

    Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression. PMID:25426869

  7. Viral suppression function of intracellular antibody against C-terminal domain of rabies virus phosphoprotein.

    PubMed

    Liu, Yang; Sun, Lina; Yu, Pengcheng; Li, Aqian; Li, Chuan; Tang, Qing; Li, Dexin; Liang, Mifang

    2015-10-01

    Rabies virus (RV) causes a fatal disease in both human and animals. The disease can be prevented by post-exposure prophylaxis in individuals exposed to RV. However, the neutralization effect is limited after the virus enters into the host cells. So, it is important to identify new targets for rabies therapy. In this study, a human antibody RV1A2 specific to RV phosphoprotein (RV-P) was generated from a human naïve immune antibody library. The antibody recognized all forms of the phosphoproteins including the full length (P1) and short length of the P proteins (P2, P3, P4, and P5). The epitope mapping and the molecular docking of antigen-antibody complex showed that the antibody targets at a conserved epitope of 'VLGWV' ranging from amino acid (aa) 262 to 266 at C-terminal domain of the P protein, which locates at a hydrophobic pocket region in the C-terminal of the RV-P. The aa W265 within the epitope is on the flat surface of the domain, suggesting that it may be a critical amino acid for the functions of the P protein. Our results further showed that intracellular antibody RV1A2 which targets at the C-terminal domain of the P protein could effectively inhibit RV propagation 2-4 days post infection. These results suggest that the conserved C-terminal domain may be used as a new target for drug discovery, which highlights an intracellular inhibition of RV propagation and provides a potential novel way to treat RV infection. PMID:26188200

  8. Dual Thermosensitive Hydrogels Assembled from the Conserved C-Terminal Domain of Spider Dragline Silk.

    PubMed

    Qian, Zhi-Gang; Zhou, Ming-Liang; Song, Wen-Wen; Xia, Xiao-Xia

    2015-11-01

    Stimuli-responsive hydrogels have great potentials in biomedical and biotechnological applications. Due to the advantages of precise control over molecular weight and being biodegradable, protein-based hydrogels and their applications have been extensively studied. However, protein hydrogels with dual thermosensitive properties are rarely reported. Here we present the first report of dual thermosensitive hydrogels assembled from the conserved C-terminal domain of spider dragline silk. First, we found that recombinant C-terminal domain of major ampullate spidroin 1 (MaSp1) of the spider Nephila clavipes formed hydrogels when cooled to approximately 2 °C or heated to 65 °C. The conformational changes and self-assembly of the recombinant protein were studied to understand the mechanism of the gelation processes using multiple methods. It was proposed that the gelation in the low-temperature regime was dominated by hydrogen bonding and hydrophobic interaction between folded protein molecules, whereas the gelation in the high-temperature regime was due to cross-linking of the exposed hydrophobic patches resulting from partial unfolding of the protein upon heating. More interestingly, genetic fusion of the C-terminal domain to a short repetitive region of N. clavipes MaSp1 resulted in a chimeric protein that formed a hydrogel with significantly improved mechanical properties at low temperatures between 2 and 10 °C. Furthermore, the formation of similar hydrogels was observed for the recombinant C-terminal domains of dragline silk of different spider species, thus demonstrating the conserved ability to form dual thermosensitive hydrogels. These findings may be useful in the design and construction of novel protein hydrogels with tunable multiple thermosensitivity for applications in the future. PMID:26457360

  9. Conserved C-Terminal Domain of Spider Tubuliform Spidroin 1 Contributes to Extensibility in Synthetic Fibers

    SciTech Connect

    Gnesa, Eric; Hsia, Yang; Yarger, Jeffery L.; Weber, Warner; Lin-Cereghino, Joan; Lin-Cereghino, Geoff; Tang, Simon; Agari, Kimiko; Vierra, Craig

    2012-05-24

    Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.

  10. The structure of the C-terminal domain of the Zaire ebolavirus nucleoprotein

    PubMed Central

    Dziubańska, Paulina J.; Derewenda, Urszula; Ellena, Jeffrey F.; Engel, Daniel A.; Derewenda, Zygmunt S.

    2014-01-01

    Ebolavirus (EBOV) causes severe hemorrhagic fever with a mortality rate of up to 90%. EBOV is a member of the order Mononegavirales and, like other viruses in this taxonomic group, contains a negative-sense single-stranded (ss) RNA. The EBOV ssRNA encodes seven distinct proteins. One of them, the nucleoprotein (NP), is the most abundant viral protein in the infected cell and within the viral nucleocapsid. Like other EBOV proteins, NP is multifunctional. It is tightly associated with the viral genome and is essential for viral transcription, RNA replication, genome packaging and nucleocapsid assembly prior to membrane encapsulation. NP is unusual among the Mononegavirales in that it contains two distinct regions, or putative domains, the C-terminal of which shows no homology to any known proteins and is purported to be a hub for protein–protein interactions within the nucleocapsid. The atomic structure of NP remains unknown. Here, the boundaries of the N- and C-terminal domains of NP from Zaire EBOV are defined, it is shown that they can be expressed as highly stable recombinant proteins in Escherichia coli, and the atomic structure of the C-terminal domain (residues 641–739) derived from analysis of two distinct crystal forms at 1.98 and 1.75 Å resolution is described. The structure reveals a novel tertiary fold that is distantly reminiscent of the β-grasp architecture. PMID:25195755

  11. Structure of the Escherichia coli RNA polymerase α subunit C-terminal domain

    SciTech Connect

    Lara-González, Samuel; Birktoft, Jens J.; Lawson, Catherine L.

    2010-07-01

    The crystal structure of the dimethyllysine derivative of the E. coli RNA polymerase α subunit C-terminal domain is reported at 2.0 Å resolution. The α subunit C-terminal domain (αCTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli αCTD (α subunit residues 245–329) determined to 2.0 Å resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P2{sub 1} and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, R{sub free} = 0.236) has improved geometry compared with prior lower resolution determinations of the αCTD structure [Jeon et al. (1995 ▶), Science, 270, 1495–1497; Benoff et al. (2002 ▶), Science, 297, 1562–1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of αCTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction.

  12. The C-terminal domain of the transcriptional corepressor CtBP is intrinsically unstructured.

    PubMed

    Nardini, Marco; Svergun, Dmitri; Konarev, Peter V; Spanò, Stefania; Fasano, Mauro; Bracco, Chiara; Pesce, Alessandra; Donadini, Alessandra; Cericola, Claudia; Secundo, Francesco; Luini, Alberto; Corda, Daniela; Bolognesi, Martino

    2006-05-01

    C-terminal binding proteins (CtBPs) are moonlighting proteins involved in nuclear transcriptional corepression and in Golgi membrane tubule fission. Structural information on CtBPs is available for their substrate-binding domain, responsible for transcriptional repressor recognition/binding, and for the nucleotide-binding domain, involved in NAD(H)-binding and dimerization. On the contrary, little is known about the structure of CtBP C-terminal region ( approximately 90 residues), hosting sites for post-translational modifications. In the present communication we apply a combined approach based on bioinformatics, nuclear magnetic resonance, circular dichroism spectroscopy, and small-angle X-ray scattering, and we show that the CtBP C-terminal region is intrinsically unstructured in the full-length CtBP and in constructs lacking the substrate- and/or the nucleotide-binding domains. The flexible nature of this protein region, and its structural transitions, may be instrumental for CtBP recognition and binding to diverse molecular partners. PMID:16597837

  13. Structure of a plant β-galactosidase C-terminal domain.

    PubMed

    Rimlumduan, Thipwarin; Hua, Yan-Ling; Tanaka, Toshiyuki; Ketudat Cairns, James R

    2016-10-01

    Most plant β-galactosidases, which belong to glycoside hydrolase family 35, have a C-terminal domain homologous to animal galactose and rhamnose-binding lectins. To investigate the structure and function of this domain, the C-terminal domain of the rice (Oryza sativa L.) β-galactosidase 1 (OsBGal1 Cter) was expressed in Escherichia coli and purified to homogeneity. The free OsBGal1 Cter is monomeric with a native molecular weight of 15kDa. NMR spectroscopy indicated that OsBGal1 Cter comprises five β-strands and one α-helix. The structure of this domain is similar to lectin domains from animals, but loops A and C of OsBGal1 Cter are longer than the corresponding loops from related animal lectins with known structures. In addition, loop A of OsBGal1 Cter was not well defined, suggesting it is flexible. Although OsBGal1 Cter was predicted to be a galactose/rhamnose-binding domain, binding with rhamnose, galactose, glucose, β-1,4-d-galactobiose and raffinose could not be observed in NMR experiments. PMID:27451952

  14. Crystallization of the C-terminal domain of the bacteriophage T7 fibre protein gp17

    PubMed Central

    Garcia-Doval, Carmela; van Raaij, Mark J.

    2012-01-01

    Bacteriophage T7 attaches to its host using the C-terminal domains of its six fibres, which are trimers of the gp17 protein. A C-terminal fragment of gp17 consisting of amino acids 371–553 has been expressed, purified and crystallized. Crystals of two forms were obtained, belonging to space group P212121 (unit-cell parameters a = 61.2, b = 86.0, c = 118.4 Å) and space group C2221 (unit-cell parameters a = 68.3, b = 145.6, c = 172.1 Å). They diffracted to 1.9 and 2.0 Å resolution, respectively. Both crystals are expected to contain one trimer in the asymmetric unit. Multiwavelength anomalous dispersion phasing with a mercury derivative is in progress. PMID:22297990

  15. Crystallization of the C-terminal domain of the bacteriophage T7 fibre protein gp17.

    PubMed

    Garcia-Doval, Carmela; van Raaij, Mark J

    2012-02-01

    Bacteriophage T7 attaches to its host using the C-terminal domains of its six fibres, which are trimers of the gp17 protein. A C-terminal fragment of gp17 consisting of amino acids 371-553 has been expressed, purified and crystallized. Crystals of two forms were obtained, belonging to space group P2(1)2(1)2(1) (unit-cell parameters a = 61.2, b = 86.0, c = 118.4 Å) and space group C222(1) (unit-cell parameters a = 68.3, b = 145.6, c = 172.1 Å). They diffracted to 1.9 and 2.0 Å resolution, respectively. Both crystals are expected to contain one trimer in the asymmetric unit. Multiwavelength anomalous dispersion phasing with a mercury derivative is in progress. PMID:22297990

  16. BS69/ZMYND11 C-Terminal Domains Bind and Inhibit EBNA2

    PubMed Central

    Shen, Chih-Lung; Gonzalez-Hurtado, Elsie; Zhang, Zhi-Min; Xu, Muyu; Martinez, Ernest; Peng, Chih-Wen; Song, Jikui

    2016-01-01

    Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) plays an important role in driving immortalization of EBV-infected B cells through regulating the expression of many viral and cellular genes. We report a structural study of the tumor suppressor BS69/ZMYND11 C-terminal region, comprised of tandem coiled-coil-MYND domains (BS69CC-MYND), in complex with an EBNA2 peptide containing a PXLXP motif. The coiled-coil domain of BS69 self-associates to bring two separate MYND domains in close proximity, thereby enhancing the BS69 MYND-EBNA2 interaction. ITC analysis of BS69CC-MYND with a C-terminal fragment of EBNA2 further suggests that the BS69CC-MYND homodimer synergistically binds to the two EBNA2 PXLXP motifs that are respectively located in the conserved regions CR7 and CR8. Furthermore, we showed that EBNA2 interacts with BS69 and down-regulates its expression at both mRNA and protein levels in EBV-infected B cells. Ectopic BS69CC-MYND is recruited to viral target promoters through interactions with EBNA2, inhibits EBNA2-mediated transcription activation, and impairs proliferation of lymphoblastoid cell lines (LCLs). Substitution of critical residues in the MYND domain impairs the BS69-EBNA2 interaction and abolishes the BS69 inhibition of the EBNA2-mediated transactivation and LCL proliferation. This study identifies the BS69 C-terminal domains as an inhibitor of EBNA2, which may have important implications in development of novel therapeutic strategies against EBV infection. PMID:26845565

  17. Membrane curvature sensing by the C-terminal domain of complexin

    NASA Astrophysics Data System (ADS)

    Snead, David; Wragg, Rachel T.; Dittman, Jeremy S.; Eliezer, David

    2014-09-01

    Complexin functions at presynaptic nerve terminals to inhibit spontaneous SNARE-mediated synaptic vesicle (SV) exocytosis, while enhancing stimulated neurotransmitter release. The C-terminal domain (CTD) of complexin is essential for its inhibitory function and has been implicated in localizing complexin to SVs via direct membrane interactions. Here we show that complexin’s CTD is highly sensitive to membrane curvature, which it senses via tandem motifs, a C-terminal motif containing a mix of bulky hydrophobic and positively charged residues, and an adjacent amphipathic region that can bind membranes in either a disordered or a helical conformation. Helix formation requires membrane packing defects found on highly curved membrane surfaces. Mutations that disrupt helix formation without disrupting membrane binding compromise complexin’s inhibitory function in vivo. Thus, this membrane curvature-dependent conformational transition, combined with curvature-sensitive binding by the adjacent C-terminal motif, constitute a novel mechanism for activating complexin’s inhibitory function on the surface of SVs.

  18. Structure of the C-terminal domain of nsp4 from feline coronavirus

    SciTech Connect

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh; Berglind, Hanna; Nordlund, Pär; Coutard, Bruno; Tucker, Paul A.

    2009-08-01

    The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.

  19. Groundnut Bud Necrosis Virus Encoded NSm Associates with Membranes via Its C-Terminal Domain

    PubMed Central

    Singh, Pratibha; Indi, Shantinath S.; Savithri, Handanahal S.

    2014-01-01

    Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200–250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell. PMID:24919116

  20. Functional analysis of the C-terminal extension of telomerase reverse transcriptase. A putative "thumb" domain.

    PubMed

    Hossain, Shabbir; Singh, Sunitha; Lue, Neal F

    2002-09-27

    Telomerase is an RNA-protein complex responsible for the extension of one strand of telomere terminal repeats. The catalytic protein subunit of telomerase, known generically as telomerase reverse transcriptase (TERT), exhibits significant homology to reverse transcriptases (RTs) encoded by retroviruses and retroelements. The mechanisms of telomerase may therefore be similar to those of the conventional reverse transcriptases. In this report, we explore potential similarity between these two classes of proteins in a region with no evident sequence similarity. Previous analysis has implicated a C-terminal domain of retroviral RTs (known as the "thumb" domain) in template-primer binding and in processivity control. The equivalent region of TERTs, although similar to one another, does not exhibit significant sequence homology to retroviral RTs. However, we found that removal of this region of yeast TERT similarly resulted in a decrease in the stability of telomerase-DNA complex and in the processivity of telomerase-mediated nucleotide addition. Moreover, the C-terminal domain of TERT exhibits a nucleic acid binding activity when recombinantly expressed and purified. Finally, amino acid substitutions of conserved residues in this region of TERT were found to impair telomerase activity and processivity. We suggest that mechanistic similarity between telomerase and retroviral RTs may extend beyond the regions with apparent sequence similarity. PMID:12151386

  1. Investigating the Roles of the C-Terminal Domain of Plasmodium falciparum GyrA

    PubMed Central

    Nagano, Soshichiro; Seki, Eiko; Lin, Ting-Yu; Shirouzu, Mikako; Yokoyama, Shigeyuki; Heddle, Jonathan G.

    2015-01-01

    Malaria remains as one of the most deadly diseases in developing countries. The Plasmodium causative agents of human malaria such as Plasmodium falciparum possess an organelle, the apicoplast, which is the result of secondary endosymbiosis and retains its own circular DNA. A type II topoisomerase, DNA gyrase, is present in the apicoplast. In prokaryotes this enzyme is a proven, effective target for antibacterial agents, and its discovery in P. falciparum opens up the prospect of exploiting it as a drug target. Basic characterisation of P. falciparum gyrase is important because there are significant sequence differences between it and the prokaryotic enzyme. However, it has proved difficult to obtain soluble protein. Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form. Biochemical analyses revealed many similarities between the C-terminal domains of GyrA from E. coli and P. falciparum, suggesting that despite its considerably larger size, the malarial protein carries out a similar DNA wrapping function. Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping. PMID:26566222

  2. The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II.

    PubMed Central

    Caron, P R; Watt, P; Wang, J C

    1994-01-01

    A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results. Images PMID:8164675

  3. N- and C-terminal Transactivation Domains of GATA1 Protein Coordinate Hematopoietic Program*

    PubMed Central

    Kaneko, Hiroshi; Kobayashi, Eri; Yamamoto, Masayuki; Shimizu, Ritsuko

    2012-01-01

    Transcription factor GATA1 regulates the expression of a cluster of genes important for hematopoietic cell differentiation toward erythroid and megakaryocytic lineages. Three functional domains have been identified in GATA1, a transactivation domain located in the N terminus (N-TAD) and two zinc finger domains located in the middle of the molecule. Although N-TAD is known as a solitary transactivation domain for GATA1, clinical observations in Down syndrome leukemia suggest that there may be additional transactivation domains. In this study, we found in reporter co-transfection assays that transactivation activity of GATA1 was markedly reduced by deletion of the C-terminal 95 amino acids without significant attenuation of the DNA binding activity or self-association potential. We therefore generated transgenic mouse lines that expressed GATA1 lacking the C-terminal region (GATA1-ΔCT). When we crossed these transgenic mouse lines to the Gata1-deficient mouse, we found that the GATA1-ΔCT transgene rescued Gata1-deficient mice from embryonic lethality. The embryos rescued with an almost similar level of GATA1-ΔCT to endogenous GATA1 developed beyond embryonic 13.5 days, showing severe anemia with accumulation of immature erythroid cells, as was the case for the embryos rescued by endogenous levels of GATA1 lacking N-TAD (GATA1-ΔNT). Distinct sets of target genes were affected in the embryos rescued by GATA1-ΔCT and GATA1-ΔNT. We also found attenuated GATA1 function in cell cycle control of immature megakaryocytes in both lines of rescued embryos. These results thus demonstrate that GATA1 has two independent transactivation domains, N-TAD and C-TAD. Both N-TAD and C-TAD retain redundant as well as specific activities for proper hematopoiesis in vivo. PMID:22556427

  4. Characterization of peptides cleaved by plasmin from the C-terminal polymerization domain of human fibrinogen.

    PubMed

    Southan, C; Thompson, E; Panico, M; Etienne, T; Morris, H R; Lane, D A

    1985-10-25

    The C-terminal region of the fibrinogen gamma chain is known to participate in several functional interactions including fibrin polymerization. This part of the molecule is retained on the gamma chain of fragment D (FgD) when fibrinogen is digested by plasmin in the presence of calcium to produce the fragment D-fragment E (FgD X FgE) complex but is lost if FgD is prepared in the absence of calcium. In an attempt to characterize the C-terminal polymerization domain we have used three techniques to examine this further degradation of FgD following the addition of EDTA and plasmin. Analysis of the digestion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a progressive cleavage of the gamma chain to two small remnants. The polymerization-inhibitory activity of the whole digest was studied using acid-solubilized fibrin. A progressive loss of inhibitory activity was associated with gamma chain shortening, reaching greater than a 120-fold reduction at the end of digestion. The cleavage of peptides was followed by reverse-phase high performance liquid chromatography and the release of a characteristic peptide triplet was associated with gamma chain cleavage. Manual sequencing, amino acid analysis, and fast atom bombardment mass spectrometry established the three peptides as gamma 303-356, 357-373, and 374-405. These peptides have sequences in common with those peptides recently reported by other investigators to be potent polymerization inhibitors. However, when a mixture of the three peptides was added in a 200-fold molar excess to polymerizing fibrin, no inhibitory activity could be demonstrated. It is concluded that the C-terminal polymerization domain of fibrinogen may be an extended region which includes the sequence gamma 303-405, when this is contiguous with the remainder of the gamma chain. PMID:2932434

  5. Structure of the Escherichia coli RNA polymerase a Subunit C-terminal Domain

    SciTech Connect

    Lara-Gonzalez, S.; Birktoft, J; Lawson, C

    2010-01-01

    The {alpha} subunit C-terminal domain ({alpha}CTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli {alpha}CTD ({alpha} subunit residues 245-329) determined to 2.0 {angstrom} resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P2{sub 1} and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, R{sub free} = 0.236) has improved geometry compared with prior lower resolution determinations of the {alpha}CTD structure [Jeon et al. (1995), Science, 270, 1495-1497; Benoff et al. (2002), Science, 297, 1562-1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of {alpha}CTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction.

  6. Structure of the Escherichia coli RNA polymerase α subunit C-terminal domain

    PubMed Central

    Lara-González, Samuel; Birktoft, Jens J.; Lawson, Catherine L.

    2010-01-01

    The α subunit C-terminal domain (αCTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli αCTD (α subunit residues 245–329) determined to 2.0 Å resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P21 and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, R free = 0.236) has improved geometry compared with prior lower resolution determinations of the αCTD structure [Jeon et al. (1995 ▶), Science, 270, 1495–1497; Benoff et al. (2002 ▶), Science, 297, 1562–1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of αCTD-containing multi-component complexes visualized at lower resolution using X-ray crystallo­graphy and electron-microscopy reconstruction. PMID:20606261

  7. Control of cytoplasmic dynein force production and processivity by its C-terminal domain

    NASA Astrophysics Data System (ADS)

    Nicholas, Matthew P.; Höök, Peter; Brenner, Sibylle; Wynne, Caitlin L.; Vallee, Richard B.; Gennerich, Arne

    2015-02-01

    Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a ‘cap’ over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function.

  8. Crystallization of the C-terminal globular domain of avian reovirus fibre

    SciTech Connect

    Raaij, Mark J. van; Hermo Parrado, X. Lois; Guardado Calvo, Pablo; Fox, Gavin C.; Llamas-Saiz, Antonio L.; Costas, Celina; Martínez-Costas, José; Benavente, Javier

    2005-07-01

    Partial proteolysis of the avian reovirus cell-attachment protein σC yields a major homotrimeric C-terminal fragment that presumably contains the receptor-binding domain. This fragment has been crystallized in the presence and absence of zinc sulfate and cadmium sulfate. One of the crystal forms diffracts synchrotron X-rays to 2.2–2.3 Å. Avian reovirus fibre, a homotrimer of the σC protein, is responsible for primary host-cell attachment. Using the protease trypsin, a C-terminal σC fragment containing amino acids 156–326 has been generated which was subsequently purified and crystallized. Two different crystal forms were obtained, one grown in the absence of divalent cations and belonging to space group P6{sub 3}22 (unit-cell parameters a = 75.6, c = 243.1 Å) and one grown in the presence of either zinc or cadmium sulfate and belonging to space group P321 (unit-cell parameters a = 74.7, c = 74.5 Å and a = 73.1, c = 69.9 Å for the Zn{sup II}- and Cd{sup II}-grown crystals, respectively). The first crystal form diffracted synchrotron radiation to 3.0 Å resolution and the second form to 2.2–2.3 Å. Its closest related structure, the C-terminal fragment of mammalian reovirus fibre, has only 18% sequence identity and molecular-replacement attempts were unsuccessful. Therefore, a search is under way for suitable heavy-atom derivatives and attempts are being made to grow protein crystals containing selenomethionine instead of methionine.

  9. Properties of the C-terminal domain of 4.1 proteins.

    PubMed

    Scott, C; Phillips, G W; Baines, A J

    2001-07-01

    At the C-terminus of all known 4.1 proteins is a sequence domain unique to these proteins, known as the C-terminal domain (CTD). Mammalian CTDs are associated with a growing number of protein-protein interactions, although such activities have yet to be associated with invertebrate CTDs. Mammalian CTDs are generally defined by sequence alignment as encoded by exons 18-21. Comparison of known vertebrate 4.1 proteins with invertebrate (Caenorhabditis elegans and Drosophila melanogaster) 4.1 proteins indicates that mammalian 4.1 exon 19 represents a vertebrate adaptation that extends the sequence of the CTD with a Ser/Thr-rich sequence. The CTD was first described as a 22/24-kDa domain by chymotryptic digestion of erythrocyte 4.1 (4.1R) [Leto, T.L. & Marchesi, V.T. (1984) J. Biol. Chem. 259, 4603-4608]. Here we show that in 4.1R the 22/24-kDa fragment is not stable but rapidly processed to a 15-kDa fragment by chymotrypsin. The 15-kDa fragment is extremely stable, being resistant to overnight digestion in chymotrypsin on ice. Analysis of this fragment indicates that it is derived from residues 709-858 (SwissProt accession no. P48193), and represents the CTD of 4.1R. The fragment behaves as a globular monomer in solution. Secondary-structure predictions indicate that this domain is composed of five or six beta strands with an alpha helix before the most C-terminal of these. Together these data indicate that the CTD probably represents an independent folding structure which has gained function since the divergence of vertebrates from invertebrates. PMID:11432737

  10. Autoinhibition of Bacteriophage T4 Mre11 by Its C-terminal Domain*

    PubMed Central

    Gao, Yang; Nelson, Scott W.

    2014-01-01

    Mre11 and Rad50 form a stable complex (MR) and work cooperatively in repairing DNA double strand breaks. In the bacteriophage T4, Rad50 (gene product 46) enhances the nuclease activity of Mre11 (gene product 47), and Mre11 and DNA in combination stimulate the ATPase activity of Rad50. The structural basis for the cross-activation of the MR complex has been elusive. Various crystal structures of the MR complex display limited protein-protein interfaces that mainly exist between the C terminus of Mre11 and the coiled-coil domain of Rad50. To test the role of the C-terminal Rad50 binding domain (RBD) in Mre11 activation, we constructed a series of C-terminal deletions and mutations in bacteriophage T4 Mre11. Deletion of the RBD in Mre11 eliminates Rad50 binding but only has moderate effect on its intrinsic nuclease activity; however, the additional deletion of the highly acidic flexible linker that lies between RBD and the main body of Mre11 increases the nuclease activity of Mre11 by 20-fold. Replacement of the acidic residues in the flexible linker with alanine elevates the Mre11 activity to the level of the MR complex when combined with deletion of RBD. Nuclease activity kinetics indicate that Rad50 association and deletion of the C terminus of Mre11 both enhance DNA substrate binding. Additionally, a short peptide that contains the flexible linker and RBD of Mre11 acts as an inhibitor of Mre11 nuclease activity. These results support a model where the Mre11 RBD and linker domain act as an autoinhibitory domain when not in complex with Rad50. Complex formation with Rad50 alleviates this inhibition due to the tight association of the RBD and the Rad50 coiled-coil. PMID:25077970

  11. Interaction of the Tim44 C-terminal domain with negatively charged phospholipids.

    PubMed

    Marom, Milit; Safonov, Roman; Amram, Shay; Avneon, Yoav; Nachliel, Esther; Gutman, Menachem; Zohary, Keren; Azem, Abdussalam; Tsfadia, Yossi

    2009-12-01

    The translocation of proteins from the cytosol into the mitochondrial matrix is mediated by the coordinated action of the TOM complex in the outer membrane, as well as the TIM23 complex and its associated protein import motor in the inner membrane. The focus of this work is the peripheral inner membrane protein Tim44. Tim44 is a vital component of the mitochondrial protein translocation motor that anchors components of the motor to the TIM23 complex. For this purpose, Tim44 associates with the import channel by direct interaction with the Tim23 protein. Additionally, it was shown in vitro that Tim44 associates with acidic model membranes, in particular those containing cardiolipin. The latter interaction was shown to be mediated by the carboxy-terminal domain of Tim44 [Weiss, C., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8890-8894]. The aim of this study was to determine the precise recognition site for negative lipids in the C-terminal domain of Tim44. In particular, we wanted to examine the recently suggested hypothesis that acidic phospholipids associate with Tim44 via a hydrophobic cavity that is observed in the high-resolution structure of the C-terminal domain of the protein [Josyula, R., et al. (2006) J. Mol. Biol. 359, 798-804]. Molecular dynamics simulations suggest that (i) the hydrophobic tail of lipids may interact with Tim44 via the latter's hydrophobic cavity and (ii) a region, located in the N-terminal alpha-helix of the C-terminal domain (helices A1 and A2), may serve as a membrane attachment site. To validate this assumption, N-terminal truncations of yeast Tim44 were examined for their ability to bind cardiolipin-containing phospholipid vesicles. The results indicate that removal of the N-terminal alpha-helix (helix A1) abolishes the capacity of Tim44 to associate with cardiolipin-containing liposomes. We suggest that helices A1 and A2, in Tim44, jointly promote the association of the protein with acidic phospholipids. PMID:19863062

  12. Structure of the C-terminal Domain of Transcription Facto IIB from Trypanosoma brucei

    SciTech Connect

    Ibrahim, B.; Kanneganti, N; Rieckhof, G; Das, A; Laurents, D; Palenchar, J; Bellofatto, V; Wah, D

    2009-01-01

    In trypanosomes, the production of mRNA relies on the synthesis of the spliced leader (SL) RNA. Expression of the SL RNA is initiated at the only known RNA polymerase II promoter in these parasites. In the pathogenic trypanosome, Trypanosoma brucei, transcription factor IIB (tTFIIB) is essential for SL RNA gene transcription and cell viability, but has a highly divergent primary sequence in comparison to TFIIB in well-studied eukaryotes. Here we describe the 2.3 A resolution structure of the C-terminal domain of tTFIIB (tTFIIBC). The tTFIIBC structure consists of 2 closely packed helical modules followed by a C-terminal extension of 32 aa. Using the structure as a guide, alanine substitutions of basic residues in regions analogous to functionally important regions of the well-studied eukaryotic TFIIB support conservation of a general mechanism of TFIIB function in eukaryotes. Strikingly, tTFIIBC contains additional loops and helices, and, in contrast to the highly basic DNA binding surface of human TFIIB, contains a neutral surface in the corresponding region. These attributes probably mediate trypanosome-specific interactions and have implications for the apparent bidirectional transcription by RNA polymerase II in protein-encoding gene expression in these organisms.

  13. Phosphorylation of a C-terminal auto-inhibitory domain increases SMARCAL1 activity.

    PubMed

    Carroll, Clinton; Bansbach, Carol E; Zhao, Runxiang; Jung, Sung Yun; Qin, Jun; Cortez, David

    2014-01-01

    SMARCAL1 promotes the repair and restart of damaged replication forks. Either overexpression or silencing SMARCAL1 causes the accumulation of replication-associated DNA damage. SMARCAL1 is heavily phosphorylated. Here we identify multiple phosphorylation sites, including S889, which is phosphorylated even in undamaged cells. S889 is highly conserved through evolution and it regulates SMARCAL1 activity. Specifically, S889 phosphorylation increases the DNA-stimulated ATPase activity of SMARCAL1 and increases its ability to catalyze replication fork regression. A phosphomimetic S889 mutant is also hyperactive when expressed in cells, while a non-phosphorylatable mutant is less active. S889 lies within a C-terminal region of the SMARCAL1 protein. Deletion of the C-terminal region also creates a hyperactive SMARCAL1 protein suggesting that S889 phosphorylation relieves an auto-inhibitory function of this SMARCAL1 domain. Thus, S889 phosphorylation is one mechanism by which SMARCAL1 activity is regulated to ensure the proper level of fork remodeling needed to maintain genome integrity during DNA synthesis. PMID:24150942

  14. The distinct C-terminal acidic domains of HMGB proteins are functionally relevant in Schistosoma mansoni.

    PubMed

    de Abreu da Silva, Isabel Caetano; Carneiro, Vitor Coutinho; Vicentino, Amanda Roberta Revoredo; Aguilera, Estefania Anahi; Mohana-Borges, Ronaldo; Thiengo, Silvana; Fernandez, Monica Ammon; Fantappié, Marcelo Rosado

    2016-04-01

    The Schistosoma mansoni High Mobility Group Box (HMGB) proteins SmHMGB1, SmHMGB2 and SmHMGB3 share highly conserved HMG box DNA binding domains but have significantly different C-terminal acidic tails. Here, we used three full-length and tailless forms of the S. mansoni HMGB proteins to examine the functional roles of their acidic tails. DNA binding assays revealed that the different lengths of the acidic tails among the three SmHMGB proteins significantly and distinctively influenced their DNA transactions. Spectroscopic analyses indicated that the longest acidic tail of SmHMGB3 contributes to the structural stabilisation of this protein. Using immunohistochemical analysis, we showed distinct patterns of SmHMGB1, SmHMGB2 and SmHMGB3 expression in different tissues of adult worms. RNA interference approaches indicated a role for SmHMGB2 and SmHMGB3 in the reproductive system of female worms, whereas for SmHMGB1 no clear phenotype was observed. Schistosome HMGB proteins can be phosphorylated, acetylated and methylated. Importantly, the acetylation and methylation of schistosome HMGBs were greatly enhanced upon removal of the acidic tail. These data support the notion that the C-terminal acidic tails dictate the differences in the structure, expression and function of schistosome HMGB proteins. PMID:26820302

  15. Export of autotransported proteins proceeds through an oligomeric ring shaped by C-terminal domains.

    PubMed

    Veiga, Esteban; Sugawara, Etsuko; Nikaido, Hiroshi; de Lorenzo, Víctor; Fernández, Luis Angel

    2002-05-01

    An investigation was made into the oligomerization, the ability to form pores and the secretion-related properties of the 45 kDa C-terminal domain of the IgA protease (C-IgAP) from Neisseria gonorrhoeae. This protease is the best studied example of the autotransporters (ATs), a large family of exoproteins from Gram-negative bacteria that includes numerous virulence factors from human pathogens. These proteins contain an N-terminal passenger domain that em bodies the secreted polypeptide, while the C-domain inserts into the outer membrane (OM) and trans locates the linked N-module into the extracellular medium. Here we report that purified C-IgAP forms an oligomeric complex of approximately 500 kDa with a ring-like structure containing a central cavity of approximately 2 nm diameter that is the conduit for the export of the N-domains. These data overcome the previous model for ATs, which postulated the passage of the N-module through the hydrophilic channel of the beta-barrel of each monomeric C-domain. Our results advocate a secretion mechanism not unlike other bacterial export systems, such as the secretins or fimbrial ushers, which rely on multimeric complexes assembled in the OM. PMID:11980709

  16. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    PubMed Central

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  17. A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

    PubMed Central

    Marshall, M S; Hill, W S; Ng, A S; Vogel, U S; Schaber, M D; Scolnick, E M; Dixon, R A; Sigal, I S; Gibbs, J B

    1989-01-01

    The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein. Images PMID:2545441

  18. The C-terminal region of Drosophila heat shock factor (HSF) contains a constitutively functional transactivation domain.

    PubMed Central

    Wisniewski, J; Orosz, A; Allada, R; Wu, C

    1996-01-01

    The heat shock transcription factor (HSF) is constitutively expressed in Drosophila cells as an inactive monomer. Upon heat shock HSF undergoes trimerization and acquires high affinity DNA binding ability leading to specific interaction with its cognate elements in heat shock promoters. Here we show that the transactivation function of HSF is conferred by the extreme C-terminal region of the protein. Deletion analysis of HSF fragments fused to the GAL4 DNA-binding domain demonstrates that transactivation is dependent on HSF residues 610-691. This domain is located beyond the C-terminal heptad repeat (leucine zipper 4) whose presence or integrity is dispensable for transactivation. The transactivation domain is functional in the absence of heat shock and can be replaced by the extreme C-terminal region of human HSF1. The Drosophila and human HSF transactivation domains are both rich in hydrophobic and acidic residues and may be structurally conserved, despite limited sequence identity. PMID:8628664

  19. The C-terminal domain of NifL is sufficient to inhibit NifA activity.

    PubMed Central

    Narberhaus, F; Lee, H S; Schmitz, R A; He, L; Kustu, S

    1995-01-01

    In Klebsiella pneumoniae, transcription of all nif (nitrogen fixation) operons except the regulatory nifLA operon itself is regulated by the proteins NifA and NifL. NifA, an enhancer-binding protein, activates transcription by RNA polymerase containing the alternative sigma factor sigma 54. The central catalytic domain of NifA is sufficient for transcriptional activation, which can occur from solution. In vivo, NifL antagonizes the action of NifA in the presence of molecular oxygen or combined nitrogen. Inhibition has also been shown in vitro, but it was not responsive to environmental signals. Assuming a two-domain structure of NifL, we localized inhibition by NifL to its carboxy (C)-terminal domain, which is more soluble than the intact protein. The first line of evidence for this is that internal deletions of NifL containing an intact C-terminal domain were able to inhibit transcriptional activation by NifA in a coupled transcription-translation system. The second line of evidence is that the isolated C-terminal domain of NifL (assayed as a fusion to the soluble maltose-binding protein [MBP]) was sufficient to inhibit transcriptional activation by the central domain of NifA in a purified transcription system. The final line of evidence is that an MBP fusion to the C-terminal domain of NifL inhibited transcriptional activation by NifA in vivo. On the basis of these data, we postulate that the inhibitory function of NifL lies in its C-terminal domain and hence infer that this domain is responsible for interaction with NifA. Gel filtration experiments with MBP-NifL fusion derivatives lacking portions of the N- or C-terminal domain of the protein revealed that the C-terminal domain is the most soluble part of NifL. Up to 50% of two MBP-NifL truncations containing only the C-terminal domain appeared to be in a defined dimeric state. PMID:7665487

  20. Structure of the C-terminal domain of nsp4 from feline coronavirus

    PubMed Central

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh; Snijder, Eric J.; Gorbalenya, Alexander E.; Berglind, Hanna; Nordlund, Pär; Coutard, Bruno; Tucker, Paul A.

    2009-01-01

    Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions. PMID:19622868

  1. Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

    SciTech Connect

    Yun, Ji-Hye; Kim, Heeyoun; Park, Jung Eun; Lee, Jung Sup; Lee, Weontae

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We have determined solution structures of vEP C-terminal regulatory domain. Black-Right-Pointing-Pointer vEP C-ter100 has a compact {beta}-barrel structure with eight anti-parallel {beta}-strands. Black-Right-Pointing-Pointer Solution structure of vEP C-ter100 shares its molecular topology with that of the collagen-binding domain of collagenase. Black-Right-Pointing-Pointer Residues in the {beta}3 region of vEP C-ter100 might be important in putative ligand/receptor binding. Black-Right-Pointing-Pointer vEP C-ter100 interacts strongly with iron ion. -- Abstract: An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel {beta}-strands with a unique fold that has a compact {beta}-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe{sup 3+}). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates

  2. A TRPV4 Channel C-terminal Folding Recognition Domain Critical for Trafficking and Function*

    PubMed Central

    Lei, Lei; Cao, Xu; Yang, Fan; Shi, Di-Jing; Tang, Yi-Quan; Zheng, Jie; Wang, KeWei

    2013-01-01

    The Ca2+-permeable transient receptor potential vanilloid subtype 4 (TRPV4) channel mediates crucial physiological functions, such as calcium signaling, temperature sensing, and maintaining cell volume and energy homeostasis. Noticeably, most disease-causing genetic mutations are concentrated in the cytoplasmic domains. In the present study, we focused on the role of the TRPV4 C terminus in modulating protein folding, trafficking, and activity. By examining a series of C-terminal deletions, we identified a 20-amino acid distal region covering residues 838–857 that is critical for channel folding, maturation, and trafficking. Surface biotinylation, confocal imaging, and fluorescence-based calcium influx assay demonstrated that mutant proteins missing this region were trapped in the endoplasmic reticulum and unglycosylated, leading to accelerated degradation and loss of channel activity. Rosetta de novo structural modeling indicated that residues 838–857 assume a defined conformation, with Gly849 and Pro851 located at critical positions. Patch clamp recordings confirmed that lowering the temperature from 37 to 30 °C rescued channel activity of folding-defective mutants. Moreover, biochemical tests demonstrated that, in addition to participating in C-C interaction, the C terminus also interacts with the N terminus. Taken together, our findings indicate that the C-terminal region of TRPV4 is critical for channel protein folding and maturation, and the short distal segment plays an essential role in this process. Therefore, selectively disrupting the folding-sensitive region may present therapeutic potential for treating overactive TRPV4-mediated diseases, such as pain and skeletal dysplasias. PMID:23457335

  3. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins

    PubMed Central

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners. PMID:22536173

  4. Interaction of chromatin with a histone H1 containing swapped N- and C-terminal domains

    PubMed Central

    Hutchinson, Jordana B.; Cheema, Manjinder S.; Wang, Jason; Missiaen, Krystal; Finn, Ron; Gonzalez Romero, Rodrigo; Th’ng, John P. H.; Hendzel, Michael; Ausió, Juan

    2015-01-01

    Although the details of the structural involvement of histone H1 in the organization of the nucleosome are quite well understood, the sequential events involved in the recognition of its binding site are not as well known. We have used a recombinant human histone H1 (H1.1) in which the N- and C-terminal domains (NTD/CTD) have been swapped and we have reconstituted it on to a 208-bp nucleosome. We have shown that the swapped version of the protein is still able to bind to nucleosomes through its structurally folded wing helix domain (WHD); however, analytical ultracentrifuge analysis demonstrates its ability to properly fold the chromatin fibre is impaired. Furthermore, FRAP analysis shows that the highly dynamic binding association of histone H1 with the chromatin fibre is altered, with a severely decreased half time of residence. All of this suggests that proper binding of histone H1 to chromatin is determined by the simultaneous and synergistic binding of its WHD–CTD to the nucleosome. PMID:26182371

  5. Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.

    PubMed Central

    Sanford, J C; Pan, Y; Wessling-Resnick, M

    1995-01-01

    Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue. Images PMID:7749197

  6. Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

    SciTech Connect

    Kamada, Katsuhiko; De Angelis, Jacqueline; Roeder, Robert G.; Burley, Stephen K.

    2012-12-13

    The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-{angstrom} resolution. The {alpha}/{beta} structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3{gamma} (HNF-3{gamma}), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3{gamma} and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the {beta} subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

  7. Structure of the C-terminal domain of the arginine repressor protein from Mycobacterium tuberculosis

    SciTech Connect

    Cherney, Leonid T.; Cherney, Maia M.; Garen, Craig R.; Lu, George J.; James, Michael N. G.

    2008-09-01

    The structure of the core domain of the arginine repressor protein from M. tuberculosis has been determined with (1.85 Å resolution) and without (2.15 Å resolution) the arginine corepressor bound. Three additional arginine molecules have been found to bind to the core domain hexamer at high (0.2 M) arginine concentration. The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the l-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of arginine repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 Å resolution with bound arginine and at 2.15 Å resolution in the unliganded form. These structures show that six molecules of MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11° upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.

  8. A helix-turn motif in the C-terminal domain of histone H1.

    PubMed

    Vila, R; Ponte, I; Jiménez, M A; Rico, M; Suau, P

    2000-04-01

    The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs. PMID:10794405

  9. Peptide library approach to uncover phosphomimetic inhibitors of the BRCA1 C-terminal domain.

    PubMed

    White, E Railey; Sun, Luxin; Ma, Zhong; Beckta, Jason M; Danzig, Brittany A; Hacker, David E; Huie, Melissa; Williams, David C; Edwards, Ross A; Valerie, Kristoffer; Glover, J N Mark; Hartman, Matthew C T

    2015-05-15

    Many intracellular protein-protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein-protein interactions mediated by serine phosphorylation. PMID:25654734

  10. Peptide Library Approach to Uncover Phosphomimetic Inhibitors of the BRCA1 C-Terminal Domain

    PubMed Central

    White, E. Railey; Sun, Luxin; Ma, Zhong; Beckta, Jason M.; Danzig, Brittany A.; Hacker, David E.; Huie, Melissa; Williams, David C.; Edwards, Ross A.; Valerie, Kristoffer; Mark Glover, J. N.; Hartman, Matthew C. T.

    2015-01-01

    Many intracellular protein–protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein–protein interactions mediated by serine phosphorylation. PMID:25654734

  11. NMR assignments of the C-terminal domain of human polypeptide release factor eRF1.

    PubMed

    Mantsyzov, Alexey B; Ivanova, Elena V; Birdsall, Berry; Kolosov, Petr M; Kisselev, Lev L; Polshakov, Vladimir I

    2007-12-01

    We report NMR assignments of the protein backbone of the C-terminal domain (163 a.a.) of human class 1 translation termination factor eRF1. It was found that several protein loop residues exist in two slowly interconverting conformational states. PMID:19636860

  12. Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

    PubMed Central

    Wiedemann, Christoph; Szambowska, Anna; Häfner, Sabine; Ohlenschläger, Oliver; Gührs, Karl-Heinz; Görlach, Matthias

    2015-01-01

    The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain. PMID:25712103

  13. Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit.

    PubMed Central

    Buratowski, S; Sharp, P A

    1990-01-01

    RNA polymerase II assembles with other factors on the adenovirus type 2 major late promoter to generate pairs of transcription initiation complexes resolvable by nondenaturing gel electrophoresis. The pairing of the complexes is caused by the presence or absence of the C-terminal domain of the largest subunit. This domain is not required for transcription stimulation by the major late transcription factor in vitro. Images PMID:2398901

  14. DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain.

    PubMed

    Morrill, Summer A; Exner, Alexandra E; Babokhov, Michael; Reinfeld, Bradley I; Fuchs, Stephen M

    2016-05-27

    The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24-26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1 In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length. PMID:27026700

  15. DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain*

    PubMed Central

    Morrill, Summer A.; Exner, Alexandra E.; Babokhov, Michael; Reinfeld, Bradley I.

    2016-01-01

    The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae. First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24–26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1. In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length. PMID:27026700

  16. The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3

    PubMed Central

    Troeberg, Linda; Fushimi, Kazunari; Scilabra, Simone D.; Nakamura, Hiroyuki; Dive, Vincent; Thøgersen, Ida B.; Enghild, Jan J.; Nagase, Hideaki

    2010-01-01

    We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better Ki value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3. PMID:19643179

  17. The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3.

    PubMed

    Troeberg, Linda; Fushimi, Kazunari; Scilabra, Simone D; Nakamura, Hiroyuki; Dive, Vincent; Thøgersen, Ida B; Enghild, Jan J; Nagase, Hideaki

    2009-10-01

    We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better K(i) value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3. PMID:19643179

  18. Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain.

    PubMed

    Alexandrovich, A; Czisch, M; Frenkiel, T A; Kelly, G P; Goosen, N; Moolenaar, G F; Chowdhry, B Z; Sanderson, M R; Lane, A N

    2001-10-01

    The solution structure, thermodynamic stability and hydrodynamic properties of the 55-residue C-terminal domain of UvrB that interacts with UvrC during excision repair in E. coli have been determined using a combination of high resolution NMR, ultracentrifugation, 15N NMR relaxation, gel permeation, NMR diffusion, circular dichroism and differential scanning calorimetry. The subunit molecular weight is 7,438 kDa., compared with 14.5+/-1.0 kDa. determined by equilibrium sedimentation, indicating a dimeric structure. The structure determined from NMR showed a stable dimer of anti-parallel helical hairpins that associate in an unusual manner, with a small and hydrophobic interface. The Stokes radius of the protein decreases from a high plateau value (ca. 22 A) at protein concentrations greater than 4 microM to about 18 A at concentrations less than 0.1 microM. The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (Tm ca. 71.5 degrees C at 36 microM). The simplest model consistent with these data was a dimer dissociating into folded monomers that then unfolds co-operatively. The van't Hoff enthalpy and dissociation constant for both transition was derived by fitting, with deltaH1=23 kJ mol(-1). K1(298)=0.4 microM and deltaH2= 184 kJ mol(-1). This is in good agreement with direct calorimetric analysis of the thermal unfolding of the protein, which gave a calorimetric enthalpy change of 181 kJ mol(-1) and a van't Hoff enthalpy change of 354 kJ mol(-1), confirming the dimer to monomer unfolding. The thermodynamic data can be reconciled with the observed mode of dimerisation. 15N NMR relaxation measurements at 14.1 T and 11.75 T confirmed that the protein behaves as an asymmetric dimer at mM concentrations, with a flexible N-terminal linker for attachment to the remainder of the UvrB protein. The role of dimerisation of this domain in the excision repair mechanism is discussed. PMID:11697728

  19. β-Subunit Binding Is Sufficient for Ligands to Open the Integrin αIIbβ3 Headpiece*

    PubMed Central

    Lin, Fu-Yang; Zhu, Jianghai; Eng, Edward T.; Hudson, Nathan E.; Springer, Timothy A.

    2016-01-01

    The platelet integrin αIIbβ3 binds to a KQAGDV motif at the fibrinogen γ-chain C terminus and to RGD motifs present in loops in many extracellular matrix proteins. These ligands bind in a groove between the integrin α and β-subunits; the basic Lys or Arg side chain hydrogen bonds to the αIIb-subunit, and the acidic Asp side chain coordinates to a metal ion held by the β3-subunit. Ligand binding induces headpiece opening, with conformational change in the β-subunit. During this opening, RGD slides in the ligand-binding pocket toward αIIb, with movement of the βI-domain β1-α1 loop toward αIIb, enabling formation of direct, charged hydrogen bonds between the Arg side chain and αIIb. Here we test whether ligand interactions with β3 suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the αIIbβ3 headpiece with affinity comparable with the RGDSP peptide from fibronectin. AGDV induced complete headpiece opening in solution as shown by increase in hydrodynamic radius. Soaking of AGDV into closed αIIbβ3 headpiece crystals induced intermediate states similarly to RGDSP. AGDV has very little contact with the α-subunit. Furthermore, as measured by epitope exposure, AGDV, like the fibrinogen γ C-terminal peptide and RGD, caused integrin extension on the cell surface. Thus, pushing by the β3-subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the αIIb subunit on Arg is required. PMID:26631735

  20. β-Subunit Binding Is Sufficient for Ligands to Open the Integrin αIIbβ3 Headpiece.

    PubMed

    Lin, Fu-Yang; Zhu, Jianghai; Eng, Edward T; Hudson, Nathan E; Springer, Timothy A

    2016-02-26

    The platelet integrin αIIbβ3 binds to a KQAGDV motif at the fibrinogen γ-chain C terminus and to RGD motifs present in loops in many extracellular matrix proteins. These ligands bind in a groove between the integrin α and β-subunits; the basic Lys or Arg side chain hydrogen bonds to the αIIb-subunit, and the acidic Asp side chain coordinates to a metal ion held by the β3-subunit. Ligand binding induces headpiece opening, with conformational change in the β-subunit. During this opening, RGD slides in the ligand-binding pocket toward αIIb, with movement of the βI-domain β1-α1 loop toward αIIb, enabling formation of direct, charged hydrogen bonds between the Arg side chain and αIIb. Here we test whether ligand interactions with β3 suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the αIIbβ3 headpiece with affinity comparable with the RGDSP peptide from fibronectin. AGDV induced complete headpiece opening in solution as shown by increase in hydrodynamic radius. Soaking of AGDV into closed αIIbβ3 headpiece crystals induced intermediate states similarly to RGDSP. AGDV has very little contact with the α-subunit. Furthermore, as measured by epitope exposure, AGDV, like the fibrinogen γ C-terminal peptide and RGD, caused integrin extension on the cell surface. Thus, pushing by the β3-subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the αIIb subunit on Arg is required. PMID:26631735

  1. The C-terminal domain is sufficient for host-binding activity of the Mu phage tail-spike protein.

    PubMed

    Suzuki, Hidetaka; Yamada, Seiko; Toyama, Yoshiharu; Takeda, Shigeki

    2010-09-01

    The Mu phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58 kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other phages; however, gp45 did not show significant sequence homology with the other phage tail-spike structures that have been identified. PMID:20478417

  2. Crystal Structure of the C-terminal Domain of Splicing Factor Prp8 Carrying Retinitis Pigmentosa Mutants

    SciTech Connect

    Zhang,L.; Shen, J.; Guarnieri, M.; Heroux, A.; Yang, K.; Zhao, R.

    2007-01-01

    Prp8 is a critical pre-mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been hindered by its large size (over 2000 amino acids) and remarkably low-sequence similarity with other proteins. Here we present the crystal structure of the C-terminal domain (the last 273 residues) of Caenorhabditis elegans Prp8 (cPrp8). The core of the C-terminal domain is an / structure that forms the MPN (Mpr1, Pad1 N-terminal) fold but without Zn{sup 2+} coordination. We propose that the C-terminal domain is a protein interaction domain instead of a Zn{sup 2+}-dependent metalloenzyme as proposed for some MPN proteins. Mapping of RP13 mutants on the Prp8 structure suggests that these residues constitute a binding surface between Prp8 and other partner(s), and the disruption of this interaction provides a plausible molecular mechanism for RP13.

  3. Crystal Structure in the Vivo-Assembled Bacillus subtilis Spx/RNA Polymerase alpha Subunit C-Terminal Domain Complex

    SciTech Connect

    Lamour, V.; Westblade, L; Campbell, E; Darst, S

    2009-01-01

    The Bacillus subtilis Spx protein is a global transcription factor that interacts with the C-terminal domain of the RNA polymerase {alpha} subunit ({alpha}CTD) and regulates transcription of genes involved in thiol-oxidative stress, sporulation, competence, and organosulfur metabolism. Here we determined the X-ray crystal structure of the Spx/{alpha}CTD complex from an entirely new crystal form than previously reported [Newberry, K.J., Nakano, S., Zuber, P., Brennan, R.G., 2005. Crystal structure of the Bacillus subtilis anti-alpha, global transcriptional regulator, Spx, in complex with the alpha C-terminal domain of RNA polymerase. Proc. Natl. Acad. Sci. USA 102, 15839-15844]. Comparison of the previously reported sulfate-bound complex and our sulfate-free complex reveals subtle conformational changes that may be important for the role of Spx in regulating organosulfur metabolism.

  4. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA.

    PubMed

    Dwivedi, Gajendradhar R; Srikanth, Kolluru D; Anand, Praveen; Naikoo, Javed; Srilatha, N S; Rao, Desirazu N

    2015-01-01

    DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA) and double stranded DNA (dsDNA). Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed. PMID:26135134

  5. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA

    PubMed Central

    Dwivedi, Gajendradhar R.; Srikanth, Kolluru D.; Anand, Praveen; Naikoo, Javed; Srilatha, N. S.; Rao, Desirazu N.

    2015-01-01

    DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA) and double stranded DNA (dsDNA). Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed. PMID:26135134

  6. Probing the Impact of the EchinT C-Terminal Domain on Structure and Catalysis

    SciTech Connect

    S Bardaweel; J Pace; T Chou; V Cody; C Wagner

    2011-12-31

    Histidine triad nucleotide binding protein (Hint) is considered as the ancestor of the histidine triad protein superfamily and is highly conserved from bacteria to humans. Prokaryote genomes, including a wide array of both Gram-negative bacteria and Gram-positive bacteria, typically encode one Hint gene. The cellular function of Hint and the rationale for its evolutionary conservation in bacteria have remained a mystery. Despite its ubiquity and high sequence similarity to eukaryote Hint1 [Escherichia coli Hint (echinT) is 48% identical with human Hint1], prokaryote Hint has been reported in only a few studies. Here we report the first conformational information on the full-length N-terminal and C-terminal residues of Hint from the E. coli complex with GMP. Structural analysis of the echinT-GMP complex reveals that it crystallizes in the monoclinic space group P2{sub 1} with four homodimers in the asymmetric unit. Analysis of electron density for both the N-terminal residues and the C-terminal residues of the echinT-GMP complex indicates that the loops in some monomers can adopt more than one conformation. The observation of conformational flexibility in terminal loop regions could explain the presence of multiple homodimers in the asymmetric unit of this structure. To explore the impact of the echinT C-terminus on protein structure and catalysis, we conducted a series of catalytic radiolabeling and kinetic experiments on the C-terminal deletion mutants of echinT. In this study, we show that sequential deletion of the C-terminus likely has no effect on homodimerization and a modest effect on the secondary structure of echinT. However, we observed a significant impact on the folding structure, as reflected by a significant lowering of the T{sub m} value. Kinetic analysis reveals that the C-terminal deletion mutants are within an order of magnitude less efficient in catalysis compared to wild type, while the overall kinetic mechanism that proceeds through a fast step

  7. Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

    SciTech Connect

    Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N.

    2012-03-15

    The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

  8. C-terminal activating and inhibitory domains determine the transactivation potential of BSAP (Pax-5), Pax-2 and Pax-8.

    PubMed Central

    Dörfler, P; Busslinger, M

    1996-01-01

    Pax-5 encodes the transcription factor BSAP which plays an essential role in early B cell development and midbrain patterning. In this study we have analysed the structural requirements for transcriptional activation by BSAP. In vitro mutagenesis and transient transfection experiments indicate that the C-terminal serine/threonine/proline-rich region of BSAP contains a potent transactivation domain of 55 amino acids which is active from promoter and enhancer positions. This transactivation domain was found to be inactivated by a naturally occurring frameshift mutation in one PAX-5 allele of the acute lymphoblastic leukemia cell line REH. The function of the transactivation domain is negatively regulated by adjacent sequences from the extreme C-terminus. The activating and inhibitory domains function together as an independent regulatory module in different cell types as shown by fusion to the GAL4 DNA binding domain. The same arrangement of positively and negatively acting sequences has been conserved in the mammalian Pax-2 and Pax-8, the zebrafish Pax-b as well as the sea urchin Pax-258 proteins. These data demonstrate that the transcriptional competence of a subfamily of Pax proteins is determined by a C-terminal regulatory module composed of activating and inhibitory sequences. Images PMID:8617244

  9. Consequences of C-terminal domains and N-terminal signal peptide deletions on LEKTI secretion, stability, and subcellular distribution.

    PubMed

    Jayakumar, Arumugam; Kang, Ya'an; Henderson, Ying; Mitsudo, Kenji; Liu, Xiaoling; Briggs, Katrina; Wang, Mary; Frederick, Mitchell J; El-Naggar, Adel K; Bebök, Zsuzsa; Clayman, Gary L

    2005-03-01

    The secretory lympho-epithelial Kazal-type-inhibitor (LEKTI) is synthesized as a pro-LEKTI protein containing an N-terminal signal peptide and 15 potentially inhibitory domains. This inhibitor is of special interest because of its pathophysiological importance for the severe congenital disease Netherton syndrome. We showed that LEKTI is a potent inhibitor of a family of serine proteinases involved in extracellular matrix remodeling and its expression is downregulated in head and neck squamous cell carcinomas. To assess the role of C-terminal domains and N-terminal signal peptide in LEKTI secretion, we constructed deletion mutants of LEKTI, expressed them in HEK 293T cells, and analyzed their secretion behavior, stability, subcellular distribution, and proteinase inhibitory function. Pro-LEKTI is processed and secreted into the medium. On the basis of partial N-terminal sequencing and immunoblotting, the cleavage products are ordered from amino- to carboxy-terminal as follows: 37, 40, and 60kDa. Inhibitors of furin lead to enhanced secretion of unprocessed LEKTI, suggesting that processing was not required for secretion. Deletion of the N-terminal signal peptide of pro-LEKTI caused altered distribution of LEKTI from endoplasmic reticulum (ER) to cytoplasm and markedly reduced its stability, consistent with its failure to become secreted into the medium. Interestingly, when we deleted the C-terminal domains, stable partial LEKTI (LD-1-6) accumulated and still retained its association with ER but was not secreted. Recombinant LD-1-6 specifically inhibited the trypsin activity. We conclude that N-terminal signal peptide is required for LEKTI import into ER and elements present in C-terminal domains may have a role in regulating LEKTI secretion. PMID:15680911

  10. Expression, purification and preliminary crystallographic studies of the C-terminal SH3 domain of human Tks4.

    PubMed

    Huang, Yuxin; Qian, Huolian; Wang, Xiaoying; Cheng, Zhong; Ren, Jixia; Zhao, Weichen; Xie, Yong

    2014-03-01

    The Src homology 3 (SH3) domain is a small, noncatalytic domain with a conserved sequence of about 60 amino-acid residues that interacts with proline-rich peptides to form a protein complex. In this study, the C-terminal SH3 domain of human Tks4 (residues 853-911) was expressed, purified and crystallized. X-ray diffraction data were collected to 2.3 Å resolution. The crystal belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = b = 83.87, c = 108.44 Å, α = β = 90, γ = 120°. Calculating the self-rotation and the native Patterson function did not lead to the detection of any noncrystallographic translational symmetry. Six, seven or eight protein molecules are likely to be present in the asymmetric unit, resulting in a Matthews coefficient and approximate solvent content of 2.71 Å(3) Da(-1) and 55%, 2.32 Å(3) Da(-1) and 47%, and 2.03 Å(3) Da(-1) and 39%, respectively. To solve the crystal structure of the C-terminal SH3 domain of human Tks4, the isomorphous replacement method is presently being utilized. PMID:24598923

  11. Structure of a bacterial putative acetyltransferase defines the fold of the human O-GlcNAcase C-terminal domain

    PubMed Central

    Rao, Francesco V.; Schüttelkopf, Alexander W.; Dorfmueller, Helge C.; Ferenbach, Andrew T.; Navratilova, Iva; van Aalten, Daan M. F.

    2013-01-01

    The dynamic modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification present in higher eukaryotes. Removal of O-GlcNAc is catalysed by O-GlcNAcase, a multi-domain enzyme that has been reported to be bifunctional, possessing both glycoside hydrolase and histone acetyltransferase (AT) activity. Insights into the mechanism, protein substrate recognition and inhibition of the hydrolase domain of human OGA (hOGA) have been obtained via the use of the structures of bacterial homologues. However, the molecular basis of AT activity of OGA, which has only been reported in vitro, is not presently understood. Here, we describe the crystal structure of a putative acetyltransferase (OgpAT) that we identified in the genome of the marine bacterium Oceanicola granulosus, showing homology to the hOGA C-terminal AT domain (hOGA-AT). The structure of OgpAT in complex with acetyl coenzyme A (AcCoA) reveals that, by homology modelling, hOGA-AT adopts a variant AT fold with a unique loop creating a deep tunnel. The structures, together with mutagenesis and surface plasmon resonance data, reveal that while the bacterial OgpAT binds AcCoA, the hOGA-AT does not, as explained by the lack of key residues normally required to bind AcCoA. Thus, the C-terminal domain of hOGA is a catalytically incompetent ‘pseudo’-AT. PMID:24088714

  12. Hevea brasiliensis prohevein possesses a conserved C-terminal domain with amyloid-like properties in vitro.

    PubMed

    Berthelot, Karine; Lecomte, Sophie; Coulary-Salin, Bénédicte; Bentaleb, Ahmed; Peruch, Frédéric

    2016-04-01

    Prohevein is a wound-induced protein and a main allergen from latex of Hevea brasiliensis (rubber tree). This 187 amino-acid protein is cleaved in two fragments: a N-terminal 43 amino-acids called hevein, a lectin bearing a chitin-binding motif with antifungal properties and a C-terminal domain (C-ter) far less characterized. We provide here new insights on the characteristics of prohevein, hevein and C-terminal domain. Using complementary biochemical (ThT/CR/chitin binding, agglutination) and structural (modeling, ATR-FTIR, TEM, WAXS) approaches, we show that this domain clearly displays all the characteristics of an amyloid-like proteins in vitro, that could confer agglutination activity in synergy with its chitin-binding activity. Additionally, this C-ter domain is highly conserved and present in numerous plant prohevein-like proteins or pathogenesis-related (PR and WIN) proteins. This could be the hallmark of the eventual presence of proteins with amyloid properties in plants, that could potentially play a role in defense through aggregation properties. PMID:26805576

  13. Crystallization of the C-terminal redox domain of the sulfur-assimilatory enzyme APR1 from Arabidopsis thaliana

    PubMed Central

    Chen, Fang-Fang; Chang, Yu-Yung; Cho, Chao-Cheng; Hsu, Chun-Hua

    2014-01-01

    Plant-type APS reductase (APR), which catalyzes the reduction of activated sulfate to sulfite in plants, consists of a reductase domain and a C-terminal redox domain showing sequence homology to thioredoxin but possessing the activity of glutaredoxin. In order to understand the structural and biochemical properties of the redox domain of plant-type APS reductase, the C-terminal domain of APR1 (APR1C) from Arabidopsis thaliana was crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to a resolution of 2.70 Å on the SPXF beamline BL13B1 at the NSRRC, Taiwan. The crystals belonged to space group P43212 or P41212, with unit-cell parameters a = b = 58.2, c = 86.7 Å. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V M) is 2.64 Å3 Da−1, which corresponds to a solvent content of approximately 53.49%. Further structure-based functional studies of APR1C would extend knowledge of the molecular mechanism and regulation of APR. PMID:25195893

  14. NMR solution structure and function of the C-terminal domain of eukaryotic class 1 polypeptide chain release factor.

    PubMed

    Mantsyzov, Alexey B; Ivanova, Elena V; Birdsall, Berry; Alkalaeva, Elena Z; Kryuchkova, Polina N; Kelly, Geoff; Frolova, Ludmila Y; Polshakov, Vladimir I

    2010-06-01

    Termination of translation in eukaryotes is triggered by two polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3. eRF1 is a three-domain protein that interacts with eukaryotic class 2 polypeptide chain release factor 3 via its C-terminal domain (C-domain). The high-resolution NMR structure of the human C-domain (residues 277-437) has been determined in solution. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure. The structure of the minidomain (residues 329-372), which was ill-defined in the crystal structure, has been determined in solution. The protein backbone dynamics, studied using (15)N-relaxation experiments, showed that the C-terminal tail 414-437 and the minidomain are the most flexible parts of the human C-domain. The minidomain exists in solution in two conformational states, slowly interconverting on the NMR timescale. Superposition of this NMR solution structure of the human C-domain onto the available crystal structure of full-length human eRF1 shows that the minidomain is close to the stop codon-recognizing N-terminal domain. Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor. The results provide new insights into the possible role of the C-domain in the process of translation termination. PMID:20553496

  15. The C-terminal domain of Cernunnos/XLF is dispensable for DNA repair in vivo.

    PubMed

    Malivert, Laurent; Callebaut, Isabelle; Rivera-Munoz, Paola; Fischer, Alain; Mornon, Jean-Paul; Revy, Patrick; de Villartay, Jean-Pierre

    2009-03-01

    The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair. PMID:19103754

  16. Folding of the C-terminal bacterial binding domain in statherin upon adsorption onto hydroxyapatite crystals

    PubMed Central

    Goobes, Gil; Goobes, Rivka; Schueler-Furman, Ora; Baker, David; Stayton, Patrick S.; Drobny, Gary P.

    2006-01-01

    Statherin is an enamel pellicle protein that inhibits hydroxyapatite (HAP) nucleation and growth, lubricates the enamel surface, and is recognized by oral bacteria in periodontal diseases. We report here from solid-state NMR measurements that the protein's C-terminal region folds into an α-helix upon adsorption to HAP crystals. This region contains the binding sites for bacterial fimbriae that mediate bacterial cell adhesion to the surface of the tooth. The helical segment is shown through long-range distance measurements to fold back onto the intermediate region (residues Y16–P28) defining the global fold of the protein. Statherin, previously shown to be unstructured in solution, undergoes conformation selection on its substrate mineral surface. This surface-induced folding of statherin can be related to its functionality in inhibiting HAP crystal growth and can explain how oral pathogens selectively recognize HAP-bound statherin. PMID:17060618

  17. Structure of the human Tim44 C-terminal domain in complex with pentaethylene glycol: ligand-bound form

    SciTech Connect

    Handa, N.; Kishishita, S.; Morita, S.; Akasaka, R.; Jin, Z.; Chrzas, J.; Chen, L.; Liu, Z.-J.; Wang, B.-C.; Sugano, S.; Tanaka, A.; Terada, T.; Shirouzu, M.; Yokoyama, S.

    2008-06-23

    Familial oncocytic thyroid carcinoma is associated with a missense mutation, P308Q, in the C-terminal domain of Tim44. Tim44 is the mitochondrial inner-membrane translocase subunit and it functions as a membrane anchor for the mitochondrial heat-shock protein 70 (mtHsp70). Here, the crystal structure of the human Tim44 C-terminal domain complexed with pentaethylene glycol has been determined at 1.9 {angstrom} resolution. The overall structure resembles that of the nuclear transport factor 2-like domain. In the crystal structure, pentaethylene glycol molecules are associated at two potential membrane-binding sites: the large hydrophobic cavity and the highly conserved loop between the {alpha} 1 and {alpha} 2 helices near Pro308. A comparison with the yeast homolog revealed that lipid binding induces conformational changes around the {alpha} 1-{alpha} 2 loop, leading to slippage of the {alpha} 1 helix along the large {beta}-sheet. These changes may play important roles in the translocation of polypeptides across the mitochondrial inner membrane.

  18. C-terminal domain of MEIS1 converts PKNOX1 (PREP1) into a HOXA9-collaborating oncoprotein.

    PubMed

    Bisaillon, Richard; Wilhelm, Brian T; Krosl, Jana; Sauvageau, Guy

    2011-10-27

    The three-amino-acid loop extension (TALE) class homeodomain proteins MEIS1 and PKNOX1 (PREP1) share the ability to interact with PBX and HOX family members and bind similar DNA sequences but appear to play opposing roles in tumor development. Elevated levels of MEIS1 accelerate development of HOX- and MLL-induced leukemias, and this pro-tumorigenic property has been associated with transcriptional activity of MEIS1. In contrast, reduction of PKNOX1 levels has been linked with cancer development despite the absence of an identifiable transactivating domain. In this report, we show that a chimeric protein generated by fusion of the MEIS1 C-terminal region encompassing the transactivating domain with the full-length PKNOX1 (PKNOX1-MC) acquired the ability to accelerate the onset of Hoxa9-induced leukemia in the mouse bone marrow transduction/transplantation model. Gene expression profiling of primary bone marrow cells transduced with Hoxa9 plus Meis1, or Hoxa9 plus Pknox1-MC revealed perturbations in overlapping functional gene subsets implicated in DNA packaging, chromosome organization, and in cell cycle regulation. Together, results presented in this report suggest that the C-terminal domain of MEIS1 confers to PKNOX1 an ectopic transactivating function that promotes leukemogenesis by regulating expression of genes involved in chromatin accessibility and cell cycle progression. PMID:21900201

  19. Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

    PubMed Central

    De Bock, Marijke; Wang, Nan; Decrock, Elke; Bultynck, Geert; Leybaert, Luc

    2015-01-01

    The coordination of tissue function is mediated by gap junctions (GJs) that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx) proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs) in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs) are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury. PMID:26424967

  20. Crystal structure of the lambda repressor C-terminal domain provides a model for cooperative operator binding.

    PubMed

    Bell, C E; Frescura, P; Hochschild, A; Lewis, M

    2000-06-23

    Interactions between transcription factors bound to separate operator sites commonly play an important role in gene regulation by mediating cooperative binding to the DNA. However, few detailed structural models for understanding the molecular basis of such cooperativity are available. The c1 repressor of bacteriophage lambda is a classic example of a protein that binds to its operator sites cooperatively. The C-terminal domain of the repressor mediates dimerization as well as a dimer-dimer interaction that results in the cooperative binding of two repressor dimers to adjacent operator sites. Here, we present the x-ray crystal structure of the lambda repressor C-terminal domain determined by multiwavelength anomalous diffraction. Remarkably, the interactions that mediate cooperativity are captured in the crystal, where two dimers associate about a 2-fold axis of symmetry. Based on the structure and previous genetic and biochemical data, we present a model for the cooperative binding of two lambda repressor dimers at adjacent operator sites. PMID:10892750

  1. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

    SciTech Connect

    Serek, Robert; Forwood, Jade K.; Hume, David A.; Martin, Jennifer L.; Kobe, Bostjan

    2006-02-01

    The C-terminal domain of the mouse long-chain acyl-CoA thioesterase has been expressed in bacteria and crystallized by vapour diffusion. The crystals diffract to 2.4 Å resolution. The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination.

  2. The C-Terminal Domain of Yeast PCNA Is Required for Physical And Functional Interactions With Cdc9 DNA Ligase

    SciTech Connect

    Vijayakumar, S.; Chapados, B.R.; Schmidt, K.H.; Kolodner, R.D.; Tainer, J.A.; Tomkinson, A.E.

    2007-07-13

    There is compelling evidence that proliferating cell nuclear antigen (PCNA), a DNA sliding clamp, co-ordinates the processing and joining of Okazaki fragments during eukaryotic DNA replication. However, a detailed mechanistic understanding of functional PCNA:ligase I interactions has been incomplete. Here we present the co-crystal structure of yeast PCNA with a peptide encompassing the conserved PCNA interaction motif of Cdc9, yeast DNA ligase I. The Cdc9 peptide contacts both the inter-domain connector loop (IDCL) and residues near the C-terminus of PCNA. Complementary mutational and biochemical results demonstrate that these two interaction interfaces are required for complex formation both in the absence of DNA and when PCNA is topologically linked to DNA. Similar to the functionally homologous human proteins, yeast RFC interacts with and inhibits Cdc9 DNA ligase whereas the addition of PCNA alleviates inhibition by RFC. Here we show that the ability of PCNA to overcome RFC-mediated inhibition of Cdc9 is dependent upon both the IDCL and the C-terminal interaction interfaces of PCNA. Together these results demonstrate the functional significance of the {beta}-zipper structure formed between the C-terminal domain of PCNA and Cdc9 and reveal differences in the interactions of FEN-1 and Cdc9 with the two PCNA interfaces that may contribute to the coordinated, sequential action of these enzymes.

  3. Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

    PubMed Central

    Scharinger, Anja; Eckrich, Stephanie; Vandael, David H.; Schönig, Kai; Koschak, Alexandra; Hecker, Dietmar; Kaur, Gurjot; Lee, Amy; Sah, Anupam; Bartsch, Dusan; Benedetti, Bruno; Lieb, Andreas; Schick, Bernhard; Singewald, Nicolas; Sinnegger-Brauns, Martina J.; Carbone, Emilio; Engel, Jutta; Striessnig, Jörg

    2015-01-01

    Cav1.3 L-type Ca2+-channel function is regulated by a C-terminal automodulatory domain (CTM). It affects channel binding of calmodulin and thereby tunes channel activity by interfering with Ca2+- and voltage-dependent gating. Alternative splicing generates short C-terminal channel variants lacking the CTM resulting in enhanced Ca2+-dependent inactivation and stronger voltage-sensitivity upon heterologous expression. However, the role of this modulatory domain for channel function in its native environment is unkown. To determine its functional significance in vivo, we interrupted the CTM with a hemagglutinin tag in mutant mice (Cav1.3DCRDHA/HA). Using these mice we provide biochemical evidence for the existence of long (CTM-containing) and short (CTM-deficient) Cav1.3 α1-subunits in brain. The long (HA-labeled) Cav1.3 isoform was present in all ribbon synapses of cochlear inner hair cells. CTM-elimination impaired Ca2+-dependent inactivation of Ca2+-currents in hair cells but increased it in chromaffin cells, resulting in hyperpolarized resting potentials and reduced pacemaking. CTM disruption did not affect hearing thresholds. We show that the modulatory function of the CTM is affected by its native environment in different cells and thus occurs in a cell-type specific manner in vivo. It stabilizes gating properties of Cav1.3 channels required for normal electrical excitability. PMID:26379493

  4. Membrane fusion by the GTPase atlastin requires a conserved C-terminal cytoplasmic tail and dimerization through the middle domain

    PubMed Central

    Moss, Tyler J.; Andreazza, Camilla; Verma, Avani; Daga, Andrea; McNew, James A.

    2011-01-01

    The biogenesis and maintenance of the endoplasmic reticulum (ER) requires membrane fusion. ER homotypic fusion is driven by the large GTPase atlastin. Domain analysis of atlastin shows that a conserved region of the C-terminal cytoplasmic tail is absolutely required for fusion activity. Atlastin in adjacent membranes must associate to bring the ER membranes into molecular contact. Drosophila atlastin dimerizes in the presence of GTPγS but is monomeric with GDP or without nucleotide. Oligomerization requires the juxtamembrane middle domain three-helix bundle, as does efficient GTPase activity. A soluble version of the N-terminal cytoplasmic domain that contains the GTPase domain and the middle domain three-helix bundle serves as a potent, concentration-dependent inhibitor of membrane fusion both in vitro and in vivo. However, atlastin domains lacking the middle domain are without effect. GTP-dependent dimerization of atlastin generates an enzymatically active protein that drives membrane fusion after nucleotide hydrolysis and conformational reorganization. PMID:21690399

  5. Solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP).

    PubMed

    Liew, Chu Kong; Crossley, Merlin; Mackay, Joel P; Nicholas, Hannah R

    2007-02-16

    The THAP (Thanatos-associated protein) domain is a recently discovered zinc-binding domain found in proteins involved in transcriptional regulation, cell-cycle control, apoptosis and chromatin modification. It contains a single zinc atom ligated by cysteine and histidine residues within a Cys-X(2-4)-Cys-X(35-53)-Cys-X(2)-His consensus. We have determined the NMR solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP) and show that it adopts a fold containing a treble clef motif, bearing similarity to the zinc finger-associated domain (ZAD) from Drosophila Grauzone. The CtBP THAP domain contains a large, positively charged surface patch and we demonstrate that this domain can bind to double-stranded DNA in an electrophoretic mobility-shift assay. These data, together with existing reports, indicate that THAP domains might exhibit a functional diversity similar to that observed for classical and GATA-type zinc fingers. PMID:17174978

  6. C-terminal domain of SMYD3 serves as a unique HSP90-regulated motif in oncogenesis

    PubMed Central

    Harriss, June; Das, Chhaya; Zhu, Li; Edwards, Melissa; Shaaban, Salam; Tucker, Haley

    2015-01-01

    The SMYD3 histone methyl transferase (HMTase) and the nuclear chaperone, HSP90, have been independently implicated as proto-oncogenes in several human malignancies. We show that a degenerate tetratricopeptide repeat (TPR)-like domain encoded in the SMYD3 C-terminal domain (CTD) mediates physical interaction with HSP90. We further demonstrate that the CTD of SMYD3 is essential for its basal HMTase activity and that the TPR-like structure is required for HSP90-enhanced enzyme activity. Loss of SMYD3-HSP90 interaction leads to SMYD3 mislocalization within the nucleus, thereby losing its chromatin association. This results in reduction of SMYD3-mediated cell proliferation and, potentially, impairment of SMYD3′s oncogenic activity. These results suggest a novel approach for blocking HSP90-driven malignancy in SMYD3-overexpressing cells with a reduced toxicity profile over current HSP90 inhibitors. PMID:25738358

  7. Functional dissection of the global repressor Tup1 in yeast: dominant role of the C-terminal repression domain.

    PubMed Central

    Zhang, Zhizhou; Varanasi, Ushasri; Trumbly, Robert J

    2002-01-01

    In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general repressor of transcription. Tup1 and Cyc8 are required for repression of diverse families of genes coordinately controlled by glucose repression, mating type, and other mechanisms. This repression is mediated by recruitment of the Cyc8-Tup1 complex to target promoters by sequence-specific DNA-binding proteins. We created a library of XhoI linker insertions and internal in-frame deletion mutations within the TUP1 coding region. Insertion mutations outside of the WD domains were wild type, while insertions within the WD domains induced mutant phenotypes with differential effects on the target genes SUC2, MFA2, RNR2, and HEM13. Deletion mutations confirmed previous findings of two separate repression domains in the N and C termini. The cumulative data suggest that the C-terminal repression domain, located near the first WD repeat, plays the dominant role in repression. Although the N-terminal repression domain is sufficient for partial repression, deletion of this region does not compromise repression. Surprisingly, deletion of the majority of the histone-binding domain of Tup1 also does not significantly reduce repression. The N-terminal region containing potential alpha-helical coiled coils is required for Tup1 oligomerization and association with Cyc8. Association with Cyc8 is required for repression of SUC2, HEM13, and RNR2 but not MFA2 and STE2. PMID:12136003

  8. C-terminal domains of a histone demethylase interact with a pair of transcription factors and mediate specific chromatin association

    PubMed Central

    Zhang, Shuaibin; Zhou, Bing; Kang, Yanyuan; Cui, Xia; Liu, Ao; Deleris, Angelique; Greenberg, Maxim V. C.; Cui, Xiekui; Qiu, Qi; Lu, Falong; Wohlschlegel, James A.; Jacobsen, Steven E.; Cao, Xiaofeng

    2015-01-01

    JmjC domain containing protein 14 (JMJ14) is an H3K4-specific histone demethylase that plays important roles in RNA-mediated gene silencing and flowering time regulation in Arabidopsis. However, how JMJ14 is recruited to its target genes remains unclear. Here, we show that the C-terminal FYRN and FYRC domains of JMJ14 are required for RNA silencing and flowering time regulation. Chromatin binding of JMJ14 is lost upon deletion of its FYRN and FYRC domains, and H3K4me3 is increased. FYRN and FYRC domains interact with a pair of NAC domain containing transcription factors, NAC050 and NAC052. Genome-wide ChIP analysis revealed that JMJ14 and NAC050/052 share a set of common target genes with CTTGNNNNNCAAG consensus sequences. Mutations in either NAC052 or NAC050 impair RNA-mediated gene silencing. Together, our findings demonstrate an important role of FYRN and FYRC domains in targeting JMJ14 through direct interaction with NAC050/052 proteins, which reveals a novel mechanism of histone demethylase recruitment. PMID:26617990

  9. The BARD1 C-Terminal Domain Structure and Interactions with Polyadenylation Factor CstF-50

    SciTech Connect

    Edwards, Ross A.; Lee, Megan S.; Tsutakawa, Susan E.; Williams, R. Scott; Tainer, John A.; Glover, J. N. Mark

    2009-07-13

    The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF- 50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins.Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

  10. Crystal structure of the C-terminal globular domain of the third paralog of the Archaeoglobus fulgidus oligosaccharyltransferases

    PubMed Central

    2013-01-01

    Background Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the asparagine residue in the N-glycosylation sequons. The catalytic subunits of the OST enzyme are STT3 in eukaryotes, AglB in archaea and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three paralogous AglB proteins. We previously solved the crystal structures of the C-terminal globular domains of two paralogs, AglB-Short 1 and AglB-Short 2. Results We determined the crystal structure of the C-terminal globular domain of the third AglB paralog, AglB-Long, at 1.9 Å resolutions. The crystallization of the fusion protein with maltose binding protein (MBP) afforded high quality protein crystals. Two MBP-AglB-L molecules formed a swapped dimer in the crystal. Since the fusion protein behaved as a monomer upon gel filtration, we reconstituted the monomer structure from the swapped dimer by exchanging the swapped segments. The C-terminal domain of A. fulgidus AglB-L includes a structural unit common to AglB-S1 and AglB-S2. This structural unit contains the evolutionally conserved WWDYG and DK motifs. The present structure revealed that A. fulgidus AglB-L contained a variant type of the DK motif with a short insertion, and confirmed that the second signature residue, Lys, of the DK motif participates in the formation of a pocket that binds to the serine and threonine residues at the +2 position of the N-glycosylation sequon. Conclusions The structure of A. fulgidus AglB-L, together with the two previously solved structures of AglB-S1 and AglB-S2, provides a complete overview of the three AglB paralogs encoded in the A. fulgidus genome. All three AglBs contain a variant type of the DK motif. This finding supports a previously proposed rule: The STT3/AglB/PglB paralogs in one organism always contain the same type of Ser/Thr-binding pocket. The present structure will be useful as a

  11. Crystallization and preliminary X-ray diffraction studies of the C-terminal domain of Chlamydia trachomatis CdsD.

    PubMed

    Meriläinen, Gitte; Wierenga, Rik K

    2014-10-01

    The inner membrane ring of the bacterial type III secretion system (TTSS) is composed of two proteins. In Chlamydia trachomatis this ring is formed by CdsD (gene name CT_664) and CdsJ (gene name CTA_0609). CdsD consists of 829 amino acids. The last 400 amino acids at its C-terminal end relate it to the type III secretion system YscD/HrpQ protein family. The C-terminal domain, consisting of amino acids 558-771, of C. trachomatis CdsD was overexpressed in Escherichia coli and purified using immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. The protein was crystallized using the vapour-diffusion method. A data set was collected to 2.26 Å resolution. The crystals have the symmetry of space group C2, with unit-cell parameters a = 106.60, b = 23.91, c = 118.65 Å, β = 104.95°. According to the data analysis there is expected to be one molecule in the asymmetric unit, with a Matthews coefficient of 3.0 Å(3) Da(-1). PMID:25286957

  12. N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition

    PubMed Central

    Hassan, Yousef I.; Moriyama, Hideaki; Olsen, Lars J.; Bi, Xin; Zempleni, Janos

    2009-01-01

    Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Carboxylases mediate essential steps in macronutrient metabolism. For example, propionyl-CoA carboxylase (PCC) catalyzes the carboxylation of propionyl-CoA in the metabolism of odd-chain fatty acids. HCS comprises four putative domains, i.e., the N-terminus, the biotin transfer/ATP binding domain, a putative linker domain, and the C-terminus. Both N- and C-termini are essential for biotinylation of carboxylases by HCS, but the exact functions of these two domains in enzyme catalysis are unknown. Here we tested the hypothesis that N- and C-termini play roles in substrate recognition by HCS. Yeast-two-hybrid (Y2H) assays were used to study interactions between the four domains of human HCS with p67, a PCC-based polypeptide and HCS substrate. Both N- and C-termini interacted with p67 in Y2H assays, whereas the biotin transfer/ATP-binding and the linker domains did not interact with p67. The essentiality of N- and C-termini for interactions with carboxylases was confirmed in rescue experiments with mutant Saccharomyces cerevisiae, using constructs of truncated human HCS. Finally, a computational biology approach was used to model the 3D structure of human HCS and identify amino acid residues that interact with p67. In silico predictions were consistent with observations from Y2H assays and yeast rescue experiments, and suggested docking of p67 near Arg508 and Ser515 within the central domain of HCS. PMID:19157941

  13. Functional synergy between the Munc13 C-terminal C1 and C2 domains.

    PubMed

    Liu, Xiaoxia; Seven, Alpay Burak; Camacho, Marcial; Esser, Victoria; Xu, Junjie; Trimbuch, Thorsten; Quade, Bradley; Su, Lijing; Ma, Cong; Rosenmund, Christian; Rizo, Josep

    2016-01-01

    Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca(2+)-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a 'primed' state that does not fuse but is ready for fast fusion upon Ca(2+) influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion. PMID:27213521

  14. Functional synergy between the Munc13 C-terminal C1 and C2 domains

    PubMed Central

    Liu, Xiaoxia; Seven, Alpay Burak; Camacho, Marcial; Esser, Victoria; Xu, Junjie; Trimbuch, Thorsten; Quade, Bradley; Su, Lijing; Ma, Cong; Rosenmund, Christian; Rizo, Josep

    2016-01-01

    Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca2+-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a ‘primed’ state that does not fuse but is ready for fast fusion upon Ca2+ influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion. DOI: http://dx.doi.org/10.7554/eLife.13696.001 PMID:27213521

  15. Role of the C-terminal domains of rice (Oryza sativa L.) bZIP proteins RF2a and RF2b in regulating transcription

    PubMed Central

    Liu, Yi; Dai, Shunhong; Beachy, Roger N.

    2007-01-01

    Rice (Oryza sativa L.) transcription factors RF2a and RF2b are bZIP (basic leucine zipper) proteins that interact with, and activate transcription from the RTBV (rice tungro bacilliform virus) promoter. Here we characterize the C-terminal domains of RF2a and RF2b: these domains are rich in glutamine and proline/glutamine, respectively. Affinity pull-down assays demonstrated that the C-terminal domains of RF2a and RF2b can associate to form either homodimers or heterodimers; however, they do not interact with other domains of RF2a or RF2b. Results of in vitro transcription assays using a rice whole-cell extract demonstrate that the C-terminal domains of both RF2a and RF2b activate transcription from the RTBV promoter. In addition, dimerization of the RF2a C-terminal domain is involved in regulating the transcription activation function of RF2a. The predicted helical region within the RF2a C-terminal glutamine-rich domain was determined to be involved in inter-molecular dimerization, and contributed to the regulatory functions of RF2a in these assays. PMID:17371296

  16. The structure of the C-terminal domain of the largest editosome interaction protein and its role in promoting RNA binding by RNA-editing ligase L2

    PubMed Central

    Park, Young-Jun; Budiarto, Tanya; Wu, Meiting; Pardon, Els; Steyaert, Jan; Hol, Wim G. J.

    2012-01-01

    Trypanosomatids, such as the sleeping sickness parasite Trypanosoma brucei, contain a ∼20S RNA-editing complex, also called the editosome, which is required for U-insertion/deletion editing of mitochondrial mRNAs. The editosome contains a core of 12 proteins including the large interaction protein A1, the small interaction protein A6, and the editing RNA ligase L2. Using biochemical and structural data, we identified distinct domains of T. brucei A1 which specifically recognize A6 and L2. We provide evidence that an N-terminal domain of A1 interacts with the C-terminal domain of L2. The C-terminal domain of A1 appears to be required for the interaction with A6 and also plays a key role in RNA binding by the RNA-editing ligase L2 in trans. Three crystal structures of the C-terminal domain of A1 have been elucidated, each in complex with a nanobody as a crystallization chaperone. These structures permitted the identification of putative dsRNA recognition sites. Mutational analysis of conserved residues of the C-terminal domain identified Arg703, Arg731 and Arg734 as key requirements for RNA binding. The data show that the editing RNA ligase activity is modulated by a novel mechanism, i.e. by the trans-acting RNA binding C-terminal domain of A1. PMID:22561373

  17. A Novel Fold in the Tral Relaxase-Helicase C-Terminal Domain Is Essential for Conjugative DNA Transfer

    SciTech Connect

    Guogas, Laura M.; Kennedy, Sarah A.; Lee, Jin-Hyup; Redinbo, Matthew R.

    2009-06-04

    TraI relaxase-helicase is the central catalytic component of the multiprotein relaxosome complex responsible for conjugative DNA transfer (CDT) between bacterial cells. CDT is a primary mechanism for the lateral propagation of microbial genetic material, including the spread of antibiotic resistance genes. The 2.4-{angstrom} resolution crystal structure of the C-terminal domain of the multifunctional Escherichia coli F (fertility) plasmid TraI protein is presented, and specific structural regions essential for CDT are identified. The crystal structure reveals a novel fold composed of a 28-residue N-terminal {alpha}-domain connected by a proline-rich loop to a compact {alpha}/{beta}-domain. Both the globular nature of the {alpha}/{beta}-domain and the presence as well as rigidity of the proline-rich loop are required for DNA transfer and single-stranded DNA binding. Taken together, these data establish the specific structural features of this noncatalytic domain that are essential to DNA conjugation.

  18. Structure and interactions of the C-terminal metal binding domain of Archaeoglobus fulgidus CopA

    SciTech Connect

    Agarwal, S.; Hong, D.; Desai, N.K.; H.Sazinsky, M.; Argüello, J.M.; Rosenzweig, A.C.

    2010-08-13

    The Cu(+)-ATPase CopA from Archaeoglobus fulgidus belongs to the P(1B) family of the P-type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P(1B-1)-type ATPases is the presence of soluble metal binding domains at the N-terminus (N-MBDs). The N-MBDs exhibit a conserved ferredoxin-like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N-MBDs enable Cu(+) regulation of turnover rates apparently through Cu-sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N-terminal MBD and a C-terminal MBD (C-MBD). The functional role of the unique C-MBD has not been established. Here, we report the crystal structure of the apo, oxidized C-MBD to 2.0 A resolution. In the structure, two C-MBD monomers form a domain-swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C-MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A-domain), has been investigated. Interestingly, the C-MBD interacts specifically with both of these domains, independent of the presence of Cu(+) or nucleotides. These data reinforce the uniqueness of the C-MBD and suggest a distinct structural role for the C-MBD in CopA transport.

  19. Triptonide Effectively Inhibits Wnt/β-Catenin Signaling via C-terminal Transactivation Domain of β-catenin.

    PubMed

    Chinison, Jessica; Aguilar, Jose S; Avalos, Alan; Huang, Ying; Wang, Zhijun; Cameron, D Joshua; Hao, Jijun

    2016-01-01

    Abnormal activation of canonical Wnt/β-catenin signaling is implicated in many diseases including cancer. As a result, therapeutic agents that disrupt this signaling pathway have been highly sought after. Triptonide is a key bioactive small molecule identified in a traditional Chinese medicine named Tripterygium wilfordii Hook F., and it has a broad spectrum of biological functions. Here we show that triptonide can effectively inhibit canonical Wnt/β-catenin signaling by targeting the downstream C-terminal transcription domain of β-catenin or a nuclear component associated with β-catenin. In addition, triptonide treatment robustly rescued the zebrafish "eyeless" phenotype induced by GSK-3β antagonist 6-bromoindirubin-30-oxime (BIO) for Wnt signaling activation during embryonic gastrulation. Finally, triptonide effectively induced apoptosis of Wnt-dependent cancer cells, supporting the therapeutic potential of triptonide. PMID:27596363

  20. Solution structure of the calmodulin-like C-terminal domain of Entamoeba α-actinin2.

    PubMed

    Karlsson, Göran; Persson, Cecilia; Mayzel, Maxim; Hedenström, Mattias; Backman, Lars

    2016-04-01

    Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross-links, or caps the filament ends have been identified and the actin cross-linker α-actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α-actinin is believed to be required for infection. To better understand the role of α-actinin in the infectious process we have determined the solution structure of the C-terminal calmodulin-like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium-binding EF-hand motifs, connected with a mobile linker. PMID:26800385

  1. Triptonide Effectively Inhibits Wnt/β-Catenin Signaling via C-terminal Transactivation Domain of β-catenin

    PubMed Central

    Chinison, Jessica; Aguilar, Jose S.; Avalos, Alan; Huang, Ying; Wang, Zhijun; Cameron, D. Joshua; Hao, Jijun

    2016-01-01

    Abnormal activation of canonical Wnt/β-catenin signaling is implicated in many diseases including cancer. As a result, therapeutic agents that disrupt this signaling pathway have been highly sought after. Triptonide is a key bioactive small molecule identified in a traditional Chinese medicine named Tripterygium wilfordii Hook F., and it has a broad spectrum of biological functions. Here we show that triptonide can effectively inhibit canonical Wnt/β-catenin signaling by targeting the downstream C-terminal transcription domain of β-catenin or a nuclear component associated with β-catenin. In addition, triptonide treatment robustly rescued the zebrafish “eyeless” phenotype induced by GSK-3β antagonist 6-bromoindirubin-30-oxime (BIO) for Wnt signaling activation during embryonic gastrulation. Finally, triptonide effectively induced apoptosis of Wnt-dependent cancer cells, supporting the therapeutic potential of triptonide. PMID:27596363

  2. Elastase inhibition by the C-terminal domains of alpha-crystallin and small heat-shock protein.

    PubMed

    Voorter, C E; de Haard-Hoekman, W; Merck, K B; Bloemendal, H; de Jong, W W

    1994-01-11

    alpha-Crystallin, an abundant eye-lens protein and a stress protein in other tissues, shows structural and functional similarities with the small heat-shock proteins. One of the properties in common is the inhibition of elastase. We now report that the separated subunits of alpha-crystallin, alpha A and alpha B, also exhibit elastase inhibition, whereas phosphorylation of these subunits apparently has no influence on the inhibitory capacity. Furthermore, for both alpha A-crystallin and mouse HSP25 the putative C-terminal structural domain, comprising the major region of homology between these proteins, is sufficient to give elastase inhibition. With database search no homology could be found between the three proteins under investigation and any of the known consensus sequences of proteinase inhibitor families. PMID:8305474

  3. Crystallization of the C-terminal domain of the fibre protein from snake adenovirus 1, an atadenovirus.

    PubMed

    Singh, Abhimanyu K; Menéndez-Conejero, Rosa; San Martín, Carmen; van Raaij, Mark J

    2013-12-01

    Adenovirus fibre proteins play an important role in determining viral tropism. The C-terminal domain of the fibre protein from snake adenovirus type 1, a member of the Atadenovirus genus, has been expressed, purified and crystallized. Crystals were obtained belonging to space groups P2(1)2(1)2(1) (two different forms), I2(1)3 and F23. The best of these diffracted synchrotron radiation to a resolution of 1.4 Å. As the protein lacks methionines or cysteines, site-directed mutagenesis was performed to change two leucine residues to methionines. Crystals of selenomethionine-derivatized crystals of the I2(1)3 form were also obtained and a multi-wavelength anomalous dispersion data set was collected. PMID:24316834

  4. Calorimetric and spectroscopic investigation of the interaction between the C-terminal domain of Enzyme I and its ligands.

    PubMed

    Yun, Young-Joo; Suh, Jeong-Yong

    2012-11-01

    Enzyme I initiates a series of phosphotransfer reactions during sugar uptake in the bacterial phosphotransferase system. Here, we have isolated a stable recombinant C-terminal domain of Enzyme I (EIC) of Escherichia coli and characterized its interaction with the N-terminal domain of Enzyme I (EIN) and also with various ligands. EIC can phosphorylate EIN, but their binding is transient regardless of the presence of phosphoenolpyruvate (PEP). Circular dichroism and NMR indicate that ligand binding to EIC induces changes near aromatic groups but not in the secondary structure of EIC. Binding of PEP to EIC is an endothermic reaction with the equilibrium dissociation constant (K(D) ) of 0.28 mM, whereas binding of the inhibitor oxalate is an exothermic reaction with K(D) of 0.66 mM from calorimetry. The binding thermodynamics of EIC and PEP compared to that of Enzyme I (EI) and PEP reveals that domain-domain motion in EI can contribute as large as ∼-3.2 kcal/mol toward PEP binding. PMID:22936614

  5. δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function.

    PubMed

    Arakel, Eric C; Richter, Kora P; Clancy, Anne; Schwappach, Blanche

    2016-06-21

    Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

  6. δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

    PubMed Central

    Arakel, Eric C.; Richter, Kora P.; Clancy, Anne; Schwappach, Blanche

    2016-01-01

    Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

  7. Clustered-charge to alanine scanning mutagenesis of the Mal63 MAL-activator C-terminal regulatory domain.

    PubMed

    Danzi, Sara E; Bali, Mehtap; Michels, Corinne A

    2003-12-01

    The MAL-activator genes of Saccharomyces cerevisiae encode regulatory proteins required for the expression of the structural genes encoding maltose permease and maltase. Residues within the C-terminal region of the Mal63 protein required for negative regulation were previously identified. Evidence suggested that the C-terminal domain is also involved in positive regulatory functions, such as inducer responsiveness and transactivation in the context of a full-length protein. Charged-cluster to alanine scanning mutagenesis of the regulatory domain of MAL63 and the constitutive MAL43-C were undertaken to identify distinct regions within Mal63p involved in positive functions and to define their roles in induction. Mutations that affect the ability to activate transcription in the inducible MAL63 but have no effect in the constitutive MAL43-C define regions that function in induction. Those that affect both the inducible and constitutive alleles define regions involved in activation more generally. Mutations in MAL63 fell into three classes, those that have little or no impact on activity, those that decrease activity, and those that enhance function. Mutations from these classes mapped to distinct regions of the protein, identifying a region of approximately 90 residues (residues 331-423) involved in maltose sensing and an approximately 50-residue region at the extreme C-terminus (residues 420-470) required for activation, such as the formation and/or maintenance of an active state. These studies support a model for MAL-activator function which involves complex protein-protein interactions and overlapping negative and positive regulatory regions. PMID:14508602

  8. NMR Determines Transient Structure and Dynamics in the Disordered C-Terminal Domain of WASp Interacting Protein

    PubMed Central

    Haba, Noam Y.; Gross, Renana; Novacek, Jiri; Shaked, Hadassa; Zidek, Lukas; Barda-Saad, Mira; Chill, Jordan H.

    2013-01-01

    WASp-interacting protein (WIP) is a 503-residue proline-rich polypeptide expressed in human T cells. The WIP C-terminal domain binds to Wiskott-Aldrich syndrome protein (WASp) and regulates its activation and degradation, and the WIP-WASp interaction has been shown to be critical for actin polymerization and implicated in the onset of WAS and X-linked thrombocytopenia. WIP is predicted to be an intrinsically disordered protein, a class of polypeptides that are of great interest because they violate the traditional structure-function paradigm. In this first (to our knowledge) study of WIP in its unbound state, we used NMR to investigate the biophysical behavior of WIPC, a C-terminal domain fragment of WIP that includes residues 407–503 and contains the WASp-binding site. In light of the poor spectral dispersion exhibited by WIPC and the high occurrence (25%) of proline residues, we employed 5D-NMR13C-detected NMR experiments with nonuniform sampling to accomplish full resonance assignment. Secondary chemical-shift analysis, 15N relaxation rates, and protection from solvent exchange all concurred in detecting transient structure located in motifs that span the WASp-binding site. Residues 446–456 exhibited a propensity for helical conformation, and an extended conformation followed by a short, capped helix was observed for residues 468–478. The 13C-detected approach allows chemical-shift assignment in the WIPC polyproline stretches and thus sheds light on their conformation and dynamics. The effects of temperature on chemical shifts referenced to a denatured sample of the polypeptide demonstrate that heating reduces the structural character of WIPC. Thus, we conclude that the disordered WIPC fragment is comprised of regions with latent structure connected by flexible loops, an architecture with implications for binding affinity and function. PMID:23870269

  9. The C-terminal domain controls the mobility of Crumbs 3 isoforms.

    PubMed

    Djuric, Ivona; Siebrasse, Jan Peter; Schulze, Ulf; Granado, Daniel; Schlüter, Marc A; Kubitscheck, Ulrich; Pavenstädt, Hermann; Weide, Thomas

    2016-06-01

    The physiological function of epithelia depends on an asymmetric distribution of their membrane domains. Polarity proteins play a crucial role for distribution processes, however, little is known about their mobility in epithelial cells. In this study, we analyzed the intracellular and plasma-membrane-associated mobility of fluorescence-labeled Crb3A and Crb3B. Both variants belong to the Crumbs protein family, which control size and identity of apical membranes in epithelial cells. Fluorescence recovery after photo-bleaching measurements revealed different mobilities for the two Crb3 variants. They also differentially affected mobility and localization of the Pals1/Mpp5 protein, which binds to Crb3A but not to Crb3B. In addition, tracking of intracellular vesicles indicated that Crb3A containing vesicles are slightly more immobile than Crb3B ones. Taken together, our data revealed different intracellular mobility patterns for Crb3A and Crb3B. PMID:26975581

  10. The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum.

    PubMed

    Nakayama, Yoshitaka; Becker, Michael; Ebrahimian, Haleh; Konishi, Tomoyuki; Kawasaki, Hisashi; Krämer, Reinhard; Martinac, Boris

    2016-01-01

    The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes. PMID:26494188

  11. Ca2+-dependent conformational changes in a C-terminal cytosolic domain of polycystin-2.

    PubMed

    Schumann, Frank; Hoffmeister, Helen; Bader, Reto; Schmidt, Maren; Witzgall, Ralph; Kalbitzer, Hans Robert

    2009-09-01

    The PKD1 and PKD2 genes are the genes that are mutated in patients suffering from autosomal dominant polycystic kidney disease. The human PKD2 gene codes for a 968-amino acid long membrane protein called polycystin-2 that represents a cation channel whose activity can be regulated by Ca(2+) ions. By CD, fluorescence, and NMR spectroscopy, we have studied a 117-amino acid-long fragment of the cytoplasmic domain of polycystin-2, polycystin-2-(680-796) that was proposed to contain a Ca(2+)-binding site. NMR structure determination reveals the existence of two Ca(2+)-binding sites in polycystin-2-(680-796) arranged in a typical and an atypical EF-hand motif. In the absence of Ca(2+) the protein forms a dimer that is dissociated by Ca(2+) binding. This dissociation may be related to the Ca(2+) inactivation observed earlier. The calcium affinity of the protein was determined by fluorescence and NMR spectroscopy. At 293 K, the K(D) values for the high and low affinity sites are 55 mum and 179 mum, respectively. PMID:19546223

  12. Thermodynamics of the binding of the C-terminal repeat domain of Streptococcus sobrinus glucosyltransferase-I to dextran.

    PubMed

    Komatsu, Hideyuki; Katayama, Motoki; Sawada, Masaki; Hirata, Yukie; Mori, Miyuki; Inoue, Tetsuyoshi; Fukui, Kazuhiro; Fukada, Harumi; Kodama, Takao

    2007-07-17

    Glucosyltransferases (GTFs) secreted by mutans streptococci and some other lactic acid bacteria catalyze glucan synthesis from sucrose, and possess a C-terminal glucan-binding domain (GBD) containing homologous, directly repeating units. We prepared a series of C-terminal truncated forms of the GBD of Streptococcus sobrinus GTF-I and studied their binding to dextran by isothermal titration calorimetry. The binding of all truncates was strongly exothermic. Their titration curves were analyzed assuming that the GBD recognizes and binds to a stretch of dextran chain, not to a whole dextran molecule. Both the number of glucose units constituting the dextran stretch (n) and the accompanying enthalpy change (DeltaH degrees ) are proportional to the molecular mass of the GBD truncate, with which the Gibbs energy change calculated by the relation DeltaG degrees = -RT ln K (R, the gas constant; T, the absolute temperature; K, the binding constant of a truncate for a dextran stretch of n glucose units) also increases linearly. For the full-length GBD (508 amino acid residues), n = 33.9, K = 4.88 x 10(7) M-1, and DeltaH degrees = -289 kJ mol-1 at 25 degrees C. These results suggest that identical, independent glucose-binding subsites, each comprising 14 amino acid residues on average, are arranged consecutively from the GBD N-terminus. Thus, the GBD binds tightly to a stretch of dextran chain through the adding up of individually weak subsite/glucose interactions. Furthermore, the entropy change accompanying the GBD/dextran interaction as given by the relation DeltaS degrees = (DeltaG degrees - DeltaH degrees)/T has a very large negative value, probably because of a loss of the conformational freedom of dextran and GBD after binding. PMID:17580962

  13. The 3D structure of Kaposi sarcoma herpesvirus LANA C-terminal domain bound to DNA

    PubMed Central

    Hellert, Jan; Weidner-Glunde, Magdalena; Krausze, Joern; Lünsdorf, Heinrich; Ritter, Christiane; Schulz, Thomas F.; Lührs, Thorsten

    2015-01-01

    Kaposi sarcoma herpesvirus (KSHV) persists as a latent nuclear episome in dividing host cells. This episome is tethered to host chromatin to ensure proper segregation during mitosis. For duplication of the latent genome, the cellular replication machinery is recruited. Both of these functions rely on the constitutively expressed latency-associated nuclear antigen (LANA) of the virus. Here, we report the crystal structure of the KSHV LANA DNA-binding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 Å resolution. In contrast to homologous proteins such as Epstein-Barr virus nuclear antigen 1 (EBNA-1) of the related γ-herpesvirus Epstein-Barr virus, specific DNA recognition by LANA is highly asymmetric. In addition to solving the crystal structure, we found that apart from the two known LANA binding sites, LBS1 and LBS2, LANA also binds to a novel site, denoted LBS3. All three sites are located in a region of the KSHV terminal repeat subunit previously recognized as a minimal replicator. Moreover, we show that the LANA DBD can coat DNA of arbitrary sequence by virtue of a characteristic lysine patch, which is absent in EBNA-1 of the Epstein-Barr virus. Likely, these higher-order assemblies involve the self-association of LANA into supermolecular spirals. One such spiral assembly was solved as a crystal structure of 3.7 Å resolution in the absence of DNA. On the basis of our data, we propose a model for the controlled nucleation of higher-order LANA oligomers that might contribute to the characteristic subnuclear KSHV microdomains (“LANA speckles”), a hallmark of KSHV latency. PMID:25947153

  14. Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase.

    PubMed

    Bornet, Olivier; Nouailler, Matthieu; Feracci, Michaël; Sebban-Kreuzer, Corinne; Byrne, Deborah; Halimi, Hubert; Morelli, Xavier; Badache, Ali; Guerlesquin, Françoise

    2014-06-01

    Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C-terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C-terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn-SH3 domain. The interaction of a kinase of the Src family with ErbB2 C-terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis. PMID:24815698

  15. Structural and Biochemical Studies of the C-Terminal Domain of Mouse Peptide-N-glycanase Identify it as a Mannose-Binding Module

    SciTech Connect

    Zhou,X.; Zhao, G.; Truglio, J.; Wang, L.; Li, G.; Lennarz, W.; Schindelin, H.

    2006-01-01

    The inability of certain N-linked glycoproteins to adopt their native conformation in the endoplasmic reticulum (ER) leads to their retrotranslocation into the cytosol and subsequent degradation by the proteasome. In this pathway the cytosolic peptide-N-glycanase (PNGase) cleaves the N-linked glycan chains off denatured glycoproteins. PNGase is highly conserved in eukaryotes and plays an important role in ER-associated protein degradation. In higher eukaryotes, PNGase has an N-terminal and a C-terminal extension in addition to its central catalytic domain, which is structurally and functionally related to transglutaminases. Although the N-terminal domain of PNGase is involved in protein-protein interactions, the function of the C-terminal domain has not previously been characterized. Here, we describe biophysical, biochemical, and crystallographic studies of the mouse PNGase C-terminal domain, including visualization of a complex between this domain and mannopentaose. These studies demonstrate that the C-terminal domain binds to the mannose moieties of N-linked oligosaccharide chains, and we further show that it enhances the activity of the mouse PNGase core domain, presumably by increasing the affinity of mouse PNGase for the glycan chains of misfolded glycoproteins.

  16. The C-Terminal RpoN Domain of sigma54 Forms an unpredictedHelix-Turn-Helix Motif Similar to domains of sigma70

    SciTech Connect

    Doucleff, Michaeleen; Malak, Lawrence T.; Pelton, Jeffrey G.; Wemmer, David E.

    2005-11-01

    The ''{delta}'' subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two {sigma} families have been identified, {sigma}{sup 70} and {sigma}{sup 54}, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the {sigma}{sup 70}-type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the {sigma}{sup 54}-type factors. Here we present the NMR derived structure of the C-terminal domain of {sigma}{sup 54} from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the {sigma}{sup 70}-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of {sigma}{sup 54} binds to DNA.

  17. Cloning, purification and preliminary X-ray analysis of the C-terminal domain of Helicobacter pylori MotB

    SciTech Connect

    Roujeinikova, Anna

    2008-04-01

    The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of a putative peptidoglycan-binding domain of H. pylori MotB, a stator component of the bacterial flagellar motor, are reported. The C-terminal domain of MotB (MotB-C) contains a putative peptidoglycan-binding motif and is believed to anchor the MotA/MotB stator unit of the bacterial flagellar motor to the cell wall. Crystals of Helicobacter pylori MotB-C (138 amino-acid residues) were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 50.8, b = 89.5, c = 66.3 Å, β = 112.5°. The crystals diffract X-rays to at least 1.6 Å resolution using a synchrotron-radiation source. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains one tetramer with 222 point-group symmetry. The anomalous difference Patterson maps calculated for an ytterbium-derivative crystal using diffraction data at a wavelength of 1.38 Å showed significant peaks on the v = 1/2 Harker section, suggesting that ab initio phase information could be derived from the MAD data.

  18. Recombinant expression, purification and preliminary biophysical and structural studies of C-terminal carbohydrate recognition domain from human galectin-4.

    PubMed

    Rustiguel, Joane K; Kumagai, Patricia S; Dias-Baruffi, Marcelo; Costa-Filho, Antonio J; Nonato, Maria Cristina

    2016-02-01

    Galectin-4 (Gal4), a tandem-repeat type galectin, is expressed in healthy epithelium of the gastrointestinal tract. Altered levels of Gal4 expression are associated with different types of cancer, suggesting its usage as a diagnostic marker as well as target for drug development. The functional data available for this class of proteins suggest that the wide spectrum of cellular activities reported for Gal4 relies on distinct glycan specificity and structural characteristics of its two carbohydrate recognition domains. In the present work, two independent constructs for recombinant expression of the C-terminal domain of human galectin-4 (hGal4-CRD2) were developed. His6-tagged and untagged recombinant proteins were overexpressed in Escherichia coli, and purified by affinity chromatography followed by gel filtration. Correct folding and activity of hGal4-CRD2 were assessed by circular dichroism and fluorescence spectroscopies, respectively. Diffraction quality crystals were obtained by vapor-diffusion sitting drop setup and the crystal structure of CRD2 was solved by molecular replacement techniques at 1.78 Å resolution. Our work describes the development of important experimental tools that will allow further studies in order to correlate structure and binding properties of hGal4-CRD2 and human galectin-4 functional activities. PMID:26432949

  19. Crystal structures of GCN2 protein kinase C-terminal domains suggest regulatory differences in yeast and mammals.

    PubMed

    He, Hongzhen; Singh, Isha; Wek, Sheree A; Dey, Souvik; Baird, Thomas D; Wek, Ronald C; Georgiadis, Millie M

    2014-05-23

    In response to amino acid starvation, GCN2 phosphorylation of eIF2 leads to repression of general translation and initiation of gene reprogramming that facilitates adaptation to nutrient stress. GCN2 is a multidomain protein with key regulatory domains that directly monitor uncharged tRNAs which accumulate during nutrient limitation, leading to activation of this eIF2 kinase and translational control. A critical feature of regulation of this stress response kinase is its C-terminal domain (CTD). Here, we present high resolution crystal structures of murine and yeast CTDs, which guide a functional analysis of the mammalian GCN2. Despite low sequence identity, both yeast and mammalian CTDs share a core subunit structure and an unusual interdigitated dimeric form, albeit with significant differences. Disruption of the dimeric form of murine CTD led to loss of translational control by GCN2, suggesting that dimerization is critical for function as is true for yeast GCN2. However, although both CTDs bind single- and double-stranded RNA, murine GCN2 does not appear to stably associate with the ribosome, whereas yeast GCN2 does. This finding suggests that there are key regulatory differences between yeast and mammalian CTDs, which is consistent with structural differences. PMID:24719324

  20. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    PubMed

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle. PMID:26405031

  1. Crystal structure and mode of helicase binding of the C-terminal domain of primase from Helicobacter pylori.

    PubMed

    Abdul Rehman, Syed Arif; Verma, Vijay; Mazumder, Mohit; Dhar, Suman K; Gourinath, S

    2013-06-01

    To better understand the poor conservation of the helicase binding domain of primases (DnaGs) among the eubacteria, we determined the crystal structure of the Helicobacter pylori DnaG C-terminal domain (HpDnaG-CTD) at 1.78 Å. The structure has a globular subdomain connected to a helical hairpin. Structural comparison has revealed that globular subdomains, despite the variation in number of helices, have broadly similar arrangements across the species, whereas helical hairpins show different orientations. Further, to study the helicase-primase interaction in H. pylori, a complex was modeled using the HpDnaG-CTD and HpDnaB-NTD (helicase) crystal structures using the Bacillus stearothermophilus BstDnaB-BstDnaG-CTD (helicase-primase) complex structure as a template. By using this model, a nonconserved critical residue Phe534 on helicase binding interface of DnaG-CTD was identified. Mutation guided by molecular dynamics, biophysical, and biochemical studies validated our model. We further concluded that species-specific helicase-primase interactions are influenced by electrostatic surface potentials apart from the critical hydrophobic surface residues. PMID:23585534

  2. A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II.

    PubMed Central

    Bourquin, J P; Stagljar, I; Meier, P; Moosmann, P; Silke, J; Baechi, T; Georgiev, O; Schaffner, W

    1997-01-01

    The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro . It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis - trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domain-containing splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcription. PMID:9153302

  3. Analysis of the C-Terminal Membrane Anchor Domains of Hepatitis C Virus Glycoproteins E1 and E2: toward a Topological Model

    PubMed Central

    Charloteaux, Benoit; Lins, Laurence; Moereels, Henri; Brasseur, Robert

    2002-01-01

    The hepatitis C virus (HCV) glycoproteins E1 and E2 should be anchored in the viral membrane by their C-terminal domains. During synthesis, they are translocated to the endoplasmic reticulum (ER) lumen where they remain. The 31 C-terminal residues of the E1 protein and the 29 C-terminal residues of the E2 protein are implicated in the ER retention. Moreover, the E1 and E2 C termini are implicated in E1-E2 heterodimerization. We studied the E1 and E2 C-terminal sequences of 25 HCV strains in silico using molecular modeling techniques. We conclude that both C-terminal domains should adopt a similar and peculiar configuration: one amphipathic α-helix followed by a pair of transmembrane β-strands. Several three-dimensional (3-D) models were generated. After energy minimization, their ability to interact with membranes was studied using the molecular hydrophobicity potentials calculation and the IMPALA procedure. The latter simulates interactions with a membrane by a Monte Carlo minimization of energy. These methods suggest that the β-hairpins could anchor the glycoproteins in the ER membrane at least transiently. Anchoring could be stabilized by the adsorption of the nearby amphipathic α-helices at the membrane surface. The 3-D models correlate with experimental results which indicate that the E1-E2 transmembrane domains are involved in the heterodimerization and have ER retention properties. PMID:11799189

  4. Structure-function analysis of the human TFIIB-related factor II protein reveals an essential role for the C-terminal domain in RNA polymerase III transcription.

    PubMed

    Saxena, Ashish; Ma, Beicong; Schramm, Laura; Hernandez, Nouria

    2005-11-01

    The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAP(c), a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II. PMID:16227591

  5. Structure-Function Analysis of the Human TFIIB-Related Factor II Protein Reveals an Essential Role for the C-Terminal Domain in RNA Polymerase III Transcription

    PubMed Central

    Saxena, Ashish; Ma, Beicong; Schramm, Laura; Hernandez, Nouria

    2005-01-01

    The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAPc, a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II. PMID:16227591

  6. Verprolin function in endocytosis and actin organization. Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin-binding domain.

    PubMed

    Thanabalu, Thirumaran; Rajmohan, Rajamuthiah; Meng, Lei; Ren, Gang; Vajjhala, Parimala R; Munn, Alan L

    2007-08-01

    Vrp1p (verprolin, End5p) is the yeast ortholog of human Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1p localizes to the cortical actin cytoskeleton, is necessary for its polarization to sites of growth and is also essential for endocytosis. At elevated temperature, Vrp1p becomes essential for growth. A C-terminal Vrp1p fragment (C-Vrp1p) retains the ability to localize to the cortical actin cytoskeleton and function in actin-cytoskeleton polarization, endocytosis and growth. Here, we demonstrate that two submodules in C-Vrp1p are required for actin-cytoskeleton polarization: a novel C-terminal actin-binding submodule (CABS) that contains a novel G-actin-binding domain, which we call a verprolin homology 2 C-terminal (VH2-C) domain; and a second submodule comprising the Las17p-binding domain (LBD) that binds Las17p (yeast WASP). The LBD localizes C-Vrp1p to membranes and the cortical actin cytoskeleton. Intriguingly, the LBD is sufficient to restore endocytosis and growth at elevated temperature to Vrp1p-deficient cells. The CABS also restores these functions, but only if modified by a lipid anchor to provide membrane association. Our findings highlight the role of Las17p binding for Vrp1p membrane association, suggest general membrane association may be more important than specific targeting to the cortical actin cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity. PMID:17635585

  7. The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer.

    PubMed

    Namadurai, Sivakumar; Jain, Deepti; Kulkarni, Dhananjay S; Tabib, Chaitanya R; Friedhoff, Peter; Rao, Desirazu N; Nair, Deepak T

    2010-01-01

    The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage. PMID:21060849

  8. Pharmacological activity of the C-terminal and N-terminal domains of secretory leukoprotease inhibitor in vitro.

    PubMed Central

    Masuda, K.; Kamimura, T.; Watanabe, K.; Suga, T.; Kanesaki, M.; Takeuchi, A.; Imaizumi, A.; Suzuki, Y.

    1995-01-01

    1. In order to characterize the physiological functions of the domain structure of secretory leukoprotease inhibitor (SLPI), the biological capacities of half-length SLPIs, (Ser1-Pro54)SLPI and (Asn55-Ala107)SLPI, were investigated and compared with those of full-length SLPI. 2. The activities of these inhibitors against several serine proteases were determined using synthetic chromogenic substrates. The inhibitory capacity of the C-terminal domain, (Asn55-Ala107)SLPI, was as strong as that of full-length SLPI against human neutrophil elastase (NE), cathepsin G and chymotrypsin. It possessed less trypsin inhibitory activity than intact SLPI. For the N-terminal domain of SLPI, (Ser1-Pro54)SLPI, no inhibitory activity could be detected against the serine proteases tested in this study. 3. The inhibitory activity of (Asn55-Ala107)SLPI against the proteolysis of the natural substrates elastin and collagen by NE was comparable with that of full-SLPI (elastin, IC50 = 907 +/- 31 nM for SLPI, 767 +/- 33 nM for (Asn55-Ala107)SLPI; collagen, IC50 = 862 +/- 36 nM for SLPI, 727 +/- 47 nM for (Asn55-Ala107)SLPI). 4. The binding affinities of full- and half-length SLPIs for heparin were measured by affinity column chromatography. Full-length SLPI showed high affinity for heparin while the binding capacities of both half-length SLPIs were lower. (Concentration of NaCl for elution, 0.45 M for SLPI, 0.24 M for (Ser1-Pro54)SLPI, 0.27 M for (Asn55-Ala107)SLPI). 5. The effects of full-SLPI and (Asn55-Ala107)SLPI on blood coagulation were measured using the activated partial thromboplastin time (APTT).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582515

  9. Crystal structure of the C-terminal globular domain of oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 Å resolution.

    PubMed

    Matsumoto, Shunsuke; Igura, Mayumi; Nyirenda, James; Matsumoto, Masaki; Yuzawa, Satoru; Noda, Nobuo; Inagaki, Fuyuhiko; Kohda, Daisuke

    2012-05-22

    Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers glycan to asparagine in the N-glycosylation sequon. The catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three AglB paralogs. Two of them are the shortest AglBs across all domains of life. We determined the crystal structure of the C-terminal globular domain of the smallest AglB to identify the minimal structural unit. The Archaeoglobus AglB lacked a β-barrel-like structure, which had been found in other AglB and PglB structures. In agreement, the deletion in a larger Pyrococcus AglB confirmed its dispensability for the activity. By contrast, the Archaeoglobus AglB contains a kinked helix bearing a conserved motif, called DK/MI motif. The lysine and isoleucine residues in the motif participate in the Ser/Thr recognition in the sequon. The Archaeoglobus AglB structure revealed that the kinked helix contained an unexpected insertion. A revised sequence alignment based on this finding identified a variant type of the DK motif with the insertion. A mutagenesis study of the Archaeoglobus AglB confirmed the contribution of this particular type of the DK motif to the activity. When taken together with our previous results, this study defined the classification of OST: one group consisting of eukaryotes and most archaea possesses the DK-type Ser/Thr pocket, and the other group consisting of eubacteria and the remaining archaea possesses the MI-type Ser/Thr pocket. This classification provides a useful framework for OST studies. PMID:22559858

  10. Functional C-TERMINALLY ENCODED PEPTIDE (CEP) plant hormone domains evolved de novo in the plant parasite Rotylenchulus reniformis.

    PubMed

    Eves-Van Den Akker, Sebastian; Lilley, Catherine J; Yusup, Hazijah B; Jones, John T; Urwin, Peter E

    2016-10-01

    Sedentary plant-parasitic nematodes (PPNs) induce and maintain an intimate relationship with their host, stimulating cells adjacent to root vascular tissue to re-differentiate into unique and metabolically active 'feeding sites'. The interaction between PPNs and their host is mediated by nematode effectors. We describe the discovery of a large and diverse family of effector genes, encoding C-TERMINALLY ENCODED PEPTIDE (CEP) plant hormone mimics (RrCEPs), in the syncytia-forming plant parasite Rotylenchulus reniformis. The particular attributes of RrCEPs distinguish them from all other CEPs, regardless of origin. Together with the distant phylogenetic relationship of R. reniformis to the only other CEP-encoding nematode genus identified to date (Meloidogyne), this suggests that CEPs probably evolved de novo in R. reniformis. We have characterized the first member of this large gene family (RrCEP1), demonstrating its significant up-regulation during the plant-nematode interaction and expression in the effector-producing pharyngeal gland cell. All internal CEP domains of multi-domain RrCEPs are followed by di-basic residues, suggesting a mechanism for cleavage. A synthetic peptide corresponding to RrCEP1 domain 1 is biologically active and capable of up-regulating plant nitrate transporter (AtNRT2.1) expression, whilst simultaneously reducing primary root elongation. When a non-CEP-containing, syncytia-forming PPN species (Heterodera schachtii) infects Arabidopsis in a CEP-rich environment, a smaller feeding site is produced. We hypothesize that CEPs of R. reniformis represent a two-fold adaptation to sustained biotrophy in this species: (i) increasing host nitrate uptake, whilst (ii) limiting the size of the syncytial feeding site produced. PMID:26996971

  11. The activity of protein phosphatase 5 towards native clients is modulated by the middle- and C-terminal domains of Hsp90

    PubMed Central

    Haslbeck, Veronika; Eckl, Julia M.; Drazic, Adrian; Rutz, Daniel A.; Lorenz, Oliver R.; Zimmermann, Kerstin; Kriehuber, Thomas; Lindemann, Claudia; Madl, Tobias; Richter, Klaus

    2015-01-01

    Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex. PMID:26593036

  12. The p53 tetramer shows an induced-fit interaction of the C-terminal domain with the DNA-binding domain

    PubMed Central

    D'Abramo, M; Bešker, N; Desideri, A; Levine, A J; Melino, G; Chillemi, G

    2016-01-01

    The Trp53 gene is the most frequently mutated gene in all human cancers. Its protein product p53 is a very powerful transcription factor that can activate different biochemical pathways and affect the regulation of metabolism, senescence, DNA damage response, cell cycle and cell death. The understanding of its function at the molecular level could be of pivotal relevance for therapy. Investigation of long-range intra- and interdomain communications in the p53 tetramer–DNA complex was performed by means of an atomistic model that included the tetramerization helices in the C-terminal domain, the DNA-binding domains and a consensus DNA-binding site of 18 base pairs. Nonsymmetric dynamics are illustrated in the four DNA-binding domains, with loop L1 switching from inward to outward conformations with respect to the DNA major groove. Direct intra- and intermonomeric long-range communications between the tetramerization and DNA-binding domains are noted. These long-distance conformational changes link the C terminus with the DNA-binding domain and provide a biophysical rationale for the reported functional regulation of the p53 C-terminal region. A fine characterization of the DNA deformation caused by p53 binding is obtained, with ‘static' deformations always present and measured by the slide parameter in the central thymine–adenine base pairs; we also detect ‘dynamic' deformations switched on and off by particular p53 tetrameric conformations and measured by the roll and twist parameters in the same base pairs. These different conformations can indeed modulate the electrostatic potential isosurfaces of the whole p53–DNA complex. These results provide a molecular/biophysical understanding of the evident role of the C terminus in post-translational modification that regulates the transcriptional function of p53. Furthermore, the unstructured C terminus is able to facilitate contacts between the core DNA-binding domains of the tetramer. PMID:26477317

  13. The p53 tetramer shows an induced-fit interaction of the C-terminal domain with the DNA-binding domain.

    PubMed

    D'Abramo, M; Bešker, N; Desideri, A; Levine, A J; Melino, G; Chillemi, G

    2016-06-23

    The Trp53 gene is the most frequently mutated gene in all human cancers. Its protein product p53 is a very powerful transcription factor that can activate different biochemical pathways and affect the regulation of metabolism, senescence, DNA damage response, cell cycle and cell death. The understanding of its function at the molecular level could be of pivotal relevance for therapy. Investigation of long-range intra- and interdomain communications in the p53 tetramer-DNA complex was performed by means of an atomistic model that included the tetramerization helices in the C-terminal domain, the DNA-binding domains and a consensus DNA-binding site of 18 base pairs. Nonsymmetric dynamics are illustrated in the four DNA-binding domains, with loop L1 switching from inward to outward conformations with respect to the DNA major groove. Direct intra- and intermonomeric long-range communications between the tetramerization and DNA-binding domains are noted. These long-distance conformational changes link the C terminus with the DNA-binding domain and provide a biophysical rationale for the reported functional regulation of the p53 C-terminal region. A fine characterization of the DNA deformation caused by p53 binding is obtained, with 'static' deformations always present and measured by the slide parameter in the central thymine-adenine base pairs; we also detect 'dynamic' deformations switched on and off by particular p53 tetrameric conformations and measured by the roll and twist parameters in the same base pairs. These different conformations can indeed modulate the electrostatic potential isosurfaces of the whole p53-DNA complex. These results provide a molecular/biophysical understanding of the evident role of the C terminus in post-translational modification that regulates the transcriptional function of p53. Furthermore, the unstructured C terminus is able to facilitate contacts between the core DNA-binding domains of the tetramer. PMID:26477317

  14. Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

    PubMed Central

    Gary, R; Bretscher, A

    1995-01-01

    Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking. Images PMID:7579708

  15. Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain

    PubMed Central

    Jakób, Michał; Kołodziejczyk, Robert; Orłowski, Marek; Krzywda, Szymon; Kowalska, Agnieszka; Dutko-Gwóźdź, Joanna; Gwóźdź, Tomasz; Kochman, Marian; Jaskólski, Mariusz; Ożyhar, Andrzej

    2007-01-01

    The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, is considered as the functional receptor for ecdysteroids initiating molting and metamorphosis in insects. Here we report the 1.95 Å structure of the complex formed by the DNA-binding domains (DBDs) the EcR and the Usp, bound to the natural pseudopalindromic response element. Comparison of the structure with that obtained previously, using an idealized response element, shows how the EcRDBD, which has been previously reported to possess extraordinary flexibility, accommodates DNA-induced structural changes. Part of the C-terminal extension (CTE) of the EcRDBD folds into an α-helix whose location in the minor groove does not match any of the locations previously observed for nuclear receptors. Mutational analyses suggest that the α-helix is a component of EcR-box, a novel element indispensable for DNA-binding and located within the nuclear receptor CTE. This element seems to be a general feature of all known EcRs. PMID:17426125

  16. A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II.

    PubMed Central

    Lee, J M; Greenleaf, A L

    1989-01-01

    The unique C-terminal repeat domain (CTD) of the largest subunit (IIa) of eukaryotic RNA polymerase II consists of multiple repeats of the heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats ranges from 26 in yeast to 42 in Drosophila to 52 in mouse. The CTD is essential in vivo, but its structure and function are not yet understood. The CTD can be phosphorylated at multiple serine and threonine residues, generating a form of the largest subunit (II0) with markedly reduced mobility in NaDodSO4/polyacrylamide gels. To investigate this extensive phosphorylation, which presumably modulates functional properties of RNA polymerase II, we began efforts to purify a specific CTD kinase. Using CTD-containing fusion proteins as substrates, we have purified a CTD kinase from the yeast Saccharomyces cerevisiae. The enzyme extensively phosphorylates the CTD portion of both the fusion proteins and intact subunit IIa, producing products with reduced electrophoretic mobilities. The properties of the CTD kinase suggest that it is distinct from previously described protein kinases. Analogous activities were also detected in Drosophila and HeLa cell extracts. Images PMID:2657724

  17. Interplay of positive and negative effectors in function of the C-terminal repeat domain of RNA polymerase II.

    PubMed Central

    Li, Y; Kornberg, R D

    1994-01-01

    RNA polymerase II lacking a C-terminal domain (CTD) was active in transcription with purified proteins from yeast but failed to support transcription in a yeast extract. CTD dependence could be reconstituted in the purified system by addition of two fractions from the extract. An inhibitory fraction abolished transcription by both wild-type and CTD-less RNA polymerases; a stimulatory fraction restored activity of the wild-type polymerase but had a much lesser effect on the CTD-less enzyme. Parallel results were obtained with the use of a kinase inhibitor that prevents phosphorylation of the CTD by RNA polymerase II initiation factor b. The kinase inhibitor abolished transcription by wild-type polymerase in yeast extract but had no significant effect in the purified system. The requirement for both the CTD and kinase action for transcription in an extract indicates that CTD phosphorylation is involved in opposing the negative effector in the extract. Factor b must play a role(s) in addition to phosphorylation of the CTD because it was still required for transcription with polymerase lacking a CTD in the purified system. Images PMID:8134400

  18. Structure of FIV capsid C-terminal domain demonstrates lentiviral evasion of genetic fragility by coevolved substitutions

    PubMed Central

    Khwaja, Aya; Galilee, Meytal; Marx, Ailie; Alian, Akram

    2016-01-01

    Viruses use a strategy of high mutational rates to adapt to environmental and therapeutic pressures, circumventing the deleterious effects of random single-point mutations by coevolved compensatory mutations, which restore protein fold, function or interactions damaged by initial ones. This mechanism has been identified as contributing to drug resistance in the HIV-1 Gag polyprotein and especially its capsid proteolytic product, which forms the viral capsid core and plays multifaceted roles in the viral life cycle. Here, we determined the X-ray crystal structure of C-terminal domain of the feline immunodeficiency virus (FIV) capsid and through interspecies analysis elucidate the structural basis of co-evolutionarily and spatially correlated substitutions in capsid sequences, which when otherwise uncoupled and individually substituted into HIV-1 capsid impair virion assembly and infectivity. The ability to circumvent the deleterious effects of single amino acid substitutions by cooperative secondary substitutions allows mutational flexibility that may afford viruses an important survival advantage. The potential of such interspecies structural analysis for preempting viral resistance by identifying such alternative but functionally equivalent patterns is discussed. PMID:27102180

  19. The C-terminal Domain Supports a Novel Function for CETPI as a New Plasma Lipopolysaccharide-Binding Protein

    PubMed Central

    García-González, Victor; Gutiérrez-Quintanar, Nadia; Mas-Oliva, Jaime

    2015-01-01

    Described by our group a few years ago, the cholesteryl-ester transfer protein isoform (CETPI), exclusively expressed in the small intestine and present in human plasma, lacked a functional identification for a role of physiological relevance. Now, this study introduces CETPI as a new protein with the potential capability to recognise, bind and neutralise lipopolysaccharides (LPS). Peptides derived from the C-terminal domain of CETPI showed that CETPI not only might interact with several LPS serotypes but also might displace LPS bound to the surface of cells. Peptide VSAK, derived from the last 18 residues of CETPI, protected against the cytotoxic effect of LPS on macrophages. At high concentrations, when different cell types were tested in culture, it did not exhibit cytotoxicity by itself and it did prevent the expression of pro-inflammatory cytokines as well as the generation of oxidative stress conditions. In a rabbit model of septic shock, the infusion of peptide VSAK exerted a protective effect against the effects of LPS and reduced the presence of tumor necrosis factor-alpha (TNFα) in plasma. Therefore, CETPI is proposed as a new protein with the capability to advance the possibilities for better understanding and treatment of the dangerous effects of LPS in vivo. PMID:26537318

  20. P13, an Integral Membrane Protein of Borrelia burgdorferi, Is C-Terminally Processed and Contains Surface-Exposed Domains

    PubMed Central

    Noppa, Laila; Östberg, Yngve; Lavrinovicha, Marija; Bergström, Sven

    2001-01-01

    To elucidate antigens present on the bacterial surface of Borrelia burgdorferi sensu lato that may be involved in pathogenesis, we characterized a protein, P13, with an apparent molecular mass of 13 kDa. The protein was immunogenic and was expressed in large amounts during in vitro cultivation compared to other known antigens. An immunofluorescence assay, immunoelectron microscopy, and protease sensitivity assays indicated that P13 is surface exposed. The deduced sequence of the P13 peptide revealed a possible signal peptidase type I cleavage site, and computer analysis predicted that P13 is an integral membrane protein with three transmembrane-spanning domains. Mass spectrometry, in vitro translation, and N- and C-terminal amino acid sequencing analyses indicated that P13 was posttranslationally processed at both ends and modified by an unknown mechanism. Furthermore, p13 belongs to a gene family with five additional members in B. burgdorferi sensu stricto. The p13 gene is located on the linear chromosome of the bacterium, in contrast to five paralogous genes, which are located on extrachromosomal plasmids. The size of the p13 transcript was consistent with a monocistronic transcript. This new gene family may be involved in functions that are specific for this spirochete and its pathogenesis. PMID:11292755

  1. Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex

    SciTech Connect

    Sampathkumar, Parthasarathy; Gheyi, Tarun; Miller, Stacy A.; Bain, Kevin T.; Dickey, Mark; Bonanno, Jeffrey B.; Kim, Seung Joong; Phillips, Jeremy; Pieper, Ursula; Fernandez-Martinez, Javier; Franke, Josef D.; Martel, Anne; Tsuruta, Hiro; Atwell, Shane; Thompson, Devon A.; Emtage, J. Spencer; Wasserman, Stephen R.; Rout, Michael P.; Sali, Andrej; Sauder, J. Michael; Burley, Stephen K.

    2012-10-23

    Nuclear pore complexes (NPCs), responsible for the nucleo-cytoplasmic exchange of proteins and nucleic acids, are dynamic macromolecular assemblies forming an eight-fold symmetric co-axial ring structure. Yeast (Saccharomyces cerevisiae) NPCs are made up of at least 456 polypeptide chains of {approx}30 distinct sequences. Many of these components (nucleoporins, Nups) share similar structural motifs and form stable subcomplexes. We have determined a high-resolution crystal structure of the C-terminal domain of yeast Nup133 (ScNup133), a component of the heptameric Nup84 subcomplex. Expression tests yielded ScNup133(944-1157) that produced crystals diffracting to 1.9{angstrom} resolution. ScNup133(944-1157) adopts essentially an all {alpha}-helical fold, with a short two stranded {beta}-sheet at the C-terminus. The 11 {alpha}-helices of ScNup133(944-1157) form a compact fold. In contrast, the previously determined structure of human Nup133(934-1156) bound to a fragment of human Nup107 has its constituent {alpha}-helices are arranged in two globular blocks. These differences may reflect structural divergence among homologous nucleoporins.

  2. Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

    SciTech Connect

    Kadohira, Ikuko; Abe, Yoichiro Nuriya, Mutsuo; Sano, Kazumi; Tsuji, Shoji; Arimitsu, Takeshi; Yoshimura, Yasunori; Yasui, Masato

    2008-12-12

    Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [{sup 32}P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition.

  3. Crystal structure of the magnetobacterial protein MtxA C-terminal domain reveals a new sequence-structure relationship

    PubMed Central

    Davidov, Geula; Müller, Frank D.; Baumgartner, Jens; Bitton, Ronit; Faivre, Damien; Schüler, Dirk; Zarivach, Raz

    2015-01-01

    Magnetotactic bacteria (MTB) are a diverse group of aquatic bacteria that have the magnetotaxis ability to align themselves along the geomagnetic field lines and to navigate to a microoxic zone at the bottom of chemically stratified natural water. This special navigation is the result of a unique linear assembly of a specialized organelle, the magnetosome, which contains a biomineralized magnetic nanocrystal enveloped by a cytoplasmic membrane. The Magnetospirillum gryphiswaldense MtxA protein (MGR_0208) was suggested to play a role in bacterial magnetotaxis due to its gene location in an operon together with putative signal transduction genes. Since no homology is found for MtxA, and to better understand the role and function of MtxA in MTBés magnetotaxis, we initiated structural and functional studies of MtxA via X-ray crystallography and deletion mutagenesis. Here, we present the crystal structure of the MtxA C-terminal domain and provide new insights into its sequence-structure relationship. PMID:26052516

  4. Pioneering Activity of the C-Terminal Domain of EBF1 Shapes the Chromatin Landscape for B Cell Programming.

    PubMed

    Boller, Sören; Ramamoorthy, Senthilkumar; Akbas, Duygu; Nechanitzky, Robert; Burger, Lukas; Murr, Rabih; Schübeler, Dirk; Grosschedl, Rudolf

    2016-03-15

    Lymphopoiesis requires the activation of lineage-specific genes embedded in naive, inaccessible chromatin or in primed, accessible chromatin. The mechanisms responsible for de novo gain of chromatin accessibility, known as "pioneer" function, remain poorly defined. Here, we showed that the EBF1 C-terminal domain (CTD) is required for the regulation of a specific gene set involved in B cell fate decision and differentiation, independently of activation and repression functions. Using genome-wide analysis of DNaseI hypersensitivity and DNA methylation in multipotent Ebf1(-/-) progenitors and derivative EBF1wt- or EBF1ΔC-expressing cells, we found that the CTD promoted chromatin accessibility and DNA demethylation in previously naive chromatin. The CTD allowed EBF1 to bind at inaccessible genomic regions that offer limited co-occupancy by other transcription factors, whereas the CTD was dispensable for EBF1 binding at regions that are occupied by multiple transcription factors. Thus, the CTD enables EBF1 to confer permissive lineage-specific changes in progenitor chromatin landscape. PMID:26982363

  5. Structure of FIV capsid C-terminal domain demonstrates lentiviral evasion of genetic fragility by coevolved substitutions.

    PubMed

    Khwaja, Aya; Galilee, Meytal; Marx, Ailie; Alian, Akram

    2016-01-01

    Viruses use a strategy of high mutational rates to adapt to environmental and therapeutic pressures, circumventing the deleterious effects of random single-point mutations by coevolved compensatory mutations, which restore protein fold, function or interactions damaged by initial ones. This mechanism has been identified as contributing to drug resistance in the HIV-1 Gag polyprotein and especially its capsid proteolytic product, which forms the viral capsid core and plays multifaceted roles in the viral life cycle. Here, we determined the X-ray crystal structure of C-terminal domain of the feline immunodeficiency virus (FIV) capsid and through interspecies analysis elucidate the structural basis of co-evolutionarily and spatially correlated substitutions in capsid sequences, which when otherwise uncoupled and individually substituted into HIV-1 capsid impair virion assembly and infectivity. The ability to circumvent the deleterious effects of single amino acid substitutions by cooperative secondary substitutions allows mutational flexibility that may afford viruses an important survival advantage. The potential of such interspecies structural analysis for preempting viral resistance by identifying such alternative but functionally equivalent patterns is discussed. PMID:27102180

  6. RNA Polymerase II C-terminal Heptarepeat Domain Ser-7 Phosphorylation Is Established in a Mediator-dependent Fashion*

    PubMed Central

    Boeing, Stefan; Rigault, Caroline; Heidemann, Martin; Eick, Dirk; Meisterernst, Michael

    2010-01-01

    The largest subunit of RNA polymerase II (RNAPII) C-terminal heptarepeat domain (CTD) is subject to phosphorylation during initiation and elongation of transcription by RNA polymerase II. Here we study the molecular mechanisms leading to phosphorylation of Ser-7 in the human enzyme. Ser-7 becomes phosphorylated before initiation of transcription at promoter regions. We identify cyclin-dependent kinase 7 (CDK7) as one responsible kinase. Phosphorylation of both Ser-5 and Ser-7 is fully dependent on the cofactor complex Mediator. A subform of Mediator associated with an active RNAPII is critical for preinitiation complex formation and CTD phosphorylation. The Mediator-RNAPII complex independently recruits TFIIB and CDK7 to core promoter regions. CDK7 phosphorylates Ser-7 selectively in the context of an intact preinitiation complex. CDK7 is not the only kinase that can modify Ser-7 of the CTD. ChIP experiments with chemical inhibitors provide evidence that other yet to be identified kinases further phosphorylate Ser-7 in coding regions. PMID:19901026

  7. A Superhelical Spiral in the Escherichia coli DNA Gyrase A C-terminal Domain Imparts Unidirectional Supercoiling Bias

    SciTech Connect

    Ruthenburg,A.; Graybosch, D.; Huetsch, J.; Verdine, G.

    2005-01-01

    DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped {beta}-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (-) DNA supercoiling.

  8. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase

    SciTech Connect

    He, Yuan; Roth, Christian; Turkenburg, Johan P.; Davies, Gideon J.

    2014-01-01

    The crystal structure of a bacterial acetyltransferase with 27% sequence identity to the C-terminal domain of human O-GlcNAcase has been solved at 1.5 Å resolution. This S. sviceus protein is compared with known GCN5-related acetyltransferases, adding to the diversity observed in this superfamily. The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP-05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain.

  9. Submolecular-Scale Imaging of α-Helices and C-Terminal Domains of Tubulins by Frequency Modulation Atomic Force Microscopy in Liquid

    PubMed Central

    Asakawa, Hitoshi; Ikegami, Koji; Setou, Mitsutoshi; Watanabe, Naoki; Tsukada, Masaru; Fukuma, Takeshi

    2011-01-01

    In this study, we directly imaged subnanometer-scale structures of tubulins by performing frequency modulation atomic force microscopy (FM-AFM) in liquid. Individual α-helices at the surface of a tubulin protofilament were imaged as periodic corrugations with a spacing of 0.53 nm, which corresponds to the common pitch of an α-helix backbone (0.54 nm). The identification of individual α-helices allowed us to determine the orientation of the deposited tubulin protofilament. As a result, C-terminal domains of tubulins were identified as protrusions with a height of 0.4 nm from the surface of the tubulin. The imaging mechanism for the observed subnanometer-scale contrasts is discussed in relation to the possible structures of the C-terminal domains. Because the C-terminal domains are chemically modified to regulate the interactions between tubulins and other biomolecules (e.g., motor proteins and microtubule-associated proteins), detailed structural information on individual C-terminal domains is valuable for understanding such regulation mechanisms. The results obtained in this study demonstrate that FM-AFM is capable of visualizing the structural variation of tubulins with subnanometer resolution. This is an important first step toward using FM-AFM to analyze the functions of tubulins. PMID:21889465

  10. Solution structure and tandem DNA recognition of the C-terminal effector domain of PmrA from Klebsiella pneumoniae

    PubMed Central

    Lou, Yuan-Chao; Wang, Iren; Rajasekaran, M.; Kao, Yi-Fen; Ho, Meng-Ru; Hsu, Shang-Te Danny; Chou, Shan-Ho; Wu, Shih-Hsiung; Chen, Chinpan

    2014-01-01

    Klebsiella pneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrAC) recognizes tandem imperfect repeat sequences on the promoters of genes to induce antimicrobial peptide resistance after phosphorylation and dimerization of its N-terminal receiver domain (PmrAN). However, structural information concerning how phosphorylation of the response regulator enhances DNA recognition remains elusive. To gain insights, we determined the nuclear magnetic resonance solution structure of PmrAC and characterized the interactions between PmrAC or BeF3−-activated full-length PmrA (PmrAF) and two DNA sequences from the pbgP promoter of K. pneumoniae. We showed that PmrAC binds to the PmrA box, which was verified to contain two half-sites, 5′-CTTAAT-3′ and 5′-CCTAAG-3′, in a head-to-tail fashion with much stronger affinity to the first than the second site without cooperativity. The structural basis for the PmrAC–DNA complex was investigated using HADDOCK docking and confirmed by paramagnetic relaxation enhancement. Unlike PmrAC, PmrAF recognizes the two sites simultaneously and specifically. In the PmrAF–DNA complex, PmrAN may maintain an activated homodimeric conformation analogous to that in the free form and the interactions between two PmrAC molecules aid in bending and binding of the DNA duplex for transcription activation. PMID:24371275

  11. Investigation of the C-terminal domain of the bacterial DNA-(adenine N6)-methyltransferase CcrM.

    PubMed

    Maier, Johannes A H; Albu, Razvan F; Jurkowski, Tomasz P; Jeltsch, Albert

    2015-12-01

    CcrM-related DNA-(adenine N6)-methyltransferases play very important roles in the biology of Caulobacter crescentus and other alpha-proteobacteria. These enzymes methylate GANTC sequences, but the molecular mechanism by which they recognize their target sequence is unknown. We carried out multiple sequence alignments and noticed that CcrM enzymes contain a conserved C-terminal domain (CTD) which is not present in other DNA-(adenine N6)-methyltransferases and we show here that deletion of this part abrogates catalytic activity and DNA binding of CcrM. A mutational study identified 7 conserved residues in the CTD (out of 13 tested), mutation of which led to a strong reduction in catalytic activity. All of these mutants showed altered DNA binding, but no change in AdoMet binding and secondary structure. Some mutants exhibited reduced DNA binding, but others showed an enhanced DNA binding. Moreover, we show that CcrM does not specifically bind to DNA containing GANTC sequences. Taken together, these findings suggest that the specific CcrM-DNA complex undergoes a conformational change, which is endergonic but essential for catalytic activity and this step is blocked by some of the mutations. Moreover, our data indicate that the CTD of CcrM is involved in DNA binding and recognition. This suggests that the CTD functions as target recognition domain of CcrM and, therefore, CcrM can be considered the first example of a δ-type DNA-(adenine N6)-methyltransferase identified so far. PMID:26475175

  12. Distributive O-GlcNAcylation on the Highly Repetitive C-Terminal Domain of RNA Polymerase II.

    PubMed

    Lu, Lei; Fan, Dacheng; Hu, Chia-Wei; Worth, Matthew; Ma, Zhi-Xiong; Jiang, Jiaoyang

    2016-02-23

    O-GlcNAcylation is a nutrient-responsive glycosylation that plays a pivotal role in transcriptional regulation. Human RNA polymerase II (Pol II) is extensively modified by O-linked N-acetylglucosamine (O-GlcNAc) on its unique C-terminal domain (CTD), which consists of 52 heptad repeats. One approach to understanding the function of glycosylated Pol II is to determine the mechanism of dynamic O-GlcNAcylation on the CTD. Here, we discovered that the Pol II CTD can be extensively O-GlcNAcylated in vitro and in cells. Efficient glycosylation requires a minimum of 20 heptad repeats of the CTD and more than half of the N-terminal domain of O-GlcNAc transferase (OGT). Under conditions of saturated sugar donor, we monitored the attachment of more than 20 residues of O-GlcNAc to the full-length CTD. Surprisingly, glycosylation on the periodic CTD follows a distributive mechanism, resulting in highly heterogeneous glycoforms. Our data suggest that initial O-GlcNAcylation can take place either on the proximal or on the distal region of the CTD, and subsequent glycosylation occurs similarly over the entire CTD with nonuniform distributions. Moreover, removal of O-GlcNAc from glycosylated CTD is also distributive and is independent of O-GlcNAcylation level. Our results suggest that O-GlcNAc cycling enzymes can employ a similar mechanism to react with other protein substrates on multiple sites. Distributive O-GlcNAcylation on Pol II provides another regulatory mechanism of transcription in response to fluctuating cellular conditions. PMID:26807597

  13. A zinc site in the C-terminal domain of RAG1 is essential for DNA cleavage activity

    PubMed Central

    Gwyn, Lori M.; Peak, Mandy M.; De, Pallabi; Rahman, Negar S.; Rodgers, Karla K.

    2009-01-01

    The recombination activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn2+ ions in its catalytically-required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well-resolved. To address this issue, we determined the stoichiometry of Zn2+ ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn2+ ions. Stripping the full complement of bound Zn2+ ions to produce apo-protein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn2+ core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn2+ ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn2+ ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active site residue E962 reduces Zn2+ coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn2+ serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962. PMID:19500590

  14. Structural and Functional Modularity of the Orange Carotenoid Protein: Distinct Roles for the N- and C-Terminal Domains in Cyanobacterial Photoprotection[C][W

    PubMed Central

    Leverenz, Ryan L.; Jallet, Denis; Li, Ming-De; Mathies, Richard A.; Kirilovsky, Diana; Kerfeld, Cheryl A.

    2014-01-01

    The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCPO, to an active red form, OCPR. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP’s photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCPR. A comparison of the spectroscopic properties of the RCP with OCPR indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCPR form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain. PMID:24399299

  15. Intrinsic disorder in the C-terminal domain of the Shaker voltage-activated K+ channel modulates its interaction with scaffold proteins

    PubMed Central

    Magidovich, Elhanan; Orr, Irit; Fass, Deborah; Abdu, Uri; Yifrach, Ofer

    2007-01-01

    The interaction of membrane-embedded voltage-activated potassium channels (Kv) with intracellular scaffold proteins, such as the postsynaptic density 95 (PSD-95) protein, is mediated by the channel C-terminal segment. This interaction underlies Kv channel clustering at unique membrane sites and is important for the proper assembly and functioning of the synapse. In the current study, we address the molecular mechanism underlying Kv/PSD-95 interaction. We provide experimental evidence, based on hydrodynamic and spectroscopic analyses, indicating that the isolated C-terminal segment of the archetypical Shaker Kv channel (ShB-C) is a random coil, suggesting that ShB-C belongs to the recently defined class of intrinsically disordered proteins. We show that isolated ShB-C is still able to bind its scaffold protein partner and support protein clustering in vivo, indicating that unfoldedness is compatible with ShB-C activity. Pulldown experiments involving C-terminal chains differing in flexibility or length further demonstrate that intrinsic disorder in the C-terminal segment of the Shaker channel modulates its interaction with the PSD-95 protein. Our results thus suggest that the C-terminal domain of the Shaker Kv channel behaves as an entropic chain and support a “fishing rod” molecular mechanism for Kv channel binding to scaffold proteins. The importance of intrinsically disordered protein segments to the complex processes of synapse assembly, maintenance, and function is discussed. PMID:17666528

  16. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    PubMed Central

    Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

    2016-01-01

    PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9. PMID:27280970

  17. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain.

    PubMed

    Poirier, Steve; Hamouda, Hocine Ait; Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

    2016-01-01

    PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9. PMID:27280970

  18. Differential Roles of C-terminal Eps15 Homology Domain Proteins as Vesiculators and Tubulators of Recycling Endosomes*

    PubMed Central

    Cai, Bishuang; Giridharan, Sai Srinivas Panapakkam; Zhang, Jing; Saxena, Sugandha; Bahl, Kriti; Schmidt, John A.; Sorgen, Paul L.; Guo, Wei; Naslavsky, Naava; Caplan, Steve

    2013-01-01

    Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 homology domain (EHD) ATPase proteins are involved in the vesiculation process. Here, we address the functional roles of the four EHD proteins. We developed a novel semipermeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess the ability of each protein to vesiculate MICAL-L1-decorated tubular recycling endosomes (TREs). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1 depletion. EHD4 serves a role similar to that of EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TREs in the semipermeabilized cells, fails to do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semipermeabilized cell system, consistent with the lack of tubulation observed upon EHD3 depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane-tubulating protein. PMID:24019528

  19. SUMOylation of the C-terminal domain of DNA topoisomerase IIα regulates the centromeric localization of Claspin

    PubMed Central

    Ryu, Hyunju; Yoshida, Makoto M; Sridharan, Vinidhra; Kumagai, Akiko; Dunphy, William G; Dasso, Mary; Azuma, Yoshiaki

    2015-01-01

    DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity. PMID:26131587

  20. Interactions of the C-terminal Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

    2007-01-01

    NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku(sub 70/80) complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The C-terminal domain of Ku70 (Ku70c, residues 559-609), contains an helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the Ku70c/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the subunit Ku70c and a 14 base pairs DNA duplex, whose starting structures are designed to be variable so as to mimic their different binding modes. By analyzing conformational changes and energetic properties of the complex during MD simulations, we found that interactions are preferred at DNA ends, and within the major groove, which is consistent with previous experimental investigations. In addition, the results indicate that cooperation of Ku70c with other subunits of Ku(sub 70/80) is necessary to explain the high affinity of binding as observed in experiments.

  1. Transient viral DNA replication and repression of viral transcription are supported by the C-terminal domain of the bovine papillomavirus type 1 E1 protein.

    PubMed

    Ferran, M C; McBride, A A

    1998-01-01

    The bovine papillomavirus type 1 E1 protein is important for viral DNA replication and transcriptional repression. It has been proposed that the full-length E1 protein consists of a small N-terminal and a larger C-terminal domain. In this study, it is shown that an E1 polypeptide containing residues 132 to 605 (which represents the C-terminal domain) is able to support transient viral DNA replication, although at a level lower than that supported by the wild-type protein. This domain can also repress E2-mediated transactivation from the P89 promoter as well as the wild-type E1 protein can. PMID:9420289

  2. Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor l-arginine

    SciTech Connect

    Garnett, James A.; Baumberg, Simon; Stockley, Peter G.; Phillips, Simon E. V.

    2007-11-01

    The crystal structure of the C-terminal domain hexameric core of AhrC, with bound corepressor (l-arginine), has been solved at 1.95 Å resolution. Binding of l-arginine results in a rotation between the two trimers of the hexamer, leading to the activation of the DNA-binding state. The arginine repressor/activator protein (AhrC) from Bacillus subtilis belongs to a large family of multifunctional transcription factors that are involved in the regulation of bacterial arginine metabolism. AhrC interacts with operator sites in the promoters of arginine biosynthetic and catabolic operons, acting as a transcriptional repressor at biosynthetic sites and an activator of transcription at catabolic sites. AhrC is a hexamer of identical subunits, each having two domains. The C-terminal domains form the core of the protein and are involved in oligomerization and l-arginine binding. The N-terminal domains lie on the outside of the compact core and play a role in binding to 18 bp DNA operators called ARG boxes. The C-terminal domain of AhrC has been expressed, purified and characterized, and also crystallized as a hexamer with the bound corepressor l-arginine. Here, the crystal structure refined to 1.95 Å is presented.

  3. Structure of the MutL C-terminal domain: a model of intact MutL and its roles in mismatch repair.

    PubMed

    Guarné, Alba; Ramon-Maiques, Santiago; Wolff, Erika M; Ghirlando, Rodolfo; Hu, Xiaojian; Miller, Jeffrey H; Yang, Wei

    2004-10-27

    MutL assists the mismatch recognition protein MutS to initiate and coordinate mismatch repair in species ranging from bacteria to humans. The MutL N-terminal ATPase domain is highly conserved, but the C-terminal region shares little sequence similarity among MutL homologs. We report here the crystal structure of the Escherichia coli MutL C-terminal dimerization domain and the likelihood of its conservation among MutL homologs. A 100-residue proline-rich linker between the ATPase and dimerization domains, which generates a large central cavity in MutL dimers, tolerates sequence substitutions and deletions of one-third of its length with no functional consequences in vivo or in vitro. Along the surface of the central cavity, residues essential for DNA binding are located in both the N- and C-terminal domains. Each domain of MutL interacts with UvrD helicase and is required for activating the helicase activity. The DNA-binding capacity of MutL is correlated with the level of UvrD activation. A model of how MutL utilizes its ATPase and DNA-binding activities to mediate mismatch-dependent activation of MutH endonuclease and UvrD helicase is proposed. PMID:15470502

  4. Leucine-rich repeat kinase 2 binds to neuronal vesicles through protein interactions mediated by its C-terminal WD40 domain.

    PubMed

    Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J O; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius; Gloeckner, Christian Johannes

    2014-06-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

  5. Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

    PubMed Central

    Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J. O.; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius

    2014-01-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

  6. Crystal Structure of the C-terminal Region of Streptococcus mutans Antigen I/II and Characterization of Salivary Agglutinin Adherence Domains

    SciTech Connect

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Crowley, Paula J.; Kelly, Charles; Mitchell, Tim J.; Brady, L. Jeannine; Deivanayagam, Champion

    2012-05-29

    The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 {angstrom} resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C{sub 1}, C{sub 2}, and C{sub 3}. Each domain adopts a DE-variant IgG fold, with two {beta}-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C{sub 1} and C{sub 2}) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C{sub 1} and C{sub 2} domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C{sub 1} and C{sub 2} domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

  7. Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation.

    PubMed

    Mei, Xiaohu; Liu, Mingjing; Herscovitz, Haya; Atkinson, David

    2016-08-01

    apoA-I plays important structural and functional roles in reverse cholesterol transport. We have described the molecular structure of the N-terminal domain, Δ(185-243) by X-ray crystallography. To understand the role of the C-terminal domain, constructs with sequential elongation of Δ(185-243), by increments of 11-residue sequence repeats were studied and compared with Δ(185-243) and WT apoA-I. Constructs up to residue 230 showed progressively decreased percent α-helix with similar numbers of helical residues, similar detergent and lipid binding affinity, and exposed hydrophobic surface. These observations suggest that the C-terminal domain is unstructured with the exception of the last 11-residue repeat (H10B). Similar monomer-dimer equilibrium suggests that the H10B region is responsible for nonspecific aggregation. Cholesterol efflux progressively increased with elongation up to ∼60% of full-length apoA-I in the absence of the H10B. In summary, the sequential repeats in the C-terminal domain are probably unstructured with the exception of H10B. This segment appears to be responsible for initiation of lipid binding and aggregation, as well as cholesterol efflux, and thus plays a vital role during HDL formation. Based on these observations and the Δ(185-243) crystal structure, we propose a lipid-free apoA-I structural model in solution and update the mechanism of HDL biogenesis. PMID:27317763

  8. Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of Ss-LrpB, a transcription regulator from Sulfolobus solfataricus

    SciTech Connect

    Peeters, Eveline; Hoa, Bach Thi Mai; Zegers, Ingrid; Charlier, Daniel; Maes, Dominique

    2005-11-01

    The C-terminal domain of the transcriptional regulator Ss-LrpB from S. solfataricus was purified by affinity chromatography and crystallized. Crystals belong to space group P2{sub 1}2{sub 1}2. A complete data set was collected to a resolution of 2 Å. Ss-LrpB from Sulfolobus solfataricus P2 belongs to the bacterial/archaeal superfamily of Lrp-like (leucine-responsive regulatory protein-like) transcription regulators. The N-terminal DNA-binding domain contains a HTH motif and the C-terminal domain contains an αβ-sandwich (βαββαβ fold). The C-terminal domain was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 59.35, b = 74.86, c = 38.08 Å and a data set was collected to 2.0 Å resolution. Structure determination using a selenomethionine derivative is under way.

  9. Localization of sequences within the C-terminal domain of the cystic fibrosis transmembrane conductance regulator which impact maturation and stability.

    PubMed

    Gentzsch, M; Riordan, J R

    2001-01-12

    Some disease-associated truncations within the 100-residue domain C-terminal of the second nucleotide-binding domain destabilize the mature protein (Haardt, M., Benharouga, M., Lechardeur, D., Kartner, N., and Lukacs, G. L. (1999) J. Biol. Chem. 274, 21873-21877). We now have identified three short oligopeptide regions in the C-terminal domain which impact cystic fibrosis transmembrane conductance regulator (CFTR) maturation and stability in different ways. A highly conserved hydrophobic patch (region I) formed by residues 1413-1416 (FLVI) was found to be crucial for the stability of the mature protein. Nascent chain stability was severely decreased by shortening the protein by 81 amino acids (1400X). This accelerated degradation was sensitive to proteasome inhibitors but not influenced by brefeldin A, indicating that it occurred at the endoplasmic reticulum. The five residues at positions 1400 to 1404 (region II) normally maintain nascent CFTR stability in a positional rather than a sequence-specific manner. A third modulating region (III) constituted by residues 1390 to 1394 destabilizes the protein. Hence the nascent form regains stability on further truncation back to residues 1390 or 1380, permitting some degree of maturation and a low level of cyclic AMP-stimulated chloride channel activity at the cell surface. Thus while not absolutely essential, the C-terminal domain strongly modulates the biogenesis and maturation of CFTR. PMID:11022033

  10. C-Terminal Domain Residues Important for Secretion and Attachment of RgpB in Porphyromonas gingivalis▿

    PubMed Central

    Slakeski, Nada; Seers, Christine A.; Ng, Kaiting; Moore, Caroline; Cleal, Steven M.; Veith, Paul D.; Lo, Alvin W.; Reynolds, Eric C.

    2011-01-01

    Porphyromonas gingivalis, a periodontal pathogen, expresses a group of surface proteins with a common C-terminal domain (CTD) that are exported by a novel secretion system to the surface, where they are covalently attached. Using RgpB as a model CTD protein, we have produced a series of site-directed mutations in the CTD sequence at conserved residues and at residues that may be modified and, hence, surface attached. The mutant RgpB proteins were expressed in a P. gingivalis host lacking functional RgpB and RgpA Arg-specific proteases. The RgpB mutants produced were Y674F, Y674F Y718F, T675Q S679Q T682Q T684Q, T693Q, F695A, D696A, N698A, G699P, G716P, T724Q, T728Q T730Q, and K732Q and a protein with a deletion of residues 692 to 702 (Δ692-702). The mutants were characterized for cell-associated Arg-specific protease activity and for cellular distribution using anti-Rgp antibodies and Western blotting of culture fractions. All the mutants exhibited cell-associated Arg-specific activity similar to that of the positive control except for the D696A and Δ692-702 mutants. For all mutants, except D696A and Δ692-702, the RgpB proteins were found modified and attached to the cell surface, which was the same profile found in the positive-control strain. Only trace amounts of the precursor form of the Δ692-702 mutant were detected in the outer membrane, with none detected in the periplasm or culture fluid although cell transcript levels were normal. The results suggest that residues 692 to 702 of the CTD, in particular, residue D696, have an important role in the attachment of RgpB at the cell surface and that without attachment secretion does not occur. PMID:20971915

  11. The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning☆

    PubMed Central

    Gherasim, Carmen; Hannibal, Luciana; Rajagopalan, Deepa; Jacobsen, Donald W.; Banerjee, Ruma

    2013-01-01

    Mutations in cobalamin or B12 trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B12 processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5′-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ~116–296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways. PMID:23415655

  12. Expression, Purification And Preliminary X-Ray Analysis of the C-Terminal Domain of An Arginine Repressor Protein From Mycobacterium Tuberculosis

    SciTech Connect

    Lu, G.J.; Garen, C.R.; Cherney, M.M.; Cherney, L.T.; Lee, C.; James, M.N.J.

    2009-06-03

    The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 {angstrom}. The crystals belong to space group P1 and the Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of six molecules per unit cell.

  13. Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal Domain of the Capsid Protein: Evaluation of 2-Arylquinazolines as Potential Antiviral Compounds.

    PubMed

    Machara, Aleš; Lux, Vanda; Kožíšek, Milan; Grantz Šašková, Klára; Štěpánek, Ondřej; Kotora, Martin; Parkan, Kamil; Pávová, Marcela; Glass, Bärbel; Sehr, Peter; Lewis, Joe; Müller, Barbara; Kräusslich, Hans-Georg; Konvalinka, Jan

    2016-01-28

    Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA to compete with the known peptide inhibitor and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA and were found to block viral replication at low micromolar concentrations. PMID:26685880

  14. Dynamics of the intrinsically disordered C-terminal domain of the nipah virus nucleoprotein and interaction with the x domain of the phosphoprotein as unveiled by NMR spectroscopy.

    PubMed

    Baronti, Lorenzo; Erales, Jenny; Habchi, Johnny; Felli, Isabella C; Pierattelli, Roberta; Longhi, Sonia

    2015-01-19

    We provide an atomic-resolution description based on NMR spectroscopy, of the intrinsically disordered C-terminal domain of the Nipah virus nucleoprotein (NTAIL ), both in its isolated state and within the nucleocapsid (NC). Within the NC the second half of NTAIL retains conformational behavior similar to that of isolated NTAIL , whereas the first half of NTAIL becomes much more rigid. In spite of the mostly disordered nature of NTAIL , chemical shifts and relaxation measurements show a significant degree of α-helical sampling in the molecular recognition element (MoRE) involved in binding to the X domain (XD) of the phosphoprotein, with this preconfiguration being more pronounced than in the NTAIL domain from the cognate Hendra virus. Outside the MoRE, an additional region exhibiting reduced flexibility was identified within NTAIL and found to be involved in binding to the XD. (1) H- and (13) C-detected titration NMR experiments support a highly dynamic binding of NTAIL at the surface of the XD. PMID:25492314

  15. High-resolution crystal structure reveals a HEPN domain at the C-terminal region of S. cerevisiae RNA endonuclease Swt1

    SciTech Connect

    Peng, Shuxia Zhou, Ke; Wang, Wenjia; Gao, Zengqiang; Dong, Yuhui; Liu, Quansheng

    2014-10-31

    Highlights: • Crystal structure of the C-terminal (CT) domain of Swt1 was determined at 2.3 Å. • Structure of the CT domain was identified as HEPN domain superfamily member. • Low-resolution envelope of Swt1 full-length in solution was analyzed by SAXS. • The middle and CT domains gave good fit to SAXS structural model. - Abstract: Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.

  16. Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

    SciTech Connect

    Pazehoski, Kristina O.; Cobine, Paul A.; Winzor, Donald J.; Dameron, Charles T.

    2011-03-11

    Research highlights: {yields} A metal-binding protein domain is directly involved in protein dimerization. {yields} Fusing the metal-binding domain to a monomeric protein induces dimerization. {yields} Frontal size-exclusion chromatography measures the strength of dimer interaction. {yields} Ultracentrifugation studies confirm the influence of metal binding on dimerization. -- Abstract: Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size-exclusion chromatography studies. The studies demonstrate that the attachment of these CopY residues to the immunoglobulin-binding domain of streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.

  17. Thrombospondin-1-N-Terminal Domain Induces a Phagocytic State and Thrombospondin-1-C-Terminal Domain Induces a Tolerizing Phenotype in Dendritic Cells

    PubMed Central

    Tabib, Adi; Krispin, Alon; Trahtemberg, Uriel; Verbovetski, Inna; Lebendiker, Mario; Danieli, Tsafi; Mevorach, Dror

    2009-01-01

    In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine the role of HBD in the clearance of apoptotic cells, and whether the phagocytic and tolerizing state of DCs is mediated by the HBD itself, or whether the entire TSP-1 is needed. Therefore, we have cloned the human HBD, and compared its interactions with DC to those with TSP-1. Here we show that rHBD by itself is not directly responsible for immune paralysis and tolerizing phenotype of DCs, at least in the monomeric form, but has a significant role in rendering DCs phagocytic. Binding of TSP-1-C-terminal domain on the other hand induces a tolerizing phenotype in dendritic cells. PMID:19721725

  18. The Crystal Structure of the C-Terminal Domain of the Salmonella enterica PduO Protein: An Old Fold with a New Heme-Binding Mode.

    PubMed

    Ortiz de Orué Lucana, Darío; Hickey, Neal; Hensel, Michael; Klare, Johann P; Geremia, Silvano; Tiufiakova, Tatiana; Torda, Andrew E

    2016-01-01

    The two-domain protein PduO, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica is an ATP:Cob(I)alamin adenosyltransferase, but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) is, however, unknown. In this study, comparative growth assays with a set of Salmonella mutant strains showed that this domain is necessary for effective in vivo catabolism of 1,2-propanediol. It was also shown that isolated, recombinantly-expressed PduOC binds heme in vivo. The structure of PduOC co-crystallized with heme was solved (1.9 Å resolution) showing an octameric assembly with four heme moieities. The four heme groups are highly solvent-exposed and the heme iron is hexa-coordinated with bis-His ligation by histidines from different monomers. Static light scattering confirmed the octameric assembly in solution, but a mutation of the heme-coordinating histidine caused dissociation into dimers. Isothermal titration calorimetry using the PduOC apoprotein showed strong heme binding (K d = 1.6 × 10(-7) M). Biochemical experiments showed that the absence of the C-terminal domain in PduO did not affect adenosyltransferase activity in vitro. The evidence suggests that PduOC:heme plays an important role in the set of cobalamin transformations required for effective catabolism of 1,2-propanediol. Salmonella PduO is one of the rare proteins which binds the redox-active metabolites heme and cobalamin, and the heme-binding mode of the C-terminal domain differs from that in other members of this protein family. PMID:27446048

  19. The Crystal Structure of the C-Terminal Domain of the Salmonella enterica PduO Protein: An Old Fold with a New Heme-Binding Mode

    PubMed Central

    Ortiz de Orué Lucana, Darío; Hickey, Neal; Hensel, Michael; Klare, Johann P.; Geremia, Silvano; Tiufiakova, Tatiana; Torda, Andrew E.

    2016-01-01

    The two-domain protein PduO, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica is an ATP:Cob(I)alamin adenosyltransferase, but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) is, however, unknown. In this study, comparative growth assays with a set of Salmonella mutant strains showed that this domain is necessary for effective in vivo catabolism of 1,2-propanediol. It was also shown that isolated, recombinantly-expressed PduOC binds heme in vivo. The structure of PduOC co-crystallized with heme was solved (1.9 Å resolution) showing an octameric assembly with four heme moieities. The four heme groups are highly solvent-exposed and the heme iron is hexa-coordinated with bis-His ligation by histidines from different monomers. Static light scattering confirmed the octameric assembly in solution, but a mutation of the heme-coordinating histidine caused dissociation into dimers. Isothermal titration calorimetry using the PduOC apoprotein showed strong heme binding (Kd = 1.6 × 10−7 M). Biochemical experiments showed that the absence of the C-terminal domain in PduO did not affect adenosyltransferase activity in vitro. The evidence suggests that PduOC:heme plays an important role in the set of cobalamin transformations required for effective catabolism of 1,2-propanediol. Salmonella PduO is one of the rare proteins which binds the redox-active metabolites heme and cobalamin, and the heme-binding mode of the C-terminal domain differs from that in other members of this protein family. PMID:27446048

  20. Extracellular Signal-Regulated Kinase 7 (ERK7), a Novel ERK with a C-Terminal Domain That Regulates Its Activity, Its Cellular Localization, and Cell Growth

    PubMed Central

    Abe, Mark K.; Kuo, Wen-Liang; Hershenson, Marc B.; Rosner, Marsha Rich

    1999-01-01

    Mitogen-activated protein (MAP) kinases play distinct roles in a variety of cellular signaling pathways and are regulated through multiple mechanisms. In this study, a novel 61-kDa member of the MAP kinase family, termed extracellular signal-regulated kinase 7 (ERK7), has been cloned and characterized. Although it has the signature TEY activation motif of ERK1 and ERK2, ERK7 is not activated by extracellular stimuli that typically activate ERK1 and ERK2 or by common activators of c-Jun N-terminal kinase (JNK) and p38 kinase. Instead, ERK7 has appreciable constitutive activity in serum-starved cells that is dependent on the presence of its C-terminal domain. Interestingly, the C-terminal tail, not the kinase domain, of ERK7 regulates its nuclear localization and inhibition of growth. Taken together, these results elucidate a novel type of MAP kinase whereby interactions via its C-terminal tail, rather than extracellular signal-mediated activation cascades, regulate its activity, localization, and function. PMID:9891064

  1. In vitro catalytic activity of N-terminal and C-terminal domains in NukM, the post-translational modification enzyme of nukacin ISK-1.

    PubMed

    Shimafuji, Chinatsu; Noguchi, Megumi; Nishie, Mami; Nagao, Jun-Ichi; Shioya, Kouki; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

    2015-12-01

    Lantibiotics are antibacterial peptides containing unique thioether cross-links termed lanthionine and methyllanthionine. NukM, the modifying enzyme of nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the dehydration of specific Ser/Thr residues in a precursor peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor peptide of nisin is modified by 2 enzymes, NisB and NisC, which mediate dehydration and cyclization, respectively. While the C-terminal domain of NukM is homologous to NisC, the N-terminal domain has no homology with other known proteins. We expressed and characterized the N- and C-terminal domains of NukM, NukMN, and NukMC, separately. In vitro reconstitution revealed that full-length NukM fully modified the substrate peptide NukA. NukMN partially phosphorylated, dehydrated, and cyclized NukA. By contrast, NukMC did not catalyze dehydration, phosphorylation, or cyclization reactions. Interaction studies using surface plasmon resonance analysis indicated that NukM and NukMN can bind NukA with high affinity, whereas NukMC has low substrate-recognition activity. These results suggest that NukMN is mainly responsible for substrate recognition and dehydration and that the whole NukM structure, including the C-terminal domain, is required for the complete modification of NukA. To the best of our knowledge, this is the first report providing insights into the in vitro catalytic activity of individual domains of a LanM-type modification enzyme. PMID:25971839

  2. Functions of the N- and C-Terminal Domains of Human RAP74 in Transcriptional Initiation, Elongation, and Recycling of RNA Polymerase II

    PubMed Central

    Lei, Lei; Ren, Delin; Finkelstein, Ann; Burton, Zachary F.

    1998-01-01

    Transcription factor IIF (TFIIF) cooperates with RNA polymerase II (pol II) during multiple stages of the transcription cycle including preinitiation complex assembly, initiation, elongation, and possibly termination and recycling. Human TFIIF appears to be an α2β2 heterotetramer of RNA polymerase II-associating protein 74- and 30-kDa subunits (RAP74 and RAP30). From inspection of its 517-amino-acid (aa) sequence, the RAP74 subunit appears to comprise separate N- and C-terminal domains connected by a flexible loop. In this study, we present functional data that strongly support this model for RAP74 architecture and further show that the N- and C-terminal domains and the central loop of RAP74 have distinct roles during separate phases of the transcription cycle. The N-terminal domain of RAP74 (minimally aa 1 to 172) is sufficient to deliver pol II into a complex formed on the adenovirus major late promoter with the TATA-binding protein, TFIIB, and RAP30. A more complete N-terminal domain fragment (aa 1 to 217) strongly stimulates both accurate initiation and elongation by pol II. The region of RAP74 between aa 172 and 205 and a subregion between aa 170 and 178 are critical for both accurate initiation and elongation, and mutations in these regions have similar effects on initiation and elongation. Based on these observations, RAP74 appears to have similar functions in initiation and elongation. The central region and the C-terminal domain of RAP74 do not contribute strongly to single-round accurate initiation or elongation stimulation but do stimulate multiple-round transcription in an extract system. PMID:9528785

  3. Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications.

    PubMed

    Gentzsch, Martina; Aleksandrov, Andrei; Aleksandrov, Luba; Riordan, John R

    2002-09-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) contains two nucleotide-binding domains (NBDs) or ATP-binding cassettes (ABCs) that characterize a large family of membrane transporters. Although the three-dimensional structures of these domains from several ABC proteins have been determined, this is not the case for CFTR, and hence the domains are defined simply on the basis of sequence alignment. The functional C-terminal boundary of NBD1 of CFTR was located by analysis of chloride channel function [Chan, Csanady, Seto-Young, Nairn and Gadsby (2000) J. Gen. Physiol. 116, 163-180]. However, the boundary between the C-terminal end of NBD2 and sequences further downstream in the whole protein, that are important for its cellular localization and endocytotic turnover, has not been defined. We have now done this by assaying the influence of progressive C-terminal truncations on photolabelling of NBD2 by 8-azido-ATP, which reflects hydrolysis, as well as binding, at that domain, and on NBD2-dependent channel gating itself. The boundary defined in this way is between residues 1420 and 1424, which corresponds to the final beta-strand in aligned NBDs whose structures have been determined. Utilization of this information should facilitate the generation of monodisperse NBD2 polypeptides for structural analysis, which until now has not been possible. The established boundary includes within NBD2 a hydrophobic patch of four residues (1413-1416) previously shown to be essential for CFTR maturation and stability [Gentzsch and Riordan (2001) J. Biol. Chem. 276, 1291-1298]. This hydrophobic cluster is conserved in most ABC proteins, and on alignment with ones of known structure constitutes the penultimate beta-strand of the domain which is likely to participate in essential structure-stabilizing beta-sheet formation. PMID:12020354

  4. Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications.

    PubMed Central

    Gentzsch, Martina; Aleksandrov, Andrei; Aleksandrov, Luba; Riordan, John R

    2002-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) contains two nucleotide-binding domains (NBDs) or ATP-binding cassettes (ABCs) that characterize a large family of membrane transporters. Although the three-dimensional structures of these domains from several ABC proteins have been determined, this is not the case for CFTR, and hence the domains are defined simply on the basis of sequence alignment. The functional C-terminal boundary of NBD1 of CFTR was located by analysis of chloride channel function [Chan, Csanady, Seto-Young, Nairn and Gadsby (2000) J. Gen. Physiol. 116, 163-180]. However, the boundary between the C-terminal end of NBD2 and sequences further downstream in the whole protein, that are important for its cellular localization and endocytotic turnover, has not been defined. We have now done this by assaying the influence of progressive C-terminal truncations on photolabelling of NBD2 by 8-azido-ATP, which reflects hydrolysis, as well as binding, at that domain, and on NBD2-dependent channel gating itself. The boundary defined in this way is between residues 1420 and 1424, which corresponds to the final beta-strand in aligned NBDs whose structures have been determined. Utilization of this information should facilitate the generation of monodisperse NBD2 polypeptides for structural analysis, which until now has not been possible. The established boundary includes within NBD2 a hydrophobic patch of four residues (1413-1416) previously shown to be essential for CFTR maturation and stability [Gentzsch and Riordan (2001) J. Biol. Chem. 276, 1291-1298]. This hydrophobic cluster is conserved in most ABC proteins, and on alignment with ones of known structure constitutes the penultimate beta-strand of the domain which is likely to participate in essential structure-stabilizing beta-sheet formation. PMID:12020354

  5. Chemical shift assignments of the C-terminal EF-hand domain of α-actinin-1.

    PubMed

    Turner, Matthew; Anderson, David E; Rajan, Sahana; Hell, Johannes W; Ames, James B

    2016-04-01

    The regulation and localization of the neuronal voltage gated Ca(2+) channel CaV1.2 is important for synaptic plasticity associated with learning and memory. The cytoskeletal protein, α-actinin-1 is known to interact with CaV1.2 and stabilize its localization at the postsynaptic membrane. Here we report both backbone and sidechain NMR assignments for the C-terminal EF-hands (EF3 and EF4) of α-actinin-1 (residues 824-892, called ACTN_EF34) bound to the IQ-motif (residues 1644-1665) from CaV1.2 (BMRB accession no. 25902). PMID:26861220

  6. C terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain

    SciTech Connect

    South, T.L.; Blake, P.R. ); Hare, D.R.; Summers, M.F. )

    1991-06-25

    Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retriviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 (Zn(HIV1-F2)). Unlike results obtained for the first retroviral-type zinc finger peptide, Zn (HIV1-F1) broad signals indicative of confomational lability were observed in the {sup 1}H NMR spectrum of An(HIV1-F2) at 25 C. The NMR signals narrowed upon cooling to {minus}2 C, enabling complete {sup 1}H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were sued to generate 30 distance geometry (DG) structures with penalties in the range 0.02-0.03 {angstrom}{sup 2}. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. These results indicate that the r.t. zinc finger sequences observed in retroviral NCPs, simple plant virus coat proteins, and in a human single-stranded nucleic acid binding protein share a common structural motif.

  7. Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity

    PubMed Central

    Faye, Audrey; Borthwick, Karen; Esnous, Catherine; Price, Nigel T.; Gobin, Stéphanie; Jackson, Vicky N.; Zammit, Victor A.; Girard, Jean; Prip-Buus, Carina

    2004-01-01

    We have previously proposed that changes in malonyl-CoA sensitivity of rat L-CPT1 (liver carnitine palmitoyltransferase 1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains. By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we have now shown the existence of such N–C (N- and C-terminal domain) intramolecular interactions both in wild-type L-CPT1 expressed in Saccharomyces cerevisiae and in the native L-CPT1 in fed rat liver mitochondria. These N–C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity. Moreover, increasing the outer membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitization, attenuated the N–C interactions. This indicates that the changes in malonyl-CoA sens-itivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N–C interactions. Finally, we show that mutations in the regulatory regions of the N-terminal domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased [S24A (Ser24→Ala)/Q30A] or abrogated (E3A) malonyl-CoA sensitivity. Moreover, we have identified the region immediately N-terminal to transmembrane domain 1 (residues 40–47) as being involved in the chemical N–C cross-linking. These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains, and that this determines its degree of malonyl-CoA sensitivity depending on the physiological state

  8. Elliptocytosis in patients with C-terminal domain mutations of protein 4.1 correlates with encoded messenger RNA levels rather than with alterations in primary protein structure.

    PubMed

    Morinière, M; Ribeiro, L; Dalla Venezia, N; Deguillien, M; Maillet, P; Cynober, T; Delhommeau, F; Almeida, H; Tamagnini, G; Delaunay, J; Baklouti, F

    2000-03-01

    Early biochemical studies defined 4 functional domains of the erythroid protein 4.1 (4.1R). From amino-terminal to carboxy-terminal, these are 30 kd, 16 kd, 10 kd, and 22/24 kd in size. Although the functional properties of both the 30-kd and the 10-kd domain have been demonstrated in red cells, no functional activities have been assigned to either the 16-kd or the 22/24-kd domain in these cells. We here describe new mutations in the sequence encoding the C-terminal 22/24-kd domain that are associated with hereditary elliptocytosis. An unusually mild phenotype observed in heterozygous and homozygous members of 1 family suggested heterogeneity in the pattern of expression of 4.1R deficiency. Using a variety of protein and messenger RNA (mRNA) quantification strategies, we showed that, regardless of the alteration in the C-terminal primary sequence, when the protein is produced, it assembles at the cell membrane. In addition, we found that alterations in red cell morphologic features and membrane function correlate with the amount of membrane-associated protein-and therefore with the amount of mRNA accumulated-rather than with the primary structure of the variant proteins. These data suggest that an intact sequence at exons 19 through 21 encoding part of the C-terminal 22/24-kd region is not required for proper protein 4.1R assembly in mature red cells. (Blood. 2000;95:1834-1841) PMID:10688845

  9. Properties and catalytic activities of MICAL1, the flavoenzyme involved in cytoskeleton dynamics, and modulation by its CH, LIM and C-terminal domains.

    PubMed

    Vitali, Teresa; Maffioli, Elisa; Tedeschi, Gabriella; Vanoni, Maria A

    2016-03-01

    MICAL1 is a cytoplasmic 119 kDa protein participating in cytoskeleton dynamics through the NADPH-dependent oxidase and F-actin depolymerizing activities of its N-terminal flavoprotein domain, which is followed by calponin homology (CH), LIM domains and a C-terminal region with Pro-, Glu-rich and coiled-coil motifs. MICAL1 and truncated forms lacking the C-terminal, LIM and/or CH regions have been produced and characterized. The CH, LIM and C-terminal regions cause an increase of Km,NADPH exhibited by the NADPH oxidase activity of the flavoprotein domain, paralleling changes in the overall protein charge. The C-terminus also determines a ∼ 10-fold decrease of kcat, revealing its role in establishing an inactive/active conformational equilibrium, which is at the heart of the regulation of MICAL1 in cells. F-actin lowers Km,NADPH (10-50 μM) and increases kcat (10-25 s(-1)) to similar values for all MICAL forms. The apparent Km,actin of MICAL1 is ∼ 10-fold higher than that of the other forms (3-5 μM), reflecting the fact that F-actin binds to the flavoprotein domain in the MICAL's active conformation and stabilizes it. Analyses of the reaction in the presence of F-actin indicate that actin depolymerization is mediated by H2O2 produced by the NADPH oxidase reaction, rather than due to direct hydroxylation of actin methionine residues. PMID:26845023

  10. The solution structure of the C-terminal domain of NfeD reveals a novel membrane-anchored OB-fold.

    PubMed

    Kuwahara, Yohta; Ohno, Ayako; Morii, Taichi; Yokoyama, Hideshi; Matsui, Ikuo; Tochio, Hidehito; Shirakawa, Masahiro; Hiroaki, Hidekazu

    2008-11-01

    Nodulation formation efficiency D (NfeD) is a member of a class of membrane-anchored ClpP-class proteases. There is a second class of NfeD homologs that lack the ClpP domain. The genes of both NfeD classes usually are part of an operon that also contains a gene for a prokaryotic homolog of stomatin. (Stomatin is a major integral-membrane protein of mammalian erythrocytes.) Such NfeD/stomatin homolog gene pairs are present in more than 290 bacterial and archaeal genomes, and their protein products may be part of the machinery used for quality control of membrane proteins. Herein, we report the structure of the isolated C-terminal domain of PH0471, a Pyrococcus horikoshii NfeD homolog, which lacks the ClpP domain. This C-terminal domain (termed NfeDC) contains a five-strand beta-barrel, which is structurally very similar to the OB-fold (oligosaccharide/oligonucleotide-binding fold) domain. However, there is little sequence similarity between it and previously characterized OB-fold domains. The NfeDC domain lacks the conserved surface residues that are necessary for the binding of an OB-fold domain to DNA/RNA, an ion. Instead, its surface is composed of residues that are uniquely conserved in NfeD homologs and that form the structurally conserved surface turns and beta-bulges. There is also a conserved tryptophan present on the surface. We propose that, in general, NfeDC domains may interact with other spatially proximal membrane proteins and thereby regulate their activities. PMID:18687870

  11. Structure of the Trichomonas vaginalis Myb3 DNA-binding domain bound to a promoter sequence reveals a unique C-terminal β-hairpin conformation.

    PubMed

    Wei, Shu-Yi; Lou, Yuan-Chao; Tsai, Jia-Yin; Ho, Meng-Ru; Chou, Chun-Chi; Rajasekaran, M; Hsu, Hong-Ming; Tai, Jung-Hsiang; Hsiao, Chwan-Deng; Chen, Chinpan

    2012-01-01

    Trichomonas vaginalis Myb3 transcription factor (tvMyb3) recognizes the MRE-1 promoter sequence and regulates ap65-1 gene, which encodes a hydrogenosomal malic enzyme that may play a role in the cytoadherence of the parasite. Here, we identified tvMyb3(53-180) as the essential fragment for DNA recognition and report the crystal structure of tvMyb3(53-180) bound to MRE-1 DNA. The N-terminal fragment adopts the classical conformation of an Myb DNA-binding domain, with the third helices of R2 and R3 motifs intercalating in the major groove of DNA. The C-terminal extension forms a β-hairpin followed by a flexible tail, which is stabilized by several interactions with the R3 motif and is not observed in other Myb proteins. Interestingly, this unique C-terminal fragment does not stably connect with DNA in the complex structure but is involved in DNA binding, as demonstrated by NMR chemical shift perturbation, (1)H-(15)N heteronuclear-nuclear Overhauser effect and intermolecular paramagnetic relaxation enhancement. Site-directed mutagenesis also revealed that this C-terminal fragment is crucial for DNA binding, especially the residue Arg(153) and the fragment K(170)KRK(173). We provide a structural basis for MRE-1 DNA recognition and suggest a possible post-translational regulation of tvMyb3 protein. PMID:21908401

  12. Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2

    SciTech Connect

    Russo, Andrew T.; Watowich, Stanley J.

    2006-06-01

    The C-terminal protease domain of Venezuelan equine encephalitis virus (VEEV) nsP2 has been overexpressed in E. coli, purified and successfully crystallized. Native crystals diffract to beyond 2.5 Å resolution and isomorphous heavy-atom derivatives suitable for phase analysis have been identified. The C-terminal region of Venezuelan equine encephalitis virus (VEEV) nsP2 is responsible for proteolytic processing of the VEEV polyprotein replication complex. This action regulates the activity of the replication complex and is essential for viral replication, thus making nsP2 a very attractive target for development of VEEV therapeutics. The 338-amino-acid C-terminal region of VEEV nsP2 has been overexpressed in Escherichia coli, purified and crystallized. Crystals diffract to beyond 2.5 Å resolution and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}. Isomorphous heavy-atom derivatives suitable for phase analysis have been obtained and work on building a complete structural model is under way.

  13. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain.

    PubMed

    de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A; Enghild, Jan J; Thøgersen, Ida B; Gao, Jinlong; Kwan, Ann H; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F Xavier; Nguyen, Ky-Anh; Potempa, Jan

    2016-01-01

    In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

  14. Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

    PubMed

    Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

    2016-10-01

    The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium. PMID:27310312

  15. Specific recognition of four-way DNA junctions by the C-terminal zinc-binding domain of HPV oncoprotein E6.

    PubMed

    Ristriani, T; Nominé, Y; Masson, M; Weiss, E; Travé, G

    2001-01-26

    E6 is an oncoprotein implicated in cervical cancers produced by " high risk " human papillomaviruses. E6 binds specifically to several cellular proteins, including the tumour suppressor p53 and the ubiquitin ligase E6-AP. However, E6 is also a DNA-binding protein which recognizes a structural motive present in four-way junctions. Here, we demonstrate that the C-terminal zinc-binding domain of E6, expressed separately from the rest of the protein, fully retains the selective four-way junction recognition activity. The domain can bind to two identical and independent sites on a single junction, whereas full-length E6 can only bind to one site. The junction bound to either one or two domains adopts an extended square conformation. These results allow us to assign the structure-dependent DNA recognition activity of E6 to its C-terminal domain, which therefore represents a new class of zinc-stabilized DNA-binding module. Comparison with the binding characteristics of other junction-specific proteins enlightens the rules which govern protein-induced deformation of four-way DNA junctions. PMID:11162088

  16. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

    PubMed Central

    de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A.; Enghild, Jan J.; Thøgersen, Ida B.; Gao, Jinlong; Kwan, Ann H.; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F. Xavier; Nguyen, Ky-Anh; Potempa, Jan

    2016-01-01

    In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

  17. Separate bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: the role of the C-terminal tail in modulating enzyme activity.

    PubMed Central

    Li, L; Ling, S; Wu, C l; Yao, W; Xu, G

    1997-01-01

    The separate bisphosphatase domain (amino acid residues 243-468) of the chicken liver bifunctional enzyme 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase was expressed in Escherichia coli and purified to homogeneity. The fructose-2, 6-bisphosphatase activity of the separate domain was 7-fold higher than that of the native bifunctional enzyme, and exhibited substrate inhibition characteristic of the native enzyme. The inhibition of the enzymes by fructose 2,6-bisphosphate could be overcome by Pi, glycerol 3-phosphate and GTP. Deletion of 30 amino acid residues from the C-terminus of the separate domain resulted in around a 5-fold increase in the Vmax of the bisphosphatase. Also, the truncated form was more accessible to chemical modification by diethyl pyrocarbonate and N-ethylmaleimide, suggesting a more open structure than the wild-type form. In addition, the mutation of cysteine-389 to alanine increased bisphosphatase activity by 20% and the Km value for fructose 2,6-bisphosphate by 3-fold, and both the point mutation at cysteine-389 and the deletional mutation led to the predominantly insoluble expression of the enzyme. The results indicated that the C-terminal tail plays a role in modulating the enzyme activity and suggested that the difference in the C-terminal tail sequence is responsible for the difference in activity of the chicken and rat liver fructose-2,6-bisphosphatases. It is postulated that an interaction between the C-terminal tail and the active site might be present. PMID:9396716

  18. Helicobacter pylori RNA polymerase α-subunit C-terminal domain shows features unique to ɛ-proteobacteria and binds NikR/DNA complexes

    PubMed Central

    Borin, Brendan N; Tang, Wei; Krezel, Andrzej M

    2014-01-01

    Bacterial RNA polymerase is a large, multi-subunit enzyme responsible for transcription of genomic information. The C-terminal domain of the α subunit of RNA polymerase (αCTD) functions as a DNA and protein recognition element localizing the polymerase on certain promoter sequences and is essential in all bacteria. Although αCTD is part of RNA polymerase, it is thought to have once been a separate transcription factor, and its primary role is the recruitment of RNA polymerase to various promoters. Despite the conservation of the subunits of RNA polymerase among bacteria, the mechanisms of regulation of transcription vary significantly. We have determined the tertiary structure of Helicobacter pylori αCTD. It is larger than other structurally determined αCTDs due to an extra, highly amphipathic helix near the C-terminal end. Residues within this helix are highly conserved among ɛ-proteobacteria. The surface of the domain that binds A/T rich DNA sequences is conserved and showed binding to DNA similar to αCTDs of other bacteria. Using several NikR dependent promoter sequences, we observed cooperative binding of H. pylori αCTD to NikR:DNA complexes. We also produced αCTD lacking the 19 C-terminal residues, which showed greatly decreased stability, but maintained the core domain structure and binding affinity to NikR:DNA at low temperatures. The modeling of H. pylori αCTD into the context of transcriptional complexes suggests that the additional amphipathic helix mediates interactions with transcriptional regulators. PMID:24442709

  19. Structural and metal binding characterization of the C-terminal metallochaperone domain of membrane fusion protein SilB from Cupriavidus metallidurans CH34.

    PubMed

    Bersch, Beate; Derfoufi, Kheiro-Mouna; De Angelis, Fabien; Auquier, Vanessa; Ekendé, Elisabeth Ngonlong; Mergeay, Max; Ruysschaert, Jean-Marie; Vandenbussche, Guy

    2011-03-29

    Detoxification of heavy metal ions in Proteobacteria is tightly controlled by various systems regulating their sequestration and transport. In Cupriavidus metallidurans CH34, a model organism for heavy metal resistance studies, the sil determinant is potentially involved in the efflux of silver and copper ions. Proteins SilA, SilB, and SilC form a resistance nodulation cell division (RND)-based transport system in which SilB is the periplasmic adaptor protein belonging to the membrane fusion protein (MFP) family. In addition to the four domains typical of known MFPs, SilB has a fifth additional C-terminal domain, called SilB(440-521), which is characterized here. Structure and backbone dynamics of SilB(440-521) have been investigated using nuclear magnetic resonance, and the residues of the metal site were identified from (15)N- and (13)C-edited HSQC spectra. The solution structure and additional metal binding experiments demonstrated that this C-terminal domain folds independently of the rest of the protein and has a conformation and a Ag(+) and Cu(+) binding specificity similar to those determined for CusF from Escherichia coli. The small protein CusF plays a role in metal trafficking in the periplasm. The similarity with CusF suggests a potential metallochaperone role for SilB(440-521) that is discussed in the context of simultaneous expression of different determinants involved in copper resistance in C. metallidurans CH34. PMID:21299248

  20. Eukaryotic N-Glycosylation Occurs via the Membrane-anchored C-terminal Domain of the Stt3p Subunit of Oligosaccharyltransferase*

    PubMed Central

    Huang, Chengdong; Bhaskaran, Rajagopalan; Mohanty, Smita

    2012-01-01

    N-Glycosylation is an essential and highly conserved protein modification. In eukaryotes, it is catalyzed by a multisubunit membrane-associated enzyme, oligosaccharyltransferase (OT). We report the high resolution structure of the C-terminal domain of eukaryotic Stt3p. Unlike its soluble β-sheet-rich prokaryotic counterparts, our model reveals that the C-terminal domain of yeast Stt3p is highly helical and has an overall oblate spheroid-shaped structure containing a membrane-embedded region. Anchoring of this protein segment to the endoplasmic reticulum membrane is likely to bring the membrane-embedded donor substrate closer, thus facilitating glycosylation efficiency. Structural comparison of the region near the WWDYG signature motif revealed that the acceptor substrate-binding site of yeast OT strikingly resembles its prokaryotic counterparts, suggesting a conserved mechanism of N-glycosylation from prokaryotes to eukaryotes. Furthermore, comparison of the NMR and cryo-EM structures of yeast OT revealed that the molecular architecture of this acceptor substrate-recognizing domain has interesting spatial specificity for interactions with other essential OT subunits. PMID:22865878

  1. MLL repression domain interacts with histone deacetylases, the polycomb group proteins HPC2 and BMI-1, and the corepressor C-terminal-binding protein

    PubMed Central

    Xia, Zhen-Biao; Anderson, Melanie; Diaz, Manuel O.; Zeleznik-Le, Nancy J.

    2003-01-01

    The MLL (mixed-lineage leukemia) gene is involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia. We previously identified a transcriptional repression domain in MLL, which contains a region with homology to DNA methyltransferase. In chromosomal translocations, the MLL repression domain is retained in the leukemogenic fusion protein and is required for transforming activity of MLL fusion proteins. We explored the mechanism of action of the MLL repression domain. Histone deacetylase 1 interacts with the MLL repression domain, partially mediating its activity; binding of Cyp33 to the adjacent MLL-PHD domain potentiates this binding. Because the MLL repression domain activity was only partially relieved with the histone deacetylase inhibitor trichostatin A, we explored other protein interactions with this domain. Polycomb group proteins HPC2 and BMI-1 and the corepressor C-terminal-binding protein also bind the MLL repression domain. Expression of exogenous BMI-1 potentiates MLL repression domain activity. Functional antagonism between Mll and Bmi-1 has been shown genetically in murine knockout models for Mll and Bmi-1. Our new data suggest a model whereby recruitment of BMI-1 to the MLL protein may be able to modulate its function. Furthermore, repression mediated by histone deacetylases and that mediated by polycomb group proteins may act either independently or together for MLL function in vivo. PMID:12829790

  2. Dandelion PPO-1/PPO-2 domain-swaps: the C-terminal domain modulates the pH optimum and the linker affects SDS-mediated activation and stability.

    PubMed

    Leufken, Christine M; Moerschbacher, Bruno M; Dirks-Hofmeister, Mareike E

    2015-02-01

    Plant polyphenol oxidases (PPOs) have a conserved three-domain structure: (i) the N-terminal domain (containing the active site) is connected via (ii) a linker to (iii) the C-terminal domain. The latter covers the active site, thereby maintaining the enzyme in a latent state. Activation can be achieved with SDS but little is known about the mechanism. We prepared domain-swap variants of dandelion PPO-1 and PPO-2 to test the specific functions of individual domains and their impact on enzyme characteristics. Our experiments revealed that the C-terminal domain modulates the pH optimum curve and has a strong influence on the optimal pH value. The linker determines the SDS concentration required for full activation. It also influences the SDS concentration required for half maximal activation (kSDS) and the stability of the enzyme during prolonged incubation in buffers containing SDS, but the N-terminal domain has the strongest effect on these parameters. The N-terminal domain also determines the IC50 of SDS and the stability in buffers containing or lacking SDS. We propose that the linker and C-terminal domain fine-tune the activation of plant PPOs. The C-terminal domain adjusts the pH optimum and the linker probably contains an SDS-binding/interaction site that influences inactivation and determines the SDS concentration required for activation. For the first time, we have determined the influence of the three PPO domains on enzyme activation and stability providing insight into the regulation and activation mechanisms of type-3 copper proteins in general. PMID:25484281

  3. Structure of the C-Terminal Half of UvrC Reveals an RNase H Endonuclease Domain with an Argonaute-like Catalytic Triad

    SciTech Connect

    Karakas,E.; Truglio, J.; Croteau, D.; Rhau, B.; Wang, L.; Van Houten, B.; Kisker, C.

    2007-01-01

    Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.

  4. Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Salmonella typhimurium

    PubMed Central

    Meshcheryakov, Vladimir A.; Samatey, Fadel A.

    2011-01-01

    FlhB is a key protein in the regulation of protein export by the bacterial flagellar secretion system. It is composed of two domains: an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhBc). FlhBc from Salmonella typhimurium has been successfully crystallized using the vapour-diffusion method. The crystals diffracted to 2.45 Å resolution and belonged to space group P42212, with unit-cell parameters a = b = 49.06, c = 142.94 Å. A selenomethionine-containing variant of FlhBc has also been crystallized in the same space group and was used for initial phase calculation by the multiwavelength anomalous dispersion (MAD) method. PMID:21795800

  5. In Sup35p filaments (the [PSI+] prion), the globular C-terminal domains are widely offset from the amyloid fibril backbone

    SciTech Connect

    Baxa, U.; Wall, J.; Keller, P. W.; Cheng, N.; Steven, A. C.

    2011-01-01

    In yeast cells infected with the [PSI+] prion, Sup35p forms aggregates and its activity in translation termination is downregulated. Transfection experiments have shown that Sup35p filaments assembled in vitro are infectious, suggesting that they reproduce or closely resemble the prion. We have used several EM techniques to study the molecular architecture of filaments, seeking clues as to the mechanism of downregulation. Sup35p has an N-terminal 'prion' domain; a highly charged middle (M-)domain; and a C-terminal domain with the translation termination activity. By negative staining, cryo-EM and scanning transmission EM (STEM), filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse 65-nm-wide cloud of globular C-domains. In diameter ({approx}8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield -1 subunit per 0.47 nm for N-fibrils, NM-filaments and Sup35p filaments, further supporting the fibril backbone model. The 30 nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations, offering an explanation for the residual Sup35p activity in infected cells, whereby the C-domains remain free enough to interact with ribosomes.

  6. Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis.

    PubMed

    Fan, Jin-Yuan; Means, John C; Bjes, Edward S; Price, Jeffrey L

    2015-07-01

    Drosophila DBT and vertebrate CKIε/δ phosphorylate the period protein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBT(C/ala)) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIδ, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBT(C/ala) did not affect circadian behavior differently from wild-type DBT (DBT(WT)), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT(WT) protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT(C/ala) did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis. PMID:25939385

  7. A Novel Bmal1 Mutant Mouse Reveals Essential Roles of the C-Terminal Domain on Circadian Rhythms

    PubMed Central

    Cheon, Solmi; Row, Hansang; Lee, Jiyeon; Han, Dong-Hee; Cho, Sehyung; Kim, Kyungjin

    2015-01-01

    The mammalian circadian clock is an endogenous biological timer comprised of transcriptional/translational feedback loops of clock genes. Bmal1 encodes an indispensable transcription factor for the generation of circadian rhythms. Here, we report a new circadian mutant mouse from gene-trapped embryonic stem cells harboring a C-terminus truncated Bmal1 (Bmal1GTΔC) allele. The homozygous mutant (Bmal1GTΔC/GTΔC) mice immediately lost circadian behavioral rhythms under constant darkness. The heterozygous (Bmal1+/GTΔC) mice displayed a gradual loss of rhythms, in contrast to Bmal1+/- mice where rhythms were sustained. Bmal1GTΔC/GTΔC mice also showed arrhythmic mRNA and protein expression in the SCN and liver. Lack of circadian reporter oscillation was also observed in cultured fibroblast cells, indicating that the arrhythmicity of Bmal1GTΔC/GTΔC mice resulted from impaired molecular clock machinery. Expression of clock genes exhibited distinct responses to the mutant allele in Bmal1+/GTΔC and Bmal1GTΔC/GTΔC mice. Despite normal cellular localization and heterodimerization with CLOCK, overexpressed BMAL1GTΔC was unable to activate transcription of Per1 promoter and BMAL1-dependent CLOCK degradation. These results indicate that the C-terminal region of Bmal1 has pivotal roles in the regulation of circadian rhythms and the Bmal1GTΔC mice constitute a novel model system to evaluate circadian functional mechanism of BMAL1. PMID:26394143

  8. Efficient DNA Transfection Mediated by the C-Terminal Domain of Human Immunodeficiency Virus Type 1 Viral Protein R

    PubMed Central

    Kichler, Antoine; Pages, Jean-Christophe; Leborgne, Christian; Druillennec, Sabine; Lenoir, Christine; Coulaud, Dominique; Delain, Etienne; Le Cam, Eric; Roques, Bernard P.; Danos, Olivier

    2000-01-01

    Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr52–96) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr52–96 was 10- to 1,000-fold more active. Vpr52–96-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection. PMID:10823846

  9. Purification, solution properties and crystallization of SIV integrase containing a continuous core and C-terminal domain.

    PubMed

    Li, Y; Yan, Y; Zugay-Murphy, J; Xu, B; Cole, J L; Witmer, M; Felock, P; Wolfe, A; Hazuda, D; Sardana, M K; Chen, Z; Kuo, L C; Sardana, V V

    1999-11-01

    The C-terminal two-thirds segment of integrase derived from the simian immunodeficiency virus has been cloned, expressed in Escherichia coli, and purified to greater than 95% homogeneity. The protein encompasses amino-acid residues 50-293 and contains a F185H substitution to enhance solubility. In dilute solutions at concentrations below 1 mg ml(-1), the enzyme is predominantly dimeric. At the higher concentrations (>10 mg ml(-1)) required to enable crystallization, the enzyme self-associates to form species with molecular weights greater than 200 kDa. Despite the apparent high aggregation in solution, the enzyme crystallizes from a 8%(v/v) polyethylene glycol (molecular weight 6000) solution in a form suitable for X-ray diffraction studies. The resulting single crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 79.76, b = 99.98, c = 150.2 A, alpha = beta = gamma = 90 degrees and Z = 4. Under X-ray irradiation generated with a rotating-anode generator, the crystals diffract to 2.8 A resolution and allow collection of a native 3 A resolution diffraction data set. PMID:10531491

  10. Crystallization of the C-terminal domain of the addiction antidote CcdA in complex with its toxin CcdB

    SciTech Connect

    Buts, Lieven; De Jonge, Natalie; Loris, Remy Wyns, Lode; Dao-Thi, Minh-Hoa

    2005-10-01

    The CcdA C-terminal domain was crystallized in complex with CcdB in two crystal forms that diffract to beyond 2.0 Å resolution. CcdA and CcdB are the antidote and toxin of the ccd addiction module of Escherichia coli plasmid F. The CcdA C-terminal domain (CcdA{sub C36}; 36 amino acids) was crystallized in complex with CcdB (dimer of 2 × 101 amino acids) in three different crystal forms, two of which diffract to high resolution. Form II belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 37.6, b = 60.5, c = 83.8 Å and diffracts to 1.8 Å resolution. Form III belongs to space group P2{sub 1}, with unit-cell parameters a = 41.0, b = 37.9, c = 69.6 Å, β = 96.9°, and diffracts to 1.9 Å resolution.

  11. Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated Cysteines

    PubMed Central

    Bischak, Connor G.; Longhi, Sonia; Snead, David M.; Costanzo, Stéphanie; Terrer, Elodie; Londergan, Casey H.

    2010-01-01

    Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) were cyanylated at cysteine and their infrared spectra in the C≡N stretching region were recorded both in the absence and in the presence of one of the physiological partners of NTAIL, namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within NTAIL, the C≡N stretching bands of the infrared probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and infrared spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level. PMID:20816082

  12. C-terminal propeptide is required for fibrillin-1 secretion and blocks premature assembly through linkage to domains cbEGF41-43.

    PubMed

    Jensen, Sacha A; Aspinall, Georgia; Handford, Penny A

    2014-07-15

    Fibrillin microfibrils are 10-12 nm diameter, extracellular matrix assemblies that provide dynamic tissues of metazoan species with many of their biomechanical properties as well as sequestering growth factors and cytokines. Assembly of fibrillin monomers into microfibrils is thought to occur at the cell surface, with initial steps including proprotein processing, multimerization driven by the C terminus, and the head-to-tail alignment of adjacent molecules. At present the mechanisms that regulate microfibril assembly are still to be elucidated. We have used structure-informed protein engineering to create a recombinant, GFP-tagged version of fibrillin-1 (GFP-Fbn) to study this process. Using HEK293T cells transiently transfected with GFP-Fbn constructs, we show that (i) the C-terminal propeptide is an essential requirement for the secretion of full-length fibrillin-1 from cells; (ii) failure to cleave off the C-terminal propeptide blocks the assembly of fibrillin-1 into microfibrils produced by dermal fibroblasts; and (iii) the requirement of the propeptide for secretion is linked to the presence of domains cbEGF41-43, because either deletion or exchange of domains in this region leads to cellular retention. Collectively, these data suggest a mechanism in which the propeptide blocks a key site at the C terminus to prevent premature microfibril assembly. PMID:24982166

  13. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    PubMed Central

    Lübker, Carolin; Dove, Stefan; Tang, Wei-Jen; Urbauer, Ramona J. Bieber; Moskovitz, Jackob; Urbauer, Jeffrey L.; Seifert, Roland

    2015-01-01

    Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils. PMID:26184312

  14. The C-terminal Kinase and ERK-binding Domains of Drosophila S6KII (RSK) Are Required for Phosphorylation of the Protein and Modulation of Circadian Behavior*

    PubMed Central

    Tangredi, Michelle M.; Ng, Fanny S.; Jackson, F. Rob

    2012-01-01

    A detailed structure/function analysis of Drosophila p90 ribosomal S6 kinase (S6KII) or its mammalian homolog RSK has not been performed in the context of neuronal plasticity or behavior. We previously reported that S6KII is required for normal circadian periodicity. Here we report a site-directed mutagenesis of S6KII and analysis of mutants, in vivo, that identifies functional domains and phosphorylation sites critical for the regulation of circadian period. We demonstrate, for the first time, a role for the S6KII C-terminal kinase that is independent of its known role in activation of the N-terminal kinase. Both S6KII C-terminal kinase activity and its ERK-binding domain are required for wild-type circadian period and normal phosphorylation status of the protein. In contrast, the N-terminal kinase of S6KII is dispensable for modulation of circadian period and normal phosphorylation of the protein. We also show that particular sites of S6KII phosphorylation, Ser-515 and Thr-732, are essential for normal circadian behavior. Surprisingly, the phosphorylation of S6KII residues, in vivo, does not follow a strict sequential pattern, as implied by certain cell-based studies of mammalian RSK protein. PMID:22447936

  15. NMR solution studies of hamster galectin-3 and electron microscopic visualization of surface-adsorbed complexes: evidence for interactions between the N- and C-terminal domains.

    PubMed

    Birdsall, B; Feeney, J; Burdett, I D; Bawumia, S; Barboni, E A; Hughes, R C

    2001-04-17

    Galectin-3, a beta-galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal domain that includes several repeats of a proline-tyrosine-glycine-rich motif. Earlier work based on a crystal structure of human galectin-3 CRD, and modeling and mutagenesis studies of the closely homologous hamster galectin-3, suggested that N-terminal tail residues immediately preceding the CRD might interfere with the canonical subunit interaction site of dimeric galectin-1 and -2, explaining the monomeric status of galectin-3 in solution. Here we describe high-resolution NMR studies of hamster galectin-3 (residues 1--245) and several of its fragments. The results indicate that the recombinant N-terminal fragment Delta 126--245 (residues 1--125) is an unfolded, extended structure. However, in the intact galectin-3 and fragment Delta 1--93 (residues 94--245), N-terminal domain residues lying between positions 94 and 113 have significantly reduced mobility values compared with those expected for bulk N-terminal tail residues, consistent with an interaction of this segment with the CRD domain. In contrast to the monomeric status of galectin-3 (and fragment Delta 1--93) in solution, electron microscopy of negatively stained and rotary shadowed samples of hamster galectin-3 as well as the CRD fragment Delta 1--103 (residues 104--245) show the presence of a significant proportion (up to 30%) of oligomers. Similar imaging of the N-terminal tail fragment Delta 126--245 reveals the presence of fibrils formed by intermolecular interactions between extended polypeptide subunits. Oligomerization of substratum-adsorbed galectin-3, through N- and C-terminal domain interactions, could be relevant to the positive cooperativity observed in binding of the lectin to immobilized multiglycosylated proteins such as laminin. PMID:11294654

  16. A conserved glutamate residue in the C-terminal deaminase domain of pentatricopeptide repeat proteins is required for RNA editing activity.

    PubMed

    Hayes, Michael L; Dang, Kim N; Diaz, Michael F; Mulligan, R Michael

    2015-04-17

    Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions. The glutamate residue of the HXE motif is catalytically required in the reaction catalyzed by cytidine deaminase. In this work, we examine the activity of the DYW deaminase domain through truncation or mutagenesis of the HXE motif. OTP84 is required for editing three chloroplast sites, and transgenes expressing OTP84 with C-terminal truncations were capable of editing only one of the three cognate sites at high efficiency. These results suggest that the deaminase domain of OTP84 is required for editing two of the sites, but another deaminase is able to supply the deamination activity for the third site. OTP84 and CREF7 transgenes were mutagenized to replace the glutamate residue of the HXE motif, and transgenic plants expressing OTP84-E824A and CREF7-E554A were unable to efficiently edit the cognate editing sites for these genes. In addition, plants expressing CREF7-E554A exhibited substantially reduced capacity to edit a non-cognate site, rpoA C200. These results indicate that the DYW deaminase domains of PPR proteins are involved in editing their cognate editing sites, and in some cases may participate in editing additional sites in the chloroplast. PMID:25739442

  17. The mode of action of centrin. Binding of Ca2+ and a peptide fragment of Kar1p to the C-terminal domain.

    PubMed

    Hu, Haitao; Sheehan, Jonathan H; Chazin, Walter J

    2004-12-01

    Centrin is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. It is found in microtubule-organizing centers of organisms ranging from algae and yeast to man. In vitro, the C-terminal domain of centrin binds to the yeast centrosomal protein Kar1p in a calcium-dependent manner, whereas the N-terminal domain does not show any appreciable affinity for Kar1p. To obtain deeper insights into the structural basis for centrin's function, we have characterized the affinities of the C-terminal domain of Chlamydomonas reinhardtii centrin for calcium and for a peptide fragment of Kar1p using CD, fluorescence, and NMR spectroscopy. Calcium binding site IV in C. reinhardtii centrin was found to bind Ca2+ approximately 100-fold more strongly than site III. In the absence of Ca2+, the protein occupies a mixture of closed conformations. Binding of a single ion in site IV is sufficient to radically alter the conformational equilibrium, promoting occupancy of an open conformation. However, an exchange between closed and open conformations remains even at saturating levels of Ca2+. The population of the open conformation is substantially stabilized by the presence of the target peptide Kar1p-(239-257) to a point where a single ion bound in site IV is sufficient to completely shift the conformational equilibrium to the open conformation. This is reflected in the enhancement of the Ca2+ affinity in this site by more than an order of magnitude. These data confirm the direct coupling of the Ca2+ binding-induced shift in the equilibrium between the closed and open conformations to the binding of the peptide. Combined with the common localization of the two proteins in the microtubule organizing center, our results suggest that centrin is constitutively bound to Kar1p through its C-terminal domain and that centrin's calcium sensor activities are mediated by the N-terminal domain. PMID:15452116

  18. Nucleocytoplasmic Shuttling of Valosin-Containing Protein (VCP/p97) Regulated by Its N domain and C-terminal Region

    PubMed Central

    Song, Changcheng; Wang, Qing; Song, Changzheng; Lockett, Stephen J.; Colburn, Nancy H.; Li, Chou-Chi H.; Wang, Ji Ming; Rogers, Thomas J.

    2014-01-01

    Valosin-containing protein (VCP or p97), a member of AAA family (ATPases associated with diverse cellular activities), plays a key role in many important cellular activities. A genetic deficiency of VCP can cause inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia (IBMPFD). Previous studies showed that the VCP N domain is essential for the regulation of nuclear entry of VCP. Here we report that IBMPFD mutations, which are mainly located in the N domain, suppress the nuclear entry of VCP. Moreover, the peptide sequence G780AGPSQ in the C-terminal region regulates the retention of VCP in the nucleus. A mutant lacking this sequence can increase the nuclear distribution of IBMPFD VCP, suggesting that this sequence is a potential molecular target for correcting the deficient nucleocytoplasmic shuttling of IBMPFD VCP proteins. PMID:25447673

  19. Crystallization and preliminary crystallographic analysis of the C-terminal domain of MamM, a magnetosome-associated protein from Magnetospirillum gryphiswaldense MSR-1

    PubMed Central

    Zeytuni, Natalie; Offer, Tal; Davidov, Geula; Zarivach, Raz

    2012-01-01

    MamM is a unique magnetosome-associated protein that shares substantial homology with cation diffusion facilitator (CDF) proteins, a group of heavy-metal-ion efflux transporters that participate in metal-ion homeostasis in all domains of life. Magnetotactic bacteria utilize CDF proteins in iron-oxide biomineralization and in magnetosome formation. Here, the crystallization and preliminary X-ray analysis of recombinant Magnetospirillum gryphiswaldense MamM is reported. The C-terminal domain of MamM was crystallized in the orthorhombic space group C2221, with unit-cell parameters a = 37.1, b = 94.0, c = 53.3 Å. X-ray diffraction data were collected to a resolution of 2.0 Å. PMID:22869124

  20. A systematic study of nuclear interactome of C-terminal domain small phosphatase-like 2 using inducible expression system and shotgun proteomics.

    PubMed

    Kang, NaNa; Koo, JaeHyung; Wang, Sen; Hur, Sun Jin; Bahk, Young Yil

    2016-06-01

    RNA polymerase II C-terminal domain phosphatases are newly emerging family of phosphatases that contain FCPH domain with Mg+2-binding DXDX(T/V) signature motif. Its subfamily includes small CTD phosphatases (SCPs). Recently, we identified several interacting partners of human SCP1 with appearance of dephosphorylation and O-GlcNAcylation. In this study, using an established cell line with inducible CTDSPL2 protein (a member of the new phosphatase family), proteomic screening was conducted to identify binding partners of CTDSPL2 in nuclear extract through immunoprecipitation of CTDSPL2 with its associated. This approach led to the identification of several interacting partners of CTDSPL2. This will provide a better understanding on CTDSPL2. [BMB Reports 2016; 49(6): 319-324]. PMID:26674342

  1. Full-length Gαq-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain

    SciTech Connect

    Lyon, Angeline M.; Dutta, Somnath; Boguth, Cassandra A.; Skiniotis, Georgios; Tesmer, John J.G.

    2014-08-21

    Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.

  2. The C-terminal region of the transcriptional regulator THAP11 forms a parallel coiled-coil domain involved in protein dimerization.

    PubMed

    Cukier, Cyprian D; Maveyraud, Laurent; Saurel, Olivier; Guillet, Valérie; Milon, Alain; Gervais, Virginie

    2016-06-01

    Thanatos associated protein 11 (THAP11) is a cell cycle and cell growth regulator differentially expressed in cancer cells. THAP11 belongs to a distinct family of transcription factors recognizing specific DNA sequences via an atypical zinc finger motif and regulating diverse cellular processes. Outside the extensively characterized DNA-binding domain, THAP proteins vary in size and predicted domains, for which structural data are still lacking. We report here the crystal structure of the C-terminal region of human THAP11 protein, providing the first 3D structure of a coiled-coil motif from a THAP family member. We further investigate the stability, dynamics and oligomeric properties of the determined structure combining molecular dynamics simulations and biophysical experiments. Our results show that the C-ter region of THAP11 forms a left-handed parallel homo-dimeric coiled-coil structure possessing several unusual features. PMID:26975212

  3. Full-length Gα(q)-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain.

    PubMed

    Lyon, Angeline M; Dutta, Somnath; Boguth, Cassandra A; Skiniotis, Georgios; Tesmer, John J G

    2013-03-01

    Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis. PMID:23377541

  4. The GSTM2 C-Terminal Domain Depresses Contractility and Ca2+ Transients in Neonatal Rat Ventricular Cardiomyocytes.

    PubMed

    Hewawasam, Ruwani P; Liu, Dan; Casarotto, Marco G; Board, Philip G; Dulhunty, Angela F

    2016-01-01

    The cardiac ryanodine receptor (RyR2) is an intracellular ion channel that regulates Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling in the heart. The glutathione transferases (GSTs) are a family of phase II detoxification enzymes with additional functions including the selective inhibition of RyR2, with therapeutic implications. The C-terminal half of GSTM2 (GSTM2C) is essential for RyR2 inhibition, and mutations F157A and Y160A within GSTM2C prevent the inhibitory action. Our objective in this investigation was to determine whether GSTM2C can enter cultured rat neonatal ventricular cardiomyocytes and influence contractility. We show that oregon green-tagged GSTM2C (at 1 μM) is internalized into the myocytes and it reduces spontaneous contraction frequency and myocyte shortening. Field stimulation of myocytes evoked contraction in the same percentage of myocytes treated either with media alone or media plus 15 μM GSTM2C. Myocyte shortening during contraction was significantly reduced by exposure to 15 μM GSTM2C, but not 5 and 10 μM GSTM2C and was unaffected by exposure to 15 μM of the mutants Y160A or F157A. The amplitude of the Ca2+ transient in the 15 μM GSTM2C - treated myocytes was significantly decreased, the rise time was significantly longer and the decay time was significantly shorter than in control myocytes. The Ca2+ transient was not altered by exposure to Y160A or F157A. The results are consistent with GSTM2C entering the myocytes and inhibiting RyR2, in a manner that indicates a possible therapeutic potential for treatment of arrhythmia in the neonatal heart. PMID:27612301

  5. Surface expression of GluR-D AMPA receptor is dependent on an interaction between its C-terminal domain and a 4.1 protein.

    PubMed

    Coleman, Sarah K; Cai, Chunlin; Mottershead, David G; Haapalahti, Jukka-Pekka; Keinänen, Kari

    2003-02-01

    Dynamic regulation of the number and activity of AMPA receptors is believed to underlie many forms of synaptic plasticity and is presumably mediated by specific protein-protein interactions involving the C-terminal domain of the receptor. Several proteins interacting with the C-terminal tails of the glutamate receptor (GluR)-A and GluR-B subunits have been identified and implicated in the regulation of endocytosis and exocytosis, clustering, and anchoring of AMPA receptors to the cytoskeleton. In contrast, little is known of the molecular interactions of the GluR-D subunit, or of the mechanisms regulating the traffic of GluR-D-containing AMPA receptors. We analyzed the subcellular localization of homomeric GluR-D receptors carrying C-terminal deletions in transfected human embryonic kidney (HEK) 293 cells and in primary neurons by immunofluorescence microscopy and ELISA. A minimal requirement for a 14-residue cytoplasmic segment for the surface expression of homomeric GluR-D receptors was identified. Previously, a similar region in the GluR-A subunit was implicated in an interaction with 4.1 family proteins. Coimmunoprecipitation demonstrated that GluR-D associated with 4.1 protein(s) in both HEK293 cells and rat brain. Moreover, glutathione S-transferase pull-down experiments showed that the same 14-residue segment is critical for 4.1 binding to GluR-A and GluR-D. Point mutations within this segment dramatically decreased the surface expression of GluR-D in HEK293 cells, with a concomitant loss of the 4.1 interaction. Our findings demonstrate a novel molecular interaction for the GluR-D subunit and suggest that the association with the 4.1 family protein(s) plays an essential role in the transport to and stabilization of GluR-D-containing AMPA receptors at the cell surface. PMID:12574408

  6. Small C-terminal domain phosphatases dephosphorylate the regulatory linker regions of Smad2 and Smad3 to enhance transforming growth factor-beta signaling.

    PubMed

    Wrighton, Katharine H; Willis, Danielle; Long, Jianyin; Liu, Fang; Lin, Xia; Feng, Xin-Hua

    2006-12-15

    Transforming growth factor-beta (TGF-beta) controls a diverse set of cellular processes, and its canonical signaling is mediated via TGF-beta-induced phosphorylation of receptor-activated Smads (2 and 3) at the C-terminal SXS motif. We recently discovered that PPM1A can dephosphorylate Smad2/3 at the C-terminal SXS motif, implicating a critical role for phosphatases in regulating TGF-beta signaling. Smad2/3 activity is also regulated by phosphorylation in the linker region (and N terminus) by a variety of intracellular kinases, making it a critical platform for cross-talk between TGF-beta and other signaling pathways. Using a functional genomic approach, we identified the small C-terminal domain phosphatase 1 (SCP1) as a specific phosphatase for Smad2/3 dephosphorylation in the linker and N terminus. A catalytically inactive SCP1 mutant (dnSCP1) had no effect on Smad2/3 phosphorylation in vitro or in vivo. Of the other FCP/SCP family members SCP2 and SCP3, but not FCP1, could also dephosphorylate Smad2/3 in the linker/N terminus. Depletion of SCP1/2/3 enhanced Smad2/3 linker phosphorylation. SCP1 increased TGF-beta-induced transcriptional activity in agreement with the idea that phosphorylation in the Smad2/3 linker must be removed for a full transcriptional response. SCP1 overexpression also counteracts the inhibitory effect of epidermal growth factor on TGF-beta-induced p15 expression. Taken together, this work identifies the first example of a Smad2/3 linker phosphatase(s) and reveals an important new substrate for SCPs. PMID:17035229

  7. Identification of Residues in the C-terminal Domain of HIV-1 Integrase That Mediate Binding to the Transportin-SR2 Protein*

    PubMed Central

    De Houwer, Stephanie; Demeulemeester, Jonas; Thys, Wannes; Taltynov, Oliver; Zmajkovicova, Katarina; Christ, Frauke; Debyser, Zeger

    2012-01-01

    Transportin-SR2 (TRN-SR2 and TNPO3) is a cellular cofactor of HIV replication that has been implicated in the nuclear import of HIV. TRN-SR2 was originally identified in a yeast two-hybrid screen as an interaction partner of HIV integrase (IN) and in two independent siRNA screens as a cofactor of viral replication. We have now studied the interaction of TRN-SR2 and HIV IN in molecular detail and identified the TRN-SR2 interacting regions of IN. A weak interaction with the catalytic core domain (CCD) and a strong interaction with the C-terminal domain (CTD) of IN were detected. By dissecting the catalytic core domain (CCD) of IN into short structural fragments, we identified a peptide (INIP1, amino acids 170EHLKTAVQMAVFIHNFKRKGGI191) retaining the ability to interact with TRN-SR2. By dissecting the C-terminal domain (CTD) of IN, we could identify two interacting peptides (amino acids 214QKQITKIQNFRVYYR228 and 262RRKVKIIRDYGK273) that come together in the CTD tertiary structure to form an exposed antiparallel β-sheet. Through site-specific mutagenesis, we defined the following sets of amino acids in IN as important for the interaction with TRN-SR2: Phe-185/Lys-186/Arg-187/Lys-188 in the CCD and Arg-262/Arg-263/Lys-264 and Lys-266/Arg-269 in the CTD. An HIV-1 strain carrying K266A/R269A in IN was replication-defective due to a block in reverse transcription, confounding the study of nuclear import. Insight into the IN/TRN-SR2 interaction interface is necessary to guide drug discovery efforts targeting the nuclear entry step of replication. PMID:22872638

  8. Cataract-causing mutation S228P promotes βB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain.

    PubMed

    Qi, Liang-Bo; Hu, Li-Dan; Liu, Huihui; Li, Hai-Yun; Leng, Xiao-Yao; Yan, Yong-Bin

    2016-07-01

    β/γ-Crystallins are predominant structural proteins in the cytoplasm of lens fiber cells and share a similar fold composing of four Greek-key motifs divided into two domains. Numerous cataract-causing mutations have been identified in various β/γ-crystallins, but the mechanisms underlying cataract caused by most mutations remains uncharacterized. The S228P mutation in βB1-crystallin has been linked to autosomal dominant congenital nuclear cataract. Here we found that the S228P mutant was prone to aggregate and degrade in both of the human and E. coli cells. The intracellular S228P aggregates could be redissolved by lanosterol. The S228P mutation modified the refolding pathway of βB1-crystallin by affecting the formation of the dimeric intermediate but not the monomeric intermediate. Compared with native βB1-crystallin, the refolded S228P protein had less packed structures, unquenched Trp fluorophores and increased hydrophobic exposure. The refolded S228P protein was prone to aggregate at the physiological temperature and decreased the protective effect of βB1-crystallin on βA3-crystallin. Molecular dynamic simulation studies indicated that the mutation decreased the subunit binding energy and modified the distribution of surface electrostatic potentials. More importantly, the mutation separated two interacting loops in the C-terminal domain, which shielded the hydrophobic core from solvent in native βB1-crystallin. These two interacting loops are highly conserved in both of the N- and C-terminal domains of all β/γ-crystallins. We propose that these two interacting loops play an important role in the folding and structural stability of β/γ-crystallin domains by protecting the hydrophobic core from solvent access. PMID:27318838

  9. Interaction between Prion Protein's Copper-Bound Octarepeat Domain and a Charged C-Terminal Pocket Suggests a Mechanism for N-Terminal Regulation.

    PubMed

    Evans, Eric G B; Pushie, M Jake; Markham, Kate A; Lee, Hsiau-Wei; Millhauser, Glenn L

    2016-07-01

    Copper plays a critical role in prion protein (PrP) physiology. Cu(2+) binds with high affinity to the PrP N-terminal octarepeat (OR) domain, and intracellular copper promotes PrP expression. The molecular details of copper coordination within the OR are now well characterized. Here we examine how Cu(2+) influences the interaction between the PrP N-terminal domain and the C-terminal globular domain. Using nuclear magnetic resonance and copper-nitroxide pulsed double electron-electron resonance, with molecular dynamics refinement, we localize the position of Cu(2+) in its high-affinity OR-bound state. Our results reveal an interdomain cis interaction that is stabilized by a conserved, negatively charged pocket of the globular domain. Interestingly, this interaction surface overlaps an epitope recognized by the POM1 antibody, the binding of which drives rapid cerebellar degeneration mediated by the PrP N terminus. The resulting structure suggests that the globular domain regulates the N-terminal domain by binding the Cu(2+)-occupied OR within a complementary pocket. PMID:27265848

  10. Deducing the functional characteristics of the human selenoprotein SELK from the structural properties of its intrinsically disordered C-terminal domain.

    PubMed

    Polo, Andrea; Colonna, Giovanni; Guariniello, Stefano; Ciliberto, Gennaro; Costantini, Susan

    2016-03-01

    The intrinsically disordered proteins (IDPs) cannot be described by a single structural representation but, due to their high structural fluctuation, through conformational ensembles. Certainly, molecular dynamics (MD) simulations represent a useful tool to study their different conformations capturing the conformational distribution. Our group is focusing on the structural characterization of proteins belonging to the seleno-proteome due to their involvement in cancer. They present disordered domains central for their biological function, and, in particular, SELK is a single-pass transmembrane protein that resides in the endoplasmic reticulum membrane (ER) with a C-terminal domain exposed to the cytoplasm that is known to interact with different components of the endoplasmic reticulum associated to the protein degradation (ERAD) pathway. This protein is found to be up-expressed in hepatocellular carcinoma and in other cancers. In this work we performed a detailed analysis of the C-terminal domain sequence of SELK and discovered that it is characterized by many prolines, and four negatively and eleven positively charged residues, which are crucial for its biological activity. This region can be considered as a weak polyelectrolyte and, specifically, a polycation, with high disordered propensity and different phosphorylation sites dislocated along the sequence. Then, we modeled its three-dimensional structure by performing MD simulations in water at neutral pH to analyze the structural stability as well as to identify the presence of HUB residues that play a key structural role as evidenced by the residue-residue interaction network analysis. Through this approach, we demonstrate that the C-terminal domain of SELK (i) presents a poor content of regular secondary structure elements, (ii) is dynamically stabilized by a network of intra-molecular H-bonds and H-bonds with water molecules, (iii) is highly fluctuating and, therefore, can be described only through a

  11. Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry.

    PubMed

    Ahrends, Robert; Kosinski, Jan; Kirsch, Dieter; Manelyte, Laura; Giron-Monzon, Luis; Hummerich, Lars; Schulz, Oliver; Spengler, Bernhard; Friedhoff, Peter

    2006-01-01

    To investigate protein-protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH-MutL-DNA complex. PMID:16772401

  12. A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation.

    PubMed

    Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie; Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique; Revers, Frédéric; Ziegler-Graff, Véronique; Brault, Véronique

    2015-12-01

    Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RTCter) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RTCter. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. PMID:26402374

  13. Recruitment of A20 by the C-terminal domain of NEMO suppresses NF-κB activation and autoinflammatory disease.

    PubMed

    Zilberman-Rudenko, Jevgenia; Shawver, Linda Monaco; Wessel, Alex W; Luo, Yongquan; Pelletier, Martin; Tsai, Wanxia Li; Lee, Younglang; Vonortas, Spiridon; Cheng, Laurence; Ashwell, Jonathan D; Orange, Jordan S; Siegel, Richard M; Hanson, Eric P

    2016-02-01

    Receptor-induced NF-κB activation is controlled by NEMO, the NF-κB essential modulator. Hypomorphic NEMO mutations result in X-linked ectodermal dysplasia with anhidrosis and immunodeficiency, also referred to as NEMO syndrome. Here we describe a distinct group of patients with NEMO C-terminal deletion (ΔCT-NEMO) mutations. Individuals harboring these mutations develop inflammatory skin and intestinal disease in addition to ectodermal dysplasia with anhidrosis and immunodeficiency. Both primary cells from these patients, as well as reconstituted cell lines with this deletion, exhibited increased IκB kinase (IKK) activity and production of proinflammatory cytokines. Unlike previously described loss-of-function mutations, ΔCT-NEMO mutants promoted increased NF-κB activation in response to TNF and Toll-like receptor stimulation. Investigation of the underlying mechanisms revealed impaired interactions with A20, a negative regulator of NF-κB activation, leading to prolonged accumulation of K63-ubiquitinated RIP within the TNFR1 signaling complex. Recruitment of A20 to the C-terminal domain of NEMO represents a novel mechanism limiting NF-κB activation by NEMO, and its absence results in autoinflammatory disease. PMID:26802121

  14. Urea Unfolding Study of E. coli Alanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

    PubMed Central

    Banerjee, Baisakhi; Banerjee, Rajat

    2015-01-01

    E. coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in the dimerization process. The truncated N-terminal containing the first 700 amino acids (1–700) forms a monomeric variant possessing similar aminoacylation activity like wild type. A point mutation in the C-terminal domain (G674D) also produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild type enzyme. Urea induced denaturation of these monomeric mutants along with another alaRS variant (N461 alaRS) was studied together with the full-length enzyme using various spectroscopic techniques such as intrinsic tryptophan fluorescence, 1-anilino-8-naphthalene-sulfonic acid binding, near- and far-UV circular dichroism, and analytical ultracentrifugation. Aminoacylation activity assay after refolding from denatured state revealed that the monomeric mutants studied here were unable to regain their activity, whereas the dimeric full-length alaRS gets back similar activity as the native enzyme. This study indicates that dimerization is one of the key regulatory factors that is important in the proper folding and stability of E. coli alaRS. PMID:26617997

  15. Assessing induced folding within the intrinsically disordered C-terminal domain of the Henipavirus nucleoproteins by site-directed spin labeling EPR spectroscopy.

    PubMed

    Martinho, Marlène; Habchi, Johnny; El Habre, Zeina; Nesme, Léo; Guigliarelli, Bruno; Belle, Valérie; Longhi, Sonia

    2013-01-01

    This work aims at characterizing structural transitions within the intrinsically disordered C-terminal domain of the nucleoprotein (NTAIL) from the Nipah and Hendra viruses, two recently emerged pathogens gathered within the Henipavirus genus. To this end, we used site-directed spin labeling combined with electron paramagnetic resonance spectroscopy to investigate the α-helical-induced folding that Henipavirus NTAIL domains undergo in the presence of the C-terminal X domain of the phosphoprotein (PXD). For each NTAIL protein, six positions located within four previously proposed molecular recognition elements (MoREs) were targeted for spin labeling, with three of these positions (475, 481, and 487) falling within the MoRE responsible for binding to PXD (Box3). A detailed analysis of the impact of the partner protein on the labeled NTAIL variants revealed a dramatic modification in the environment of the spin labels grafted within Box3, with the observed modifications supporting the formation of an induced α-helix within this region. In the free state, the slightly lower mobility of the spin labels grafted within Box3 as compared to the other positions suggests the existence of a transiently populated α-helix, as already reported for measles virus (MeV) NTAIL. Comparison with the well-characterized MeV NTAIL-PXD system, allowed us to validate the structural models of Henipavirus NTAIL-PXD complexes that we previously proposed. In addition, this study highlighted a few notable differences between the Nipah and Hendra viruses. In particular, the observation of composite spectra for the free form of the Nipah virus NTAIL variants spin labeled in Box3 supports conformational heterogeneity of this partly pre-configured α-helix, with the pre-existence of stable α-helical segments. Altogether these results provide insights into the molecular mechanisms of the Henipavirus NTAIL-PXD binding reaction. PMID:22881220

  16. The catalytic subunit of shiga-like toxin 1 interacts with ribosomal stalk proteins and is inhibited by their conserved C-terminal domain.

    PubMed

    McCluskey, Andrew J; Poon, Gregory M K; Bolewska-Pedyczak, Eleonora; Srikumar, Tharan; Jeram, Stanley M; Raught, Brian; Gariépy, Jean

    2008-04-25

    Shiga-like toxin 1 (SLT-1) is a type II ribosome-inactivating protein; its A(1) domain blocks protein synthesis in eukaryotic cells by catalyzing the depurination of a single adenine base in 28 S rRNA. The molecular mechanism leading to this site-specific depurination event is thought to involve interactions with eukaryotic ribosomal proteins. Here, we present evidence that the A(1) chain of SLT-1 binds to the ribosomal proteins P0, P1, and P2. These proteins were identified from a HeLa cell lysate by tandem mass spectrometry, and subsequently confirmed to bind to SLT-1 A(1) chain by yeast-two-hybrid and pull-down experiments using candidate full-length proteins. Moreover, the removal of the last 17 amino acids of either protein P1 or P2 abolishes the interaction with the A(1) chain, whereas P0, lacking this common C terminus, still binds to the A(1) domain. In vitro pull-down experiments using fusion protein-tagged C-terminal peptides corresponding to the common 7, 11, and 17 terminal residues of P1 and P2 confirmed that the A(1) chain of SLT-1 as well as the A chain of ricin bind to this shared C-terminal peptide motif. More importantly, a synthetic peptide corresponding to the 17 amino acid C terminus of P1 and P2 was shown to inhibit the ribosome-inactivating function of the A(1) chain of SLT-1 in an in vitro transcription and translation-coupled assay. These results suggest a role for the ribosomal stalk in aiding the A(1) chain of SLT-1 and other type II ribosome-inactivating proteins in localizing its catalytic domain near the site of depurination in the 28 S rRNA. PMID:18358491

  17. Crystal Structure of Mouse Elf3 C-terminal DNA-binding Domain in Complex with Type II TGF-[beta] Receptor Promoter DNA

    SciTech Connect

    Agarkar, Vinod B.; Babayeva, Nigar D.; Wilder, Phillip J.; Rizzino, Angie; Tahirov, Tahir H.

    2010-08-18

    The Ets family of transcription factors is composed of more than 30 members. One of its members, Elf3, is expressed in virtually all epithelial cells as well as in many tumors, including breast tumors. Several studies observed that the promoter of the type II TGF-{beta} receptor gene (T{beta}R-II) is strongly stimulated by Elf3 via two adjacent Elf3 binding sites, the A-site and the B-site. Here, we report the 2.2 {angstrom} resolution crystal structure of a mouse Elf3 C-terminal fragment, containing the DNA-binding Ets domain, in complex with the B-site of mouse type II TGF-{beta} receptor promoter DNA (mT{beta}R-II{sub DNA}). Elf3 contacts the core GGAA motif of the B-site from a major groove similar to that of known Ets proteins. However, unlike other Ets proteins, Elf3 also contacts sequences of the A-site from the minor groove of the DNA. DNA binding experiments and cell-based transcription studies indicate that minor groove interaction by Arg349 located in the Ets domain is important for Elf3 function. Equally interesting, previous studies have shown that the C-terminal region of Elf3, which flanks the Ets domain, is required for Elf3 binding to DNA. In this study, we determined that Elf3 amino acid residues within this flanking region, including Trp361, are important for the structural integrity of the protein as well as for the Efl3 DNA binding and transactivation activity.

  18. The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.

    PubMed

    Alderwick, Luke J; Lloyd, Georgina S; Ghadbane, Hemza; May, John W; Bhatt, Apoorva; Eggeling, Lothar; Fütterer, Klaus; Besra, Gurdyal S

    2011-02-01

    The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT)) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT), linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis. PMID:21383969

  19. Crystal structure of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-β receptor promoter DNA

    PubMed Central

    Agarkar, Vinod B.; Babayeva, Nigar D.; Wilder, Phillip J.; Rizzino, Angie; Tahirov, Tahir H.

    2010-01-01

    The Ets family of transcription factors is composed of more than 30 members. One of its members, Elf3, is expressed in virtually all epithelial cells as well as in many tumors, including breast tumors. Several studies observed that the promoter of the type II TGF-β receptor gene (TβR-II) is strongly stimulated by Elf3 via two adjacent Elf3 binding sites, A-site and B-site. Here we report the 2.2 Å resolution crystal structure of a mouse Elf3 C-terminal fragment, containing the DNA-binding Ets domain, in complex with the B-site of mouse type II TGF-β receptor promoter DNA (mTβR-IIDNA). Elf3 contacts the core GGAA motif of the B-site from major groove similar to that of known Ets proteins. However, unlike other Ets proteins, Elf3 also contacts sequences of the A-site from the minor groove of the DNA. DNA binding experiments and cell-based transcription studies indicate that minor groove interaction by Arg349 located in the Ets domain is important for Elf3 function. Equally interesting, previous studies have shown that the C-terminal region of Elf3, which flanks the Ets domain, is required for Elf3 binding to DNA. In this study, we determined that Elf3 amino acid residues within this flanking region, including Trp361, are important for the structural integrity of the protein as well as for the Efl3 DNA binding and transactivation activity. PMID:20079749

  20. The structure of the C-terminal domain of the pro-apoptotic protein Bak and its interaction with model membranes.

    PubMed Central

    Martínez-Senac, María del Mar; Corbalán-García, Senena; Gómez-Fernández, Juan C

    2002-01-01

    Bak is a pro-apoptotic protein widely distributed in different cell types that is associated with the mitochondrial outer membrane, apparently through a C-terminal hydrophobic domain. We used infrared spectroscopy to study the secondary structure of a synthetic peptide ((+)(3)HN-(188)ILNVLVVLGVVLLGQFVVRRFFKS(211)-COO(-)) with the same sequence as the C-terminal domain of Bak. The spectrum of this peptide in D(2)O buffer shows an amide I' band with a maximum at 1636 cm(-1), which clearly indicates the predominance of an extended beta-structure in aqueous solvent. However, the peptide incorporated in multilamellar dimyristoylphosphatidylcholine (DMPC) membranes shows a different amide I' band spectrum, with a maximum at 1658 cm(-1), indicating a predominantly alpha-helical structure induced by its interaction with the membrane. It was observed that through differential scanning calorimetry the transition of the phospholipid model membrane was broadened in the presence of the peptide. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) in fluid DMPC vesicles showed that increasing concentrations of the peptide produced increased polarization values, which is compatible with the peptide being inserted into the membrane. High concentrations of the peptide considerably broaden the phase transition of DMPC multilamellar vesicles, and DPH polarization increased, especially at temperatures above the T(c) transition temperature of the pure phospholipid. The addition of peptide destabilized unilamellar vesicles and released encapsulated carboxyfluorescein. These results indicate that this domain is able to insert itself into membranes, where it adopts an alpha-helical structure and considerably perturbs the physical properties of the membrane. PMID:11751312

  1. Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo.

    PubMed

    Lageix, Sebastien; Zhang, Jinwei; Rothenburg, Stefan; Hinnebusch, Alan G

    2015-02-01

    The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction. PMID:25695491

  2. Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

    PubMed Central

    Lageix, Sebastien; Zhang, Jinwei; Rothenburg, Stefan; Hinnebusch, Alan G.

    2015-01-01

    The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction. PMID:25695491

  3. Role of C-Terminal Domains in Surface Attachment of the Fructosyltransferase of Streptococcus salivarius ATCC 25975

    PubMed Central

    Rathsam, Catherine; Jacques, Nicholas A.

    1998-01-01

    The cell-associated β-d-fructosyltransferase of Streptococcus salivarius, which is devoid of the cell wall anchoring motif, LPXTG, is released on exposure to its substrate, sucrose. Deletions within the C terminus of the enzyme implicated both the hydrophobic and the proline-glycine-serine-threonine-rich wall-associated domain in stabilizing the enzyme on the cell surface. PMID:9829954

  4. Crystallization of the C-terminal domain of the addiction antidote CcdA in complex with its toxin CcdB.

    PubMed

    Buts, Lieven; De Jonge, Natalie; Loris, Remy; Wyns, Lode; Dao-Thi, Minh-Hoa

    2005-10-01

    CcdA and CcdB are the antidote and toxin of the ccd addiction module of Escherichia coli plasmid F. The CcdA C-terminal domain (CcdAC36; 36 amino acids) was crystallized in complex with CcdB (dimer of 2 x 101 amino acids) in three different crystal forms, two of which diffract to high resolution. Form II belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 37.6, b = 60.5, c = 83.8 A and diffracts to 1.8 A resolution. Form III belongs to space group P2(1), with unit-cell parameters a = 41.0, b = 37.9, c = 69.6 A, beta = 96.9 degrees, and diffracts to 1.9 A resolution. PMID:16511204

  5. Preliminary crystallographic analysis of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-[beta] receptor promoter DNA

    SciTech Connect

    Agarkar, Vinod B.; Babayeva, Nigar D.; Rizzino, Angie; Tahirov, Tahir H.

    2010-10-08

    Ets proteins are transcription factors that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. Like other Ets-family members, Elf3 functions as a sequence-specific DNA-binding transcriptional factor. A mouse Elf3 C-terminal fragment (amino-acid residues 269-371) containing the DNA-binding domain has been crystallized in complex with mouse type II TGF-{beta} receptor promoter (TR-II) DNA. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 42.66, b = 52, c = 99.78 {angstrom}, and diffracted to a resolution of 2.2 {angstrom}.

  6. SUMO modification of TBK1 at the adaptor-binding C-terminal coiled-coil domain contributes to its antiviral activity.

    PubMed

    Saul, Vera V; Niedenthal, Rainer; Pich, Andreas; Weber, Friedemann; Schmitz, M Lienhard

    2015-01-01

    The non-canonical IKK kinase TBK1 serves as an important signal transmitter of the antiviral interferon response, but is also involved in the regulation of further processes such as autophagy. The activity of TBK1 is regulated by posttranslational modifications comprising phosphorylation and ubiquitination. This study identifies SUMOylation as a novel posttranslational TBK1 modification. TBK1 kinase activity is required to allow the attachment of SUMO1 or SUMO2/3 proteins. Since TBK1 does not bind to the E2 enzyme Ubc9, this modification most likely proceeds via trans-SUMOylation. Mass spectrometry allowed identifying K694 as the SUMO acceptor site, a residue located in the C-terminal coiled-coil domain which is exclusively responsible for the association with the adaptor proteins NAP1, Sintbad and TANK. SUMO modification at K694 contributes to the antiviral function of TBK1 and accordingly the viral protein Gam1 antagonizes this posttranslational modification. PMID:25409927

  7. Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490963–1138 domain of TamB from Escherichia coli

    PubMed Central

    Josts, Inokentijs; Grinter, Rhys; Kelly, Sharon M.; Mosbahi, Khedidja; Roszak, Aleksander; Cogdell, Richard; Smith, Brian O.; Byron, Olwyn; Walker, Daniel

    2014-01-01

    TamB is a recently described inner membrane protein that, together with its partner protein TamA, is required for the efficient secretion of a subset of autotransporter proteins in Gram-negative bacteria. In this study, the C-terminal DUF490963–1138 domain of TamB was overexpressed in Escherichia coli K-12, purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the primitive trigonal space group P3121, with unit-cell parameters a = b = 57.34, c = 220.74 Å, and diffracted to 2.1 Å resolution. Preliminary secondary-structure and X-ray diffraction analyses are reported. Two molecules are predicted to be present in the asymmetric unit. Experimental phasing using selenomethionine-labelled protein will be undertaken in the future. PMID:25195908

  8. Complete integrin headpiece opening in eight steps

    PubMed Central

    Zhu, Jieqing; Zhu, Jianghai

    2013-01-01

    Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the αIIbβ3 integrin headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca2+, α1 helix, α1’ helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the βI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward αIIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening. PMID:23798730

  9. The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain.

    PubMed

    Cruz-Gallardo, Isabel; Aroca, Ángeles; Gunzburg, Menachem J; Sivakumaran, Andrew; Yoon, Je-Hyun; Angulo, Jesús; Persson, Cecilia; Gorospe, Myriam; Karlsson, B Göran; Wilce, Jacqueline A; Díaz-Moreno, Irene

    2014-01-01

    T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein-nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5' TOPs (5' terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations. PMID:24824036

  10. The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain

    PubMed Central

    Cruz-Gallardo, Isabel; Aroca, Ángeles; Gunzburg, Menachem J; Sivakumaran, Andrew; Yoon, Je-Hyun; Angulo, Jesús; Persson, Cecilia; Gorospe, Myriam; Karlsson, B Göran; Wilce, Jacqueline A; Díaz-Moreno, Irene

    2014-01-01

    T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein–nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5′ TOPs (5′ terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations. PMID:24824036