Cardiolipin synthesizing enzymes form a complex that interacts with cardiolipin-dependent membrane organizing proteins.

PubMed

Serricchio, Mauro; Vissa, Adriano; Kim, Peter K; Yip, Christopher M; McQuibban, G Angus

2018-04-01

The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology. Most notably, cardiolipin directly binds to mitochondrial proteins and helps assemble and stabilize mitochondrial multi-protein complexes. Despite their importance for mitochondrial health, how the proteins involved in cardiolipin biosynthesis are organized and embedded in mitochondrial membranes has not been investigated in detail. Here we show that human PGS1 and CLS1 are constituents of large protein complexes. We show that PGS1 forms oligomers and associates with CLS1 and PTPMT1. Using super-resolution microscopy, we observed well-organized nanoscale structures formed by PGS1. Together with the observation that cardiolipin and CLS1 are not required for PGS1 to assemble in the complex we predict the presence of a PGS1-centered cardiolipin-synthesizing scaffold within the mitochondrial inner membrane. Using an unbiased proteomic approach we found that PGS1 and CLS1 interact with multiple cardiolipin-binding mitochondrial membrane proteins, including prohibitins, stomatin-like protein 2 and the MICOS components MIC60 and MIC19. We further mapped the protein-protein interaction sites between PGS1 and itself, CLS1, MIC60 and PHB. Overall, this study provides evidence for the presence of a cardiolipin synthesis structure that transiently interacts with cardiolipin-dependent protein complexes. Copyright © 2018 Elsevier B.V. All rights reserved.

Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography.

PubMed

Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S; Kent, Stephen B H

2012-09-11

Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

Structure of homeodomain-leucine zipper/DNA complexes studied using hydroxyl radical cleavage of DNA and methylation interference.

PubMed

Tron, Adriana E; Comelli, Raúl N; Gonzalez, Daniel H

2005-12-27

Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3' end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence.

Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

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

Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in

2015-07-28

Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging themore » ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.« less

Structure of a group II intron in complex with its reverse transcriptase.

PubMed

Qu, Guosheng; Kaushal, Prem Singh; Wang, Jia; Shigematsu, Hideki; Piazza, Carol Lyn; Agrawal, Rajendra Kumar; Belfort, Marlene; Wang, Hong-Wei

2016-06-01

Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice, yielding mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein that performs multiple activities including reverse transcription, in which intron RNA is copied into the DNA target. Here we report cryo-EM structures of an endogenously spliced Lactococcus lactis group IIA intron in its ribonucleoprotein complex form at 3.8-Å resolution and in its protein-depleted form at 4.5-Å resolution, revealing functional coordination of the intron RNA with the protein. Remarkably, the protein structure reveals a close relationship between the reverse transcriptase catalytic domain and telomerase, whereas the active splicing center resembles the spliceosomal Prp8 protein. These extraordinary similarities hint at intricate ancestral relationships and provide new insights into splicing and retromobility.

Stabilization of model beverage cloud emulsions using protein-polysaccharide electrostatic complexes formed at the oil-water interface.

PubMed

Harnsilawat, Thepkunya; Pongsawatmanit, Rungnaphar; McClements, David J

2006-07-26

The potential of utilizing interfacial complexes, formed through the electrostatic interactions of proteins and polysaccharides at oil-water interfaces, to stabilize model beverage cloud emulsions has been examined. These interfacial complexes were formed by mixing charged polysaccharides with oil-in-water emulsions containing oppositely charged protein-coated oil droplets. Model beverage emulsions were prepared that consisted of 0.1 wt % corn oil droplets coated by beta-lactoglobulin (beta-Lg), beta-Lg/alginate, beta-Lg/iota-carrageenan, or beta-Lg/gum arabic interfacial layers (pH 3 or 4). Stable emulsions were formed when the polysaccharide concentration was sufficient to saturate the protein-coated droplets. The emulsions were subjected to variations in pH (from 3 to 7), ionic strength (from 0 to 250 mM NaCl), and thermal processing (from 30 or 90 degrees C), and the influence on their stability was determined. The emulsions containing alginate and carrageenan had the best stability to ionic strength and thermal processing. This study shows that the controlled formation of protein-polysaccharide complexes at droplet surfaces may be used to produce stable beverage emulsions, which may have important implications for industrial applications.

Nanoparticle-protein complexes mimicking corona formation in ocular environment.

PubMed

Jo, Dong Hyun; Kim, Jin Hyoung; Son, Jin Gyeong; Dan, Ki Soon; Song, Sang Hoon; Lee, Tae Geol; Kim, Jeong Hun

2016-12-01

Nanoparticles adsorb biomolecules to form corona upon entering the biological environment. In this study, tissue-specific corona formation is provided as a way of controlling protein interaction with nanoparticles in vivo. In the vitreous, the composition of the corona was determined by the electrostatic and hydrophobic properties of the associated proteins, regardless of the material (gold and silica) or size (20- and 100-nm diameter) of the nanoparticles. To control protein adsorption, we pre-incubate 20-nm gold nanoparticles with 5 selectively enriched proteins from the corona, formed in the vitreous, to produce nanoparticle-protein complexes. Compared to bare nanoparticles, nanoparticle-protein complexes demonstrate improved binding to vascular endothelial growth factor (VEGF) in the vitreous. Furthermore, nanoparticle-protein complexes retain in vitro anti-angiogenic properties of bare nanoparticles. In particular, priming the nanoparticles (gold and silica) with tissue-specific corona proteins allows nanoparticle-protein complexes to exert better in vivo therapeutic effects by higher binding to VEGF than bare nanoparticles. These results suggest that controlled corona formation that mimics in vivo processes may be useful in the therapeutic use of nanomaterials in local environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography

PubMed Central

Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S.; Kent, Stephen B.H.

2012-01-01

Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF165 to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form ofVEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å2 in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2. PMID:22927390

Whirlin and PDZ domain-containing 7 (PDZD7) proteins are both required to form the quaternary protein complex associated with Usher syndrome type 2.

PubMed

Chen, Qian; Zou, Junhuang; Shen, Zuolian; Zhang, Weiping; Yang, Jun

2014-12-26

Usher syndrome (USH) is the leading genetic cause of combined hearing and vision loss. Among the three USH clinical types, type 2 (USH2) occurs most commonly. USH2A, GPR98, and WHRN are three known causative genes of USH2, whereas PDZD7 is a modifier gene found in USH2 patients. The proteins encoded by these four USH genes have been proposed to form a multiprotein complex, the USH2 complex, due to interactions found among some of these proteins in vitro, their colocalization in vivo, and mutual dependence of some of these proteins for their normal in vivo localizations. However, evidence showing the formation of the USH2 complex is missing, and details on how this complex is formed remain elusive. Here, we systematically investigated interactions among the intracellular regions of the four USH proteins using colocalization, yeast two-hybrid, and pull-down assays. We show that multiple domains of the four USH proteins interact among one another. Importantly, both WHRN and PDZD7 are required for the complex formation with USH2A and GPR98. In this USH2 quaternary complex, WHRN prefers to bind to USH2A, whereas PDZD7 prefers to bind to GPR98. Interaction between WHRN and PDZD7 is the bridge between USH2A and GPR98. Additionally, the USH2 quaternary complex has a variable stoichiometry. These findings suggest that a non-obligate, short term, and dynamic USH2 quaternary protein complex may exist in vivo. Our work provides valuable insight into the physiological role of the USH2 complex in vivo and informs possible reconstruction of the USH2 complex for future therapy. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Metazoans evolved by taking domains from soluble proteins to expand intercellular communication network

PubMed Central

Nam, Hyun-Jun; Kim, Inhae; Bowie, James U.; Kim, Sanguk

2015-01-01

A central question in animal evolution is how multicellular animals evolved from unicellular ancestors. We hypothesize that membrane proteins must be key players in the development of multicellularity because they are well positioned to form the cell-cell contacts and to provide the intercellular communication required for the creation of complex organisms. Here we find that a major mechanism for the necessary increase in membrane protein complexity in the transition from non-metazoan to metazoan life was the new incorporation of domains from soluble proteins. The membrane proteins that have incorporated soluble domains in metazoans are enriched in many of the functions unique to multicellular organisms such as cell-cell adhesion, signaling, immune defense and developmental processes. They also show enhanced protein-protein interaction (PPI) network complexity and centrality, suggesting an important role in the cellular diversification found in complex organisms. Our results expose an evolutionary mechanism that contributed to the development of higher life forms. PMID:25923201

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

NASA Astrophysics Data System (ADS)

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

2018-03-01

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

Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex.

PubMed

Sorusch, Nasrin; Bauß, Katharina; Plutniok, Janet; Samanta, Ananya; Knapp, Barbara; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe

2017-03-15

The Usher syndrome (USH) is the most common form of inherited deaf-blindness, accompanied by vestibular dysfunction. Due to the heterogeneous manifestation of the clinical symptoms, three USH types (USH1-3) and additional atypical forms are distinguished. USH1 and USH2 proteins have been shown to function together in multiprotein networks in photoreceptor cells and hair cells. Mutations in USH proteins are considered to disrupt distinct USH protein networks and finally lead to the development of USH.To get novel insights into the molecular pathomechanisms underlying USH, we further characterize the periciliary USH protein network in photoreceptor cells. We show the direct interaction between the scaffold protein SANS (USH1G) and the transmembrane adhesion protein ush2a and that both assemble into a ternary USH1/USH2 complex together with the PDZ-domain protein whirlin (USH2D) via mutual interactions. Immunohistochemistry and proximity ligation assays demonstrate co-localization of complex partners and complex formation, respectively, in the periciliary region, the inner segment and at the synapses of rodent and human photoreceptor cells. Protein-protein interaction assays and co-expression of complex partners reveal that pathogenic mutations in USH1G severely affect formation of the SANS/ush2a/whirlin complex. Translational read-through drug treatment, targeting the c.728C > A (p.S243X) nonsense mutation, restored SANS scaffold function. We conclude that USH1 and USH2 proteins function together in higher order protein complexes. The maintenance of USH1/USH2 protein complexes depends on multiple USH1/USH2 protein interactions, which are disrupted by pathogenic mutations in USH1G protein SANS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins

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

Sosa, Brian A.; Rothballer, Andrea; Kutay, Ulrike

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansivemore » grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.« less

An asymmetric structure of the Bacillus subtilis replication terminator protein in complex with DNA.

PubMed

Vivian, J P; Porter, C J; Wilce, J A; Wilce, M C J

2007-07-13

In Bacillus subtilis, the termination of DNA replication via polar fork arrest is effected by a specific protein:DNA complex formed between the replication terminator protein (RTP) and DNA terminator sites. We report the crystal structure of a replication terminator protein homologue (RTP.C110S) of B. subtilis in complex with the high affinity component of one of its cognate DNA termination sites, known as the TerI B-site, refined at 2.5 A resolution. The 21 bp RTP:DNA complex displays marked structural asymmetry in both the homodimeric protein and the DNA. This is in contrast to the previously reported complex formed with a symmetrical TerI B-site homologue. The induced asymmetry is consistent with the complex's solution properties as determined using NMR spectroscopy. Concomitant with this asymmetry is variation in the protein:DNA binding pattern for each of the subunits of the RTP homodimer. It is proposed that the asymmetric "wing" positions, as well as other asymmetrical features of the RTP:DNA complex, are critical for the cooperative binding that underlies the mechanism of polar fork arrest at the complete terminator site.

Tic20 forms a channel independent of Tic110 in chloroplasts

PubMed Central

2011-01-01

Background The Tic complex (Translocon at the inner envelope membrane of chloroplasts) mediates the translocation of nuclear encoded chloroplast proteins across the inner envelope membrane. Tic110 forms one prominent protein translocation channel. Additionally, Tic20, another subunit of the complex, was proposed to form a protein import channel - either together with or independent of Tic110. However, no experimental evidence for Tic20 channel activity has been provided so far. Results We performed a comprehensive biochemical and electrophysiological study to characterize Tic20 in more detail and to gain a deeper insight into its potential role in protein import into chloroplasts. Firstly, we compared transcript and protein levels of Tic20 and Tic110 in both Pisum sativum and Arabidopsis thaliana. We found the Tic20 protein to be generally less abundant, which was particularly pronounced in Arabidopsis. Secondly, we demonstrated that Tic20 forms a complex larger than 700 kilodalton in the inner envelope membrane, which is clearly separate from Tic110, migrating as a dimer at about 250 kilodalton. Thirdly, we defined the topology of Tic20 in the inner envelope, and found its N- and C-termini to be oriented towards the stromal side. Finally, we successfully reconstituted overexpressed and purified full-length Tic20 into liposomes. Using these Tic20-proteoliposomes, we could demonstrate for the first time that Tic20 can independently form a cation selective channel in vitro. Conclusions The presented data provide first biochemical evidence to the notion that Tic20 can act as a channel protein within the chloroplast import translocon complex. However, the very low abundance of Tic20 in the inner envelope membranes indicates that it cannot form a major protein translocation channel. Furthermore, the independent complex formation of Tic20 and Tic110 argues against a joint channel formation. Thus, based on the observed channel activity of Tic20 in proteoliposomes, we speculate that the chloroplast inner envelope contains multiple (at least two) translocation channels: Tic110 as the general translocation pore, whereas Tic20 could be responsible for translocation of a special subset of proteins. PMID:21961525

Proteins required for lipopolysaccharide assembly in Escherichia coli form a transenvelope complex.

PubMed

Chng, Shu-Sin; Gronenberg, Luisa S; Kahne, Daniel

2010-06-08

The viability of Gram-negative organisms is dependent on the proper placement of lipopolysaccharide (LPS) in the outer leaflet of its outer membrane. LPS is synthesized inside the cell and transported to the surface by seven essential lipopolysaccharide transport (Lpt) proteins. How these proteins cooperate to transport LPS is unknown. We show that these Lpt proteins can be found in a membrane fraction that contains inner and outer membranes and that they copurify. This constitutes the first evidence that the Lpt proteins form a transenvelope complex. We suggest that this protein bridge provides a route for LPS transport across the cell envelope.

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

PubMed

Drissi, Romain; Dubois, Marie-Line; Douziech, Mélanie; Boisvert, François-Michel

2015-07-01

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

TelAP1 links telomere complexes with developmental expression site silencing in African trypanosomes

PubMed Central

Reis, Helena; Schwebs, Marie; Dietz, Sabrina; Janzen, Christian J; Butter, Falk

2018-01-01

Abstract During its life cycle, Trypanosoma brucei shuttles between a mammalian host and the tsetse fly vector. In the mammalian host, immune evasion of T. brucei bloodstream form (BSF) cells relies on antigenic variation, which includes monoallelic expression and periodic switching of variant surface glycoprotein (VSG) genes. The active VSG is transcribed from only 1 of the 15 subtelomeric expression sites (ESs). During differentiation from BSF to the insect-resident procyclic form (PCF), the active ES is transcriptionally silenced. We used mass spectrometry-based interactomics to determine the composition of telomere protein complexes in T. brucei BSF and PCF stages to learn more about the structure and functions of telomeres in trypanosomes. Our data suggest a different telomere complex composition in the two forms of the parasite. One of the novel telomere-associated proteins, TelAP1, forms a complex with telomeric proteins TbTRF, TbRAP1 and TbTIF2 and influences ES silencing kinetics during developmental differentiation. PMID:29385523

Oligomeric protein complexes of apolipoproteins stabilize the internal fluid environment of organism in redfins of the Tribolodon genus [Pisces; Cypriniformes, Cyprinidae].

PubMed

Andreeva, Alla M; Serebryakova, Marina V; Lamash, Nina E

2017-06-01

One of the most important functions of plasma proteins in vertebrates is their participation in osmotic homeostasis in the organism. Modern concepts about plasma proteins and their capillary filtration are based on a model of large monomeric proteins that are able to penetrate the interstitial space. At the same time, it was revealed that a considerable amount of oligomeric complexes are present in the low-molecular-weight (LM) protein fraction in the extracellular fluids of fishes. The functions of these complexes are unknown. In the present study, we investigated the LM-fraction proteins in the plasma and interstitial fluid (IF) of redfins of the genus Tribolodon. This fish alternatively spends parts of its life cycle in saline and fresh waters. We identified the protein Wap65, serpins and apolipoproteins in this fraction. By combining the methods of 2D-E under native and denaturing conditions with MALDI, we demonstrated that only apolipoproteins formed complexes. We showed that serum apolipoproteins (АроА-I, Аро-14) were present in the form of homooligomeric complexes that were dissociated with the release of monomeric forms of proteins in the course of capillary filtration to IF. Dissociation of homooligomers is not directly correlated with the change in salinity but is correlated with seasonal dynamics. We found that there was a significant decrease in the total protein concentration in IF relative to plasma. Therefore, we suggested that dissociation of homooligomeric complexes from various apolipoproteins supports the isoosmoticity of extracellular fluids relative to capillary wall stabilization through a fluid medium in fish. Copyright © 2017 Elsevier Inc. All rights reserved.

Immune complexes in chronic Chagas disease patients are formed by exovesicles from Trypanosoma cruzi carrying the conserved MASP N-terminal region

PubMed Central

Díaz Lozano, Isabel María; De Pablos, Luis Miguel; Longhi, Silvia Andrea; Zago, María Paola; Schijman, Alejandro Gabriel; Osuna, Antonio

2017-01-01

The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease. PMID:28294160

Immune complexes in chronic Chagas disease patients are formed by exovesicles from Trypanosoma cruzi carrying the conserved MASP N-terminal region

NASA Astrophysics Data System (ADS)

Díaz Lozano, Isabel María; de Pablos, Luis Miguel; Longhi, Silvia Andrea; Zago, María Paola; Schijman, Alejandro Gabriel; Osuna, Antonio

2017-03-01

The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease.

Oxidative stress/damage induces multimerization and interaction of Fanconi anemia proteins.

PubMed

Park, Su-Jung; Ciccone, Samantha L M; Beck, Brian D; Hwang, Byounghoon; Freie, Brian; Clapp, D Wade; Lee, Suk-Hee

2004-07-16

Fanconi anemia (FANC) is a heterogeneous genetic disorder characterized by a hypersensitivity to DNA-damaging agents, chromosomal instability, and defective DNA repair. Eight FANC genes have been identified so far, and five of them (FANCA, -C, -E, -F, and -G) assemble in a multinuclear complex and function at least in part in a complex to activate FANCD2 by monoubiquitination. Here we show that FANCA and FANCG are redox-sensitive proteins that are multimerized and/or form a nuclear complex in response to oxidative stress/damage. Both FANCA and FANCG proteins exist as monomers under non-oxidizing conditions, whereas they become multimers following H2O2 treatment. Treatment of cells with oxidizing agent not only triggers the multimeric complex of FANCA and FANCG in vivo but also induces the interaction between FANCA and FANCG. N-Ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro. Taken together, our results lead us to conclude that FANCA and FANCG uniquely respond to oxidative damage by forming complex(es) via intermolecular disulfide linkage(s), which may be crucial in forming such complexes and in determining their function.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

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

Klopffleisch, Karsten; Phan, Nguyen; Chen, Jay

2011-01-01

The heterotrimeric G-protein complex is minimally composed of G{alpha}, G{beta}, and G{gamma} subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, wemore » detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.« less

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

PubMed

Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-09-27

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

PubMed Central

Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-01-01

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification. PMID:21952135

Structural symmetry and protein function.

PubMed

Goodsell, D S; Olson, A J

2000-01-01

The majority of soluble and membrane-bound proteins in modern cells are symmetrical oligomeric complexes with two or more subunits. The evolutionary selection of symmetrical oligomeric complexes is driven by functional, genetic, and physicochemical needs. Large proteins are selected for specific morphological functions, such as formation of rings, containers, and filaments, and for cooperative functions, such as allosteric regulation and multivalent binding. Large proteins are also more stable against denaturation and have a reduced surface area exposed to solvent when compared with many individual, smaller proteins. Large proteins are constructed as oligomers for reasons of error control in synthesis, coding efficiency, and regulation of assembly. Symmetrical oligomers are favored because of stability and finite control of assembly. Several functions limit symmetry, such as interaction with DNA or membranes, and directional motion. Symmetry is broken or modified in many forms: quasisymmetry, in which identical subunits adopt similar but different conformations; pleomorphism, in which identical subunits form different complexes; pseudosymmetry, in which different molecules form approximately symmetrical complexes; and symmetry mismatch, in which oligomers of different symmetries interact along their respective symmetry axes. Asymmetry is also observed at several levels. Nearly all complexes show local asymmetry at the level of side chain conformation. Several complexes have reciprocating mechanisms in which the complex is asymmetric, but, over time, all subunits cycle through the same set of conformations. Global asymmetry is only rarely observed. Evolution of oligomeric complexes may favor the formation of dimers over complexes with higher cyclic symmetry, through a mechanism of prepositioned pairs of interacting residues. However, examples have been found for all of the crystallographic point groups, demonstrating that functional need can drive the evolution of any symmetry.

Binding regularities in complexes of transcription factors with operator DNA: homeodomain family.

PubMed

Chirgadze, Yu N; Zheltukhin, E I; Polozov, R V; Sivozhelezov, V S; Ivanov, V V

2009-06-01

In order to disclose general regularities of binding in homeodomain-DNA complexes we considered five of them and extended the observed regularities over the entire homeodomain family. The five complexes have been selected by similarity of protein structures and patterns of contacting residues. Their long range interactions and interfaces were compared. The long-range stage of the recognition process was characterized by electrostatic potentials about 5 Angstrom away from molecular surfaces of protein or DNA. For proteins, clear positive potential is displayed only at the side contacting the DNA. The double-chained DNA molecule displays a rather strong negative potential, especially in their grooves. Thus, a functional role of electrostatics is a guiding of the protein into the DNA major groove, so the protein and DNA could form a loose non-specific complex. At the close-range stage, neutralization of the phosphate charges by positively charged residues is necessary for decreasing the strong electrostatic potential of DNA, allowing nucleotide bases to participate in the formation of protein-DNA atomic contacts in the interface. The recognizing alpha-helix of protein was shown to form both invariant and variable groups of contacts with DNA by means of certain specific side groups. The invariant contacts included highly specific protein-DNA hydrogen bonds between asparagine and adenine, nonpolar contacts of hydrophobic amino acids serving as a stereochemical barrier for fixing the protein factor on DNA, and an interface cluster of water molecules providing local conformational mobility necessary for the dissociation process. There is a unique water molecule within the interface that is conservative and located at the interface center. Invariant contacts of the proteins are mostly formed with the TAAT motif of the promoter DNA forward strand. While the invariant contacts specify the family of homeodomains, the variable contacts that are formed with the reverse strand of DNA provide specificity of individual complexes within the homeodomain family.

Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1.

PubMed

Lalev, A I; Abeyrathne, P D; Nazar, R N

2000-09-08

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed. Copyright 2000 Academic Press.

Protein-Protein Interactions of Azurin Complex by Coarse-Grained Simulations with a Gō-Like Model

NASA Astrophysics Data System (ADS)

Rusmerryani, Micke; Takasu, Masako; Kawaguchi, Kazutomo; Saito, Hiroaki; Nagao, Hidemi

Proteins usually perform their biological functions by forming a complex with other proteins. It is very important to study the protein-protein interactions since these interactions are crucial in many processes of a living organism. In this study, we develop a coarse grained model to simulate protein complex in liquid system. We carry out molecular dynamics simulations with topology-based potential interactions to simulate dynamical properties of Pseudomonas Aeruginosa azurin complex systems. Azurin is known to play an essential role as an anticancer agent and bind many important intracellular molecules. Some physical properties are monitored during simulation time to get a better understanding of the influence of protein-protein interactions to the azurin complex dynamics. These studies will provide valuable insights for further investigation on protein-protein interactions in more realistic system.

Can misfolded proteins be beneficial? The HAMLET case.

PubMed

Pettersson-Kastberg, Jenny; Aits, Sonja; Gustafsson, Lotta; Mossberg, Anki; Storm, Petter; Trulsson, Maria; Persson, Filip; Mok, K Hun; Svanborg, Catharina

2009-01-01

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.

Genetics Home Reference: autoimmune Addison disease

MedlinePlus

... is the most common form in developed countries, accounting for up to 90 percent of cases. Related ... HLA) complex . The HLA complex helps the immune system distinguish the body's own proteins from proteins made ...

Capture of unstable protein complex on the streptavidin-coated single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Liu, Zunfeng; Voskamp, Patrick; Zhang, Yue; Chu, Fuqiang; Abrahams, Jan Pieter

2013-04-01

Purification of unstable protein complexes is a bottleneck for investigation of their 3D structure and in protein-protein interaction studies. In this paper, we demonstrate that streptavidin-coated single-walled carbon nanotubes (Strep•SWNT) can be used to capture the biotinylated DNA- EcoRI complexes on a 2D surface and in solution using atomic force microscopy and electrophoresis analysis, respectively. The restriction enzyme EcoRI forms unstable complexes with DNA in the absence of Mg2+. Capturing the EcoRI-DNA complexes on the Strep•SWNT succeeded in the absence of Mg2+, demonstrating that the Strep•SWNT can be used for purifying unstable protein complexes.

Molecular mechanisms of the cytotoxicity of human α-lactalbumin made lethal to tumor cells (HAMLET) and other protein-oleic acid complexes.

PubMed

Nakamura, Takashi; Aizawa, Tomoyasu; Kariya, Ryusho; Okada, Seiji; Demura, Makoto; Kawano, Keiichi; Makabe, Koki; Kuwajima, Kunihiro

2013-05-17

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.

Molecular Mechanisms of the Cytotoxicity of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET) and Other Protein-Oleic Acid Complexes*

PubMed Central

Nakamura, Takashi; Aizawa, Tomoyasu; Kariya, Ryusho; Okada, Seiji; Demura, Makoto; Kawano, Keiichi; Makabe, Koki; Kuwajima, Kunihiro

2013-01-01

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane. PMID:23580643

Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

PubMed Central

He, Yi-Ming; Ma, Bin-Guang

2016-01-01

Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions. PMID:27220911

Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

NASA Astrophysics Data System (ADS)

He, Yi-Ming; Ma, Bin-Guang

2016-05-01

Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed Central

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-01-01

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions. Images PMID:2556266

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-12-20

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

Super-complexes of adhesion GPCRs and neural guidance receptors

NASA Astrophysics Data System (ADS)

Jackson, Verity A.; Mehmood, Shahid; Chavent, Matthieu; Roversi, Pietro; Carrasquero, Maria; Del Toro, Daniel; Seyit-Bremer, Goenuel; Ranaivoson, Fanomezana M.; Comoletti, Davide; Sansom, Mark S. P.; Robinson, Carol V.; Klein, Rüdiger; Seiradake, Elena

2016-04-01

Latrophilin adhesion-GPCRs (Lphn1-3 or ADGRL1-3) and Unc5 cell guidance receptors (Unc5A-D) interact with FLRT proteins (FLRT1-3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger `super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.

Iron-sulfur Proteins Are the Major Source of Protein-bound Dinitrosyl Iron Complexes Formed in Escherichia coli Cells under Nitric Oxide Stress

PubMed Central

Landry, Aaron P.; Duan, Xuewu; Huang, Hao; Ding, Huangen

2011-01-01

Protein-bound dinitrosyl iron complexes (DNICs) have been observed in prokaryotic and eukaryotic cells under nitric oxide (NO) stress. The identity of proteins that bind DNICs, however, still remains elusive. Here we demonstrate that iron-sulfur proteins are the major source of protein-bound DNICs formed in Escherichia coli cells under NO stress. Expression of recombinant iron-sulfur proteins, but not the proteins without iron-sulfur clusters, almost doubles the amount of protein-bound DNICs formed in E. coli cells after NO exposure. Purification of recombinant proteins from the NO-exposed E. coli cells further confirms that iron-sulfur proteins, but not the proteins without iron-sulfur clusters, are modified forming protein-bound DINCs. Deletion of the iron-sulfur cluster assembly proteins IscA and SufA to block the [4Fe-4S] cluster biogenesis in E. coli cells largely eliminates the NO-mediated formation of protein-bound DNICs, suggesting that iron-sulfur clusters are mainly responsible for the NO-mediated formation of protein-bound DNICs in cells. Furthermore, depletion of “chelatable iron pool” in the wild-type E. coli cells effectively removes iron-sulfur clusters from proteins and concomitantly diminishes the NO-mediated formation of protein-bound DNICs, indicating that iron-sulfur clusters in proteins constitute at least part of “chelatable iron pool” in cells. PMID:21420489

Structural study of surfactant-dependent interaction with protein

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

Mehan, Sumit; Aswal, Vinod K., E-mail: vkaswal@barc.gov.in; Kohlbrecher, Joachim

2015-06-24

Small-angle neutron scattering (SANS) has been used to study the complex structure of anionic BSA protein with three different (cationic DTAB, anionic SDS and non-ionic C12E10) surfactants. These systems form very different surfactant-dependent complexes. We show that the structure of protein-surfactant complex is initiated by the site-specific electrostatic interaction between the components, followed by the hydrophobic interaction at high surfactant concentrations. It is also found that hydrophobic interaction is preferred over the electrostatic interaction in deciding the resultant structure of protein-surfactant complexes.

Structural study of surfactant-dependent interaction with protein

NASA Astrophysics Data System (ADS)

Mehan, Sumit; Aswal, Vinod K.; Kohlbrecher, Joachim

2015-06-01

Small-angle neutron scattering (SANS) has been used to study the complex structure of anionic BSA protein with three different (cationic DTAB, anionic SDS and non-ionic C12E10) surfactants. These systems form very different surfactant-dependent complexes. We show that the structure of protein-surfactant complex is initiated by the site-specific electrostatic interaction between the components, followed by the hydrophobic interaction at high surfactant concentrations. It is also found that hydrophobic interaction is preferred over the electrostatic interaction in deciding the resultant structure of protein-surfactant complexes.

Molecular dynamics simulations show altered secondary structure of clawless in binary complex with DNA providing insights into aristaless-clawless-DNA ternary complex formation.

PubMed

Kachhap, Sangita; Priyadarshini, Pragya; Singh, Balvinder

2017-05-01

Aristaless (Al) and clawless (Cll) homeodomains that are involved in leg development in Drosophila melanogaster are known to bind cooperatively to 5'-(T/C)TAATTAA(T/A)(T/A)G-3' DNA sequence, but the mechanism of their binding to DNA is unknown. Molecular dynamics (MD) studies have been carried out on binary, ternary, and reconstructed protein-DNA complexes involving Al, Cll, and DNA along with binding free energy analysis of these complexes. Analysis of MD trajectories of Cll-3A01, binary complex reveals that C-terminal end of helixIII of Cll, unwind in the absence of Al and remains so in reconstructed ternary complex, Cll-3A01-Al. In addition, this change in secondary structure of Cll does not allow it to form protein-protein interactions with Al in the ternary reconstructed complex. However, secondary structure of Cll and its interactions are maintained in other reconstructed ternary complex, Al-3A01-Cll where Cll binds to Al-3A01, binary complex to form ternary complex. These interactions as observed during MD simulations compare well with those observed in ternary crystal structure. Thus, this study highlights the role of helixIII of Cll and protein-protein interactions while proposing likely mechanism of recognition in ternary complex, Al-Cll-DNA.

A-Kinase Anchoring Proteins: From protein complexes to physiology and disease

PubMed Central

Carnegie, Graeme K.; Means, Christopher K.; Scott, John D.

2009-01-01

Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review we focus on recent advances in the elucidation of AKAP function. PMID:19319965

A-kinase anchoring proteins: from protein complexes to physiology and disease.

PubMed

Carnegie, Graeme K; Means, Christopher K; Scott, John D

2009-04-01

Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review, we focus on recent advances in the elucidation of AKAP function.

Categorizing Biases in High-Confidence High-Throughput Protein-Protein Interaction Data Sets

DTIC Science & Technology

2011-01-01

may partially explain why we did not observe any of the interactions between RNA polymerase II compo- nents in any of the Y2H set (11). Methodological...DNA. Fig. 5 shows that RNA syn- thesis complexes formed a highly interconnected cluster, in- cluding RNA polymerases I, II , and III, Transcription...factor complexes II F (TFIIF) and III C (TFIIIC), which were connected via direct protein-protein interactions with many other func- tional complexes. Fig

Peculiarities of hemoglobin interaction with serum proteins of mice with Ehrlich carcinoma.

PubMed

Sitdikova, S M; Amandzholov, B S; Serebryakova, M V; Zhdanovich, M Yu; Kiselevskii, M V; Donenko, F V

2006-05-01

In male C57Bl/6 mice with transplanted Ehrlich carcinoma, hemoglobin forms a complex with serum proteins characterized by a molecular weight of about 300 kDa. The complex incorporates proteins weighing 100, 68, 65, and 15 kDa identified by MALDI-TOF mass spectrometry as haptoglobin, serum albumin, gi/26341396 nameless protein Mus musculus, and alpha-hemoglobin, respectively. This complex can possess biological activity and contribute to the control of tumor growth.

Isolation and Compositional Analysis of a CP12-Associated Complex of Calvin Cycle Enzymes from Nicotiana tabacum

USDA-ARS?s Scientific Manuscript database

CP12 is a small intrinsically unstructured protein that forms a multiprotein complex with two Calvin Cycle enzymes, phosphoribulokinase (PRK) and NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The complex can be reconstituted in vitro from recombinant proteins under conditions t...

Detection of the HA-33 protein in botulinum neurotoxin type G complex by mass spectrometry.

PubMed

Kalb, Suzanne R; Baudys, Jakub; Barr, John R

2015-10-23

The disease botulism is caused by intoxication with botulinum neurotoxins (BoNTs), extremely toxic proteins which cause paralysis. This neurotoxin is produced by some members of the Clostridium botulinum and closely related species, and is produced as a protein complex consisting of the neurotoxin and neurotoxin-associated proteins (NAPs). There are seven known serotypes of BoNT, A-G, and the composition of the NAPs can differ between these serotypes. It was previously published that the BoNT/G complex consisted of BoNT/G, nontoxic-nonhemagglutinin (NTNH), Hemagglutinin 70 (HA-70), and HA-17, but that HA-33, a component of the protein complex of other serotypes of BoNT, was not found. Components of the BoNT/G complex were first separated by SDS-PAGE, and bands corresponding to components of the complex were digested and analyzed by LC-MS/MS. Gel bands were identified with sequence coverages of 91% for BoNT/G, 91% for NTNH, 89% for HA-70, and 88% for HA-17. Notably, one gel band was also clearly identified as HA-33 with 93% sequence coverage. The BoNT/G complex consists of BoNT/G, NTNH, HA-70, HA-17, and HA-33. These proteins form the progenitor form of BoNT/G, similar to all other HA positive progenitor toxin complexes.

ComplexContact: a web server for inter-protein contact prediction using deep learning.

PubMed

Zeng, Hong; Wang, Sheng; Zhou, Tianming; Zhao, Feifeng; Li, Xiufeng; Wu, Qing; Xu, Jinbo

2018-05-22

ComplexContact (http://raptorx2.uchicago.edu/ComplexContact/) is a web server for sequence-based interfacial residue-residue contact prediction of a putative protein complex. Interfacial residue-residue contacts are critical for understanding how proteins form complex and interact at residue level. When receiving a pair of protein sequences, ComplexContact first searches for their sequence homologs and builds two paired multiple sequence alignments (MSA), then it applies co-evolution analysis and a CASP-winning deep learning (DL) method to predict interfacial contacts from paired MSAs and visualizes the prediction as an image. The DL method was originally developed for intra-protein contact prediction and performed the best in CASP12. Our large-scale experimental test further shows that ComplexContact greatly outperforms pure co-evolution methods for inter-protein contact prediction, regardless of the species.

A conserved protein interaction interface on the type 5 G protein beta subunit controls proteolytic stability and activity of R7 family regulator of G protein signaling proteins.

PubMed

Porter, Morwenna Y; Xie, Keqiang; Pozharski, Edwin; Koelle, Michael R; Martemyanov, Kirill A

2010-12-24

Regulators of G protein signaling (RGS) proteins of the R7 subfamily limit signaling by neurotransmitters in the brain and by light in the retina. They form obligate complexes with the Gβ5 protein that are subject to proteolysis to control their abundance and alter signaling. The mechanisms that regulate this proteolysis, however, remain unclear. We used genetic screens to find mutations in Gβ5 that selectively destabilize one of the R7 RGS proteins in Caenorhabditis elegans. These mutations cluster at the binding interface between Gβ5 and the N terminus of R7 RGS proteins. Equivalent mutations within mammalian Gβ5 allowed the interface to still bind the N-terminal DEP domain of R7 RGS proteins, and mutant Gβ5-R7 RGS complexes initially formed in cells but were then rapidly degraded by proteolysis. Molecular dynamics simulations suggest the mutations weaken the Gβ5-DEP interface, thus promoting dynamic opening of the complex to expose determinants of proteolysis known to exist on the DEP domain. We propose that conformational rearrangements at the Gβ5-DEP interface are key to controlling the stability of R7 RGS protein complexes.

The PsB glycoprotein complex is secreted as a preassembled precursor of the spore coat in Dictyostelium discoideum.

PubMed

Watson, N; McGuire, V; Alexander, S

1994-09-01

The PsB glycoprotein in Dictyostelium discoideum is one of a diverse group of developmentally regulated, prespore-cell-specific proteins, that contain a common O-linked oligosaccharide. This post-translational modification is dependent on the wild-type modB allele. The PsB protein exists as part of a multiprotein complex of six different proteins, which have different post-translational modifications and are held together by both covalent and non-covalent interactions (Watson et al. (1993). J. Biol. Chem. 268, 22634-22641). In this study we have used microscopic and biochemical analyses to examine the cellular localization and function of the PsB complex during development. We found that the PsB complex first accumulates in prespore vesicles in slug cells and is secreted later during culmination and becomes localized to both the extracellular matrix of the apical spore mass of mature fruiting bodies and to the inner layer of the spore coat. The PsB associated with the spore coat is covalently bound by disulfide bridges. The PsB protein always exists in a multiprotein complex, but the composition of the PsB complex changes during secretion and spore maturation. Some of the PsB complex proteins have been identified as spore coat proteins. These data demonstrate that some of the proteins that form the spore coat exist as a preassembled precursor complex. The PsB complex is secreted in a developmentally regulated manner during the process of spore differentiation, at which time proteins of the complex, as well as additional spore coat proteins, become covalently associated in at least two forms of extracellular matrix: the interspore matrix and the spore coat. These and other studies show that proteins with modB dependent O-linked oligosaccharides are involved in a wide variety of processes underlying morphogenesis in this organism. These developmental processes are the direct result of cellular mechanisms regulating protein targeting, assembly and secretion, and the assembly of specific extracellular matrices.

The Network Organization of Cancer-associated Protein Complexes in Human Tissues

PubMed Central

Zhao, Jing; Lee, Sang Hoon; Huss, Mikael; Holme, Petter

2013-01-01

Differential gene expression profiles for detecting disease genes have been studied intensively in systems biology. However, it is known that various biological functions achieved by proteins follow from the ability of the protein to form complexes by physically binding to each other. In other words, the functional units are often protein complexes rather than individual proteins. Thus, we seek to replace the perspective of disease-related genes by disease-related complexes, exemplifying with data on 39 human solid tissue cancers and their original normal tissues. To obtain the differential abundance levels of protein complexes, we apply an optimization algorithm to genome-wide differential expression data. From the differential abundance of complexes, we extract tissue- and cancer-selective complexes, and investigate their relevance to cancer. The method is supported by a clustering tendency of bipartite cancer-complex relationships, as well as a more concrete and realistic approach to disease-related proteomics. PMID:23567845

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

Assembly of the Herpes Simplex Virus Capsid: Preformed Triplexes Bind to the Nascent Capsid

PubMed Central

Spencer, Juliet V.; Newcomb, William W.; Thomsen, Darrell R.; Homa, Fred L.; Brown, Jay C.

1998-01-01

The herpes simplex virus type 1 (HSV-1) capsid is a T=16 icosahedral shell that forms in the nuclei of infected cells. Capsid assembly also occurs in vitro in reaction mixtures created from insect cell extracts containing recombinant baculovirus-expressed HSV-1 capsid proteins. During capsid formation, the major capsid protein, VP5, and the scaffolding protein, pre-VP22a, condense to form structures that are extended into procapsids by addition of the triplex proteins, VP19C and VP23. We investigated whether triplex proteins bind to the major capsid-scaffold protein complexes as separate polypeptides or as preformed triplexes. Assembly products from reactions lacking one triplex protein were immunoprecipitated and examined for the presence of the other. The results showed that neither triplex protein bound unless both were present, suggesting that interaction between VP19C and VP23 is required before either protein can participate in the assembly process. Sucrose density gradient analysis was employed to determine the sedimentation coefficients of VP19C, VP23, and VP19C-VP23 complexes. The results showed that the two proteins formed a complex with a sedimentation coefficient of 7.2S, a value that is consistent with formation of a VP19C-VP232 heterotrimer. Furthermore, VP23 was observed to have a sedimentation coefficient of 4.9S, suggesting that this protein exists as a dimer in solution. Deletion analysis of VP19C revealed two domains that may be required for attachment of the triplex to major capsid-scaffold protein complexes; none of the deletions disrupted interaction of VP19C with VP23. We propose that preformed triplexes (VP19C-VP232 heterotrimers) interact with major capsid-scaffold protein complexes during assembly of the HSV-1 capsid. PMID:9557680

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

PubMed

Howard, Thomas P; Lloyd, Julie C; Raines, Christine A

2011-07-01

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes.

PubMed

Bastiani, Michele; Liu, Libin; Hill, Michelle M; Jedrychowski, Mark P; Nixon, Susan J; Lo, Harriet P; Abankwa, Daniel; Luetterforst, Robert; Fernandez-Rojo, Manuel; Breen, Michael R; Gygi, Steven P; Vinten, Jorgen; Walser, Piers J; North, Kathryn N; Hancock, John F; Pilch, Paul F; Parton, Robert G

2009-06-29

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer-based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.

WAVE2 forms a complex with PKA and is involved in PKA enhancement of membrane protrusions.

PubMed

Yamashita, Hiroshi; Ueda, Kazumitsu; Kioka, Noriyuki

2011-02-04

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation.

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

PubMed Central

Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins. PMID:26544075

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography.

PubMed

Park, Seong-Jun; Ahn, Hee-Sung; Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins.

Coevolution study of mitochondria respiratory chain proteins: toward the understanding of protein--protein interaction.

PubMed

Yang, Ming; Ge, Yan; Wu, Jiayan; Xiao, Jingfa; Yu, Jun

2011-05-20

Coevolution can be seen as the interdependency between evolutionary histories. In the context of protein evolution, functional correlation proteins are ever-present coordinated evolutionary characters without disruption of organismal integrity. As to complex system, there are two forms of protein--protein interactions in vivo, which refer to inter-complex interaction and intra-complex interaction. In this paper, we studied the difference of coevolution characters between inter-complex interaction and intra-complex interaction using "Mirror tree" method on the respiratory chain (RC) proteins. We divided the correlation coefficients of every pairwise RC proteins into two groups corresponding to the binary protein--protein interaction in intra-complex and the binary protein--protein interaction in inter-complex, respectively. A dramatical discrepancy is detected between the coevolution characters of the two sets of protein interactions (Wilcoxon test, p-value = 4.4 × 10(-6)). Our finding reveals some critical information on coevolutionary study and assists the mechanical investigation of protein--protein interaction. Furthermore, the results also provide some unique clue for supramolecular organization of protein complexes in the mitochondrial inner membrane. More detailed binding sites map and genome information of nuclear encoded RC proteins will be extraordinary valuable for the further mitochondria dynamics study. Copyright © 2011. Published by Elsevier Ltd.

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

PubMed Central

Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

2013-01-01

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

Recent progress in biopolymer nanoparticle and microparticle formation by heat-treating electrostatic protein-polysaccharide complexes.

PubMed

Jones, Owen G; McClements, David Julian

2011-09-14

Functional biopolymer nanoparticles or microparticles can be formed by heat treatment of globular protein-ionic polysaccharide electrostatic complexes under appropriate solution conditions. These biopolymer particles can be used as encapsulation and delivery systems, fat mimetics, lightening agents, or texture modifiers. This review highlights recent progress in the design and fabrication of biopolymer particles based on heating globular protein-ionic polysaccharide complexes above the thermal denaturation temperature of the proteins. The influence of biopolymer type, protein-polysaccharide ratio, pH, ionic strength, and thermal history on the characteristics of the biopolymer particles formed is reviewed. Our current understanding of the underlying physicochemical mechanisms of particle formation and properties is given. The information provided in this review should facilitate the rational design of biopolymer particles with specific physicochemical and functional attributes, as well as stimulate further research in identifying the physicochemical origin of particle formation. Copyright © 2010 Elsevier B.V. All rights reserved.

Triangle network motifs predict complexes by complementing high-error interactomes with structural information.

PubMed

Andreopoulos, Bill; Winter, Christof; Labudde, Dirk; Schroeder, Michael

2009-06-27

A lot of high-throughput studies produce protein-protein interaction networks (PPINs) with many errors and missing information. Even for genome-wide approaches, there is often a low overlap between PPINs produced by different studies. Second-level neighbors separated by two protein-protein interactions (PPIs) were previously used for predicting protein function and finding complexes in high-error PPINs. We retrieve second level neighbors in PPINs, and complement these with structural domain-domain interactions (SDDIs) representing binding evidence on proteins, forming PPI-SDDI-PPI triangles. We find low overlap between PPINs, SDDIs and known complexes, all well below 10%. We evaluate the overlap of PPI-SDDI-PPI triangles with known complexes from Munich Information center for Protein Sequences (MIPS). PPI-SDDI-PPI triangles have ~20 times higher overlap with MIPS complexes than using second-level neighbors in PPINs without SDDIs. The biological interpretation for triangles is that a SDDI causes two proteins to be observed with common interaction partners in high-throughput experiments. The relatively few SDDIs overlapping with PPINs are part of highly connected SDDI components, and are more likely to be detected in experimental studies. We demonstrate the utility of PPI-SDDI-PPI triangles by reconstructing myosin-actin processes in the nucleus, cytoplasm, and cytoskeleton, which were not obvious in the original PPIN. Using other complementary datatypes in place of SDDIs to form triangles, such as PubMed co-occurrences or threading information, results in a similar ability to find protein complexes. Given high-error PPINs with missing information, triangles of mixed datatypes are a promising direction for finding protein complexes. Integrating PPINs with SDDIs improves finding complexes. Structural SDDIs partially explain the high functional similarity of second-level neighbors in PPINs. We estimate that relatively little structural information would be sufficient for finding complexes involving most of the proteins and interactions in a typical PPIN.

Triangle network motifs predict complexes by complementing high-error interactomes with structural information

PubMed Central

Andreopoulos, Bill; Winter, Christof; Labudde, Dirk; Schroeder, Michael

2009-01-01

Background A lot of high-throughput studies produce protein-protein interaction networks (PPINs) with many errors and missing information. Even for genome-wide approaches, there is often a low overlap between PPINs produced by different studies. Second-level neighbors separated by two protein-protein interactions (PPIs) were previously used for predicting protein function and finding complexes in high-error PPINs. We retrieve second level neighbors in PPINs, and complement these with structural domain-domain interactions (SDDIs) representing binding evidence on proteins, forming PPI-SDDI-PPI triangles. Results We find low overlap between PPINs, SDDIs and known complexes, all well below 10%. We evaluate the overlap of PPI-SDDI-PPI triangles with known complexes from Munich Information center for Protein Sequences (MIPS). PPI-SDDI-PPI triangles have ~20 times higher overlap with MIPS complexes than using second-level neighbors in PPINs without SDDIs. The biological interpretation for triangles is that a SDDI causes two proteins to be observed with common interaction partners in high-throughput experiments. The relatively few SDDIs overlapping with PPINs are part of highly connected SDDI components, and are more likely to be detected in experimental studies. We demonstrate the utility of PPI-SDDI-PPI triangles by reconstructing myosin-actin processes in the nucleus, cytoplasm, and cytoskeleton, which were not obvious in the original PPIN. Using other complementary datatypes in place of SDDIs to form triangles, such as PubMed co-occurrences or threading information, results in a similar ability to find protein complexes. Conclusion Given high-error PPINs with missing information, triangles of mixed datatypes are a promising direction for finding protein complexes. Integrating PPINs with SDDIs improves finding complexes. Structural SDDIs partially explain the high functional similarity of second-level neighbors in PPINs. We estimate that relatively little structural information would be sufficient for finding complexes involving most of the proteins and interactions in a typical PPIN. PMID:19558694

Recording information on protein complexes in an information management system.

PubMed

Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

2011-08-01

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. Copyright © 2011 Elsevier Inc. All rights reserved.

Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

PubMed

Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

2016-02-01

Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation. © 2015 Wiley Periodicals, Inc.

Why Congo red binding is specific for amyloid proteins - model studies and a computer analysis approach.

PubMed

Roterman, I; KrUl, M; Nowak, M; Konieczny, L; Rybarska, J; Stopa, B; Piekarska, B; Zemanek, G

2001-01-01

The complexing of Congo red in two different ligand forms - unimolecular and supramolecular (seven molecules in a micelle) - with eight deca-peptides organized in a b-sheet was tested by computational analysis to identify its dye-binding preferences. Polyphenylananine and polylysine peptides were selected to represent the specific side chain interactions expected to ensure particularly the stabilization of the dye-protein complex. Polyalanine was used to verify the participation of non-specific backbone-derived interactions. The initial complexes for calculation were constructed by intercalating the dye between the peptides in the middle of the beta-sheet. The long axis of the dye molecule (in the case of unimolecular systems) or the long axis of the ribbon-like micelle (in the case of the supramolecular dye form) was oriented parallel to the peptide backbone. This positioning maximally reduced the exposure of the hydrophobic diphenyl (central dye fragment) to water. In general the complexes of supramolecular Congo red ligands appeared more stable than those formed by individual dye molecules. Specific interactions (electrostatic and/or ring stacking) dominated as binding forces in the case of the single molecule, while non-specific surface adsorption seemed decisive in complexing with the supramolecular ligand. Both the unimolecular and supramolecular versions of the dye ligand were found to be likely to form complexes of sufficient stability with peptides. The low stability of the protein and the gap accessible to penetration in the peptide sheet seem sufficient for supramolecular ligand binding, but the presence of positively charged or hydrophobic amino acids may strengthen binding significantly. The need for specific interaction makes single-molecule Congo red binding rather unusual as a general amyloid protein ligand. The structural feature of Congo red, which enables specific and common interaction with amyloid proteins, probably derives from the ribbon-like self-assembled form of the dye.

Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.

PubMed

Kerppola, Tom K

2006-01-01

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can form a fluorescent complex and that the association of the fragments can be facilitated when they are fused to two proteins that interact with each other. BiFC must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other because they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at levels comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.

Reversible Association of the Hemagglutinin Subcomplex, HA-33/HA-17 Trimer, with the Botulinum Toxin Complex.

PubMed

Sagane, Yoshimasa; Mutoh, Shingo; Koizumi, Ryosuke; Suzuki, Tomonori; Miyashita, Shin-Ichiro; Miyata, Keita; Ohyama, Tohru; Niwa, Koichi; Watanabe, Toshihiro

2017-10-01

Botulinum neurotoxin (BoNT) associates with nontoxic proteins, either a nontoxic nonhemagglutinin (NTNHA) or the complex of NTNHA and hemagglutinin (HA), to form M- or L-toxin complexes (TCs). Single BoNT and NTNHA molecules are associated and form M-TC. A trimer of the 70-kDa HA protein (HA-70) attaches to the M-TC to form M-TC/HA-70. Further, 1-3 arm-like 33- and 17-kDa HA molecules (HA-33/HA-17 trimer), consisting of 1 HA-17 protein and 2 HA-33 proteins, can attach to the M-TC/HA-70 complex, yielding 1-, 2-, and 3-arm L-TC. In this study, the purified 1- and 2-arm L-TCs spontaneously converted into another L-TC species after acquiring the HA-33/HA-17 trimer from other TCs during long-term storage and freezing/thawing. Transmission electron microscopy analysis provided evidence of the formation of detached HA-33/HA-17 trimers in the purified TC preparation. These findings provide evidence of reversible association/dissociation of the M-TC/HA-70 complex with the HA-33/HA-17 trimers, as well as dynamic conversion of the quaternary structure of botulinum TC in culture.

Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain

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

Singh, Rajesh Kumar; Palm, Gottfried J.; Panjikar, Santosh

2007-04-01

Crystal structure analysis of the apo form of catabolite control protein A reveals the three-helix bundle of the DNA-binding domain. In the crystal packing, this domain interacts with the binding site for the corepressor protein. Crystal structure determination of catabolite control protein A (CcpA) at 2.6 Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. Inmore » the crystal packing of apo-CcpA, the protein–protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer.« less

The RSV F and G glycoproteins interact to form a complex on the surface of infected cells

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

Low, Kit-Wei; Tan, Timothy; Ng, Ken

2008-02-08

In this study, the interaction between the respiratory syncytial virus (RSV) fusion (F) protein, attachment (G) protein, and small hydrophobic (SH) proteins was examined. Immunoprecipitation analysis suggested that the F and G proteins exist as a protein complex on the surface of RSV-infected cells, and this conclusion was supported by ultracentrifugation analysis that demonstrated co-migration of surface-expressed F and G proteins. Although our analysis provided evidence for an interaction between the G and SH proteins, no evidence was obtained for a single protein complex involving all three of the virus proteins. These data suggest the existence of multiple virus glycoproteinmore » complexes within the RSV envelope. Although the stimulus that drives RSV-mediated membrane fusion is unknown, the association between the G and F proteins suggest an indirect role for the G protein in this process.« less

Modular assembly of chimeric phi29 packaging RNAs that support DNA packaging.

PubMed

Fang, Yun; Shu, Dan; Xiao, Feng; Guo, Peixuan; Qin, Peter Z

2008-08-08

The bacteriophage phi29 DNA packaging motor is a protein/RNA complex that can produce strong force to condense the linear-double-stranded DNA genome into a pre-formed protein capsid. The RNA component, called the packaging RNA (pRNA), utilizes magnesium-dependent inter-molecular base-pairing interactions to form ring-shaped complexes. The pRNA is a class of non-coding RNA, interacting with phi29 motor proteins to enable DNA packaging. Here, we report a two-piece chimeric pRNA construct that is fully competent in interacting with partner pRNA to form ring-shaped complexes, in packaging DNA via the motor, and in assembling infectious phi29 virions in vitro. This is the first example of a fully functional pRNA assembled using two non-covalently interacting fragments. The results support the notion of modular pRNA architecture in the phi29 packaging motor.

Modular assembly of chimeric phi29 packaging RNAs that support DNA packaging

PubMed Central

Fang, Yun; Shu, Dan; Xiao, Feng; Guo, Peixuan; Qin, Peter Z.

2008-01-01

The bacteriophage phi29 DNA packaging motor is a protein/RNA complex that can produce strong force to condense the linear-double stranded DNA genome into a pre-formed protein capsid. The RNA component, called the packaging RNA (pRNA), utilizes magnesium-dependent intermolecular base-pairing interactions to form ring-shaped complexes. The pRNA is a class of non-coding RNA, interacting with phi29 motor proteins to enable DNA packaging. Here, we report a 2-piece chimeric pRNA construct that is fully competent in interacting with partner pRNA to form ring-shaped complexes, in packaging DNA via the motor, and in assembling infectious phi29 virions in vitro. This is the first example of a fully functional pRNA assembled using two non-covalently interacting fragments. The results support the notion of modular pRNA architecture in the phi29 packaging motor. PMID:18514064

Evidence for a ternary complex formed between flavodoxin and cytochrome c3: 1H-NMR and molecular modeling studies.

PubMed

Palma, P N; Moura, I; LeGall, J; Van Beeumen, J; Wampler, J E; Moura, J J

1994-05-31

Small electron-transfer proteins such as flavodoxin (16 kDa) and the tetraheme cytochrome c3 (13 kDa) have been used to mimic, in vitro, part of the complex electron-transfer chain operating between substrate electron donors and respiratory electron acceptors, in sulfate-reducing bacteria (Desulfovibrio species). The nature and properties of the complex formed between these proteins are revealed by 1H-NMR and molecular modeling approaches. Our previous study with the Desulfovibrio vulgaris proteins [Moura, I., Moura, J.J. G., Santos, M.H., & Xavier, A. V. (1980) Cienc. Biol. (Portugal) 5, 195-197; Stewart, D.E. LeGall, J., Moura, I., Moura, J. J. G., Peck, H.D. Jr., Xavier, A. V., Weiner, P. K., & Wampler, J.E. (1988) Biochemistry 27, 2444-2450] indicated that the complex between cytochrome c3 and flavodoxin could be monitored by changes in the NMR signals of the heme methyl groups of the cytochrome and that the electrostatic surface charge (Coulomb's law) on the two proteins favored interaction between one unique heme of the cytochrome with flavodoxin. If the interaction is indeed driven by the electrostatic complementarity between the acidic flavodoxin and a unique positive region of the cytochrome c3, other homologous proteins from these two families of proteins might be expected to interact similarly. In this study, three homologous Desulfovibrio cytochromes c3 were used, which show a remarkable variation in their individual isoelectric points (ranging from 5.5 to 9.5). On the basis of data obtained from protein-protein titrations followed at specific proton NMR signals (i.e., heme methyl resonances), a binding model for this complex has been developed with evaluation of stoichiometry and binding constants. This binding model involves one site on the cytochromes c3 and two sites on the flavodoxin, with formation of a ternary complex at saturation. In order to understand the potential chemical form of the binding model, a structural model for the hypothetical ternary complex, formed between one molecule of Desulfovibrio salexigens flavodoxin and two molecules of cytochrome c3, is proposed. These molecular models of the complexes were constructed on the basis of complementarity of Coulombic electrostatic surface potentials, using the available X-ray structures of the isolated proteins and, when required, model structures (D. salexigens flavodoxin and Desulfovibrio desulfuricans ATCC 27774 cytochrome c3) predicted by homology modeling.

Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

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

Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi

Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing,more » Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.« less

Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis.

PubMed

Godlewska, Renata; Wiśniewska, Katarzyna; Pietras, Zbigniew; Jagusztyn-Krynicka, Elzbieta Katarzyna

2009-09-01

The protein Pal (peptidoglycan-associated lipoprotein) is anchored in the outer membrane (OM) of Gram-negative bacteria and interacts with Tol proteins. Tol-Pal proteins form two complexes: the first is composed of three inner membrane Tol proteins (TolA, TolQ and TolR); the second consists of the TolB and Pal proteins linked to the cell's OM. These complexes interact with one another forming a multiprotein membrane-spanning system. It has recently been demonstrated that Pal is essential for bacterial survival and pathogenesis, although its role in virulence has not been clearly defined. This review summarizes the available data concerning the structure and function of Pal and its role in pathogenesis.

Multi-Dimensional Scaling based grouping of known complexes and intelligent protein complex detection.

PubMed

Rehman, Zia Ur; Idris, Adnan; Khan, Asifullah

2018-06-01

Protein-Protein Interactions (PPI) play a vital role in cellular processes and are formed because of thousands of interactions among proteins. Advancements in proteomics technologies have resulted in huge PPI datasets that need to be systematically analyzed. Protein complexes are the locally dense regions in PPI networks, which extend important role in metabolic pathways and gene regulation. In this work, a novel two-phase protein complex detection and grouping mechanism is proposed. In the first phase, topological and biological features are extracted for each complex, and prediction performance is investigated using Bagging based Ensemble classifier (PCD-BEns). Performance evaluation through cross validation shows improvement in comparison to CDIP, MCode, CFinder and PLSMC methods Second phase employs Multi-Dimensional Scaling (MDS) for the grouping of known complexes by exploring inter complex relations. It is experimentally observed that the combination of topological and biological features in the proposed approach has greatly enhanced prediction performance for protein complex detection, which may help to understand various biological processes, whereas application of MDS based exploration may assist in grouping potentially similar complexes. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Pigment-protein complexes nd the number of the reaction photosystem centers in pea chlorophyll mutants].

PubMed

Ladygin, V G

2004-01-01

We studied fluorescent and absorption properties of the chloroplasts and pigment-protein complexes isolated by gel electrophoresis from the leaves of pea, the initial cultivar Torsdag and mutants chlorotica 2004 and 2014. Specific maxima of fluorescence and chlorophyll forms in individual complexes have been determined from the absorption and fluorescence spectra of the chloroplast chlorophyll and their secondary derivatives at 23 and -196 degrees C. Chlorotica 2004 mutant proved to have an increased intensity of a long-wave band at both 23 degrees C (745 nm) and -196 degrees C (728 nm) of the light-harvesting complex I. At the same time, this mutant featured a decreased accumulation of chlorophyll forms at 690, 697, and 708 nm forming the nearest-neighbor antenna of PSI reaction center. No spectral differences have been revealed between chlorotica 2014 mutant and the initial cultivar. Gel electrophoresis demonstrated synthesis of all chlorophyll-protein complexes in both mutants. At the same time, analysis of photochemical activity of PSI and PSII reaction centers and evaluation of the light-harvesting antenna as well as the number of reaction centers of the photosystems suggest that chlorotica 2004 mutant has 1.7 times less PSI reaction centers due to a mutation-disturbed chlorophyll a-protein complex of PSI. The primary effect of chlorotica 2014 mutation remains unclear. The proportional changes in the photosystem complexes in this mutant suggest that they are secondary and result from a 50% decrease in chlorophyll content.

Architectures of Lipid Transport Systems for the Bacterial Outer Membrane.

PubMed

Ekiert, Damian C; Bhabha, Gira; Isom, Georgia L; Greenan, Garrett; Ovchinnikov, Sergey; Henderson, Ian R; Cox, Jeffery S; Vale, Ronald D

2017-04-06

How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles. Copyright © 2017 Elsevier Inc. All rights reserved.

Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2–7 Helicase to Reveal Essential Features of Structure and Function

PubMed Central

Miller, Justin M.; Enemark, Eric J.

2015-01-01

In eukaryotes, the replicative helicase is the large multisubunit CMG complex consisting of the Mcm2–7 hexameric ring, Cdc45, and the tetrameric GINS complex. The Mcm2–7 ring assembles from six different, related proteins and forms the core of this complex. In archaea, a homologous MCM hexameric ring functions as the replicative helicase at the replication fork. Archaeal MCM proteins form thermostable homohexamers, facilitating their use as models of the eukaryotic Mcm2–7 helicase. Here we review archaeal MCM helicase structure and function and how the archaeal findings relate to the eukaryotic Mcm2–7 ring. PMID:26539061

Channelopathies from Mutations in the Cardiac Sodium Channel Protein Complex

PubMed Central

Adsit, Graham S.; Vaidyanathan, Ravi; Galler, Carla M.; Kyle, John W.; Makielski, Jonathan C.

2013-01-01

The cardiac sodium current underlies excitability in heart, and inherited abnormalities of the proteins regulating and conducting this current cause inherited arrhythmia syndromes. This review focuses on inherited mutations in non-pore forming proteins of sodium channel complexes that cause cardiac arrhythmia, and the deduced mechanisms by which they affect function and dysfunction of the cardiac sodium current. Defining the structure and function of these complexes and how they are regulated will contribute to understanding the possible roles for this complex in normal and abnormal physiology and homeostasis. PMID:23557754

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

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

Shetty, Nishant D.; Reddy, Manchi C.M.; Palaninathan, Satheesh K.

2010-10-11

PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 {angstrom} resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T-loop ofmore » one subunit and the C-loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T-loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T-loop makes direct contact with the {gamma}-phosphate of the ATP molecule replacing the Mg{sup 2+} position seen in the Methanococcus jannaschii GlnK1 structure. The C-loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C-terminal 3{sub 10} helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.« less

The central element of the synaptonemal complex in mice is organized as a bilayered junction structure.

PubMed

Hernández-Hernández, Abrahan; Masich, Sergej; Fukuda, Tomoyuki; Kouznetsova, Anna; Sandin, Sara; Daneholt, Bertil; Höög, Christer

2016-06-01

The synaptonemal complex transiently stabilizes pairing interactions between homologous chromosomes during meiosis. Assembly of the synaptonemal complex is mediated through integration of opposing transverse filaments into a central element, a process that is poorly understood. We have, here, analyzed the localization of the transverse filament protein SYCP1 and the central element proteins SYCE1, SYCE2 and SYCE3 within the central region of the synaptonemal complex in mouse spermatocytes using immunoelectron microscopy. Distribution of immuno-gold particles in a lateral view of the synaptonemal complex, supported by protein interaction data, suggest that the N-terminal region of SYCP1 and SYCE3 form a joint bilayered central structure, and that SYCE1 and SYCE2 localize in between the two layers. We find that disruption of SYCE2 and TEX12 (a fourth central element protein) localization to the central element abolishes central alignment of the N-terminal region of SYCP1. Thus, our results show that all four central element proteins, in an interdependent manner, contribute to stabilization of opposing N-terminal regions of SYCP1, forming a bilayered transverse-filament-central-element junction structure that promotes synaptonemal complex formation and synapsis. © 2016. Published by The Company of Biologists Ltd.

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

PubMed

Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

2014-08-01

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes

PubMed Central

Bastiani, Michele; Liu, Libin; Hill, Michelle M.; Jedrychowski, Mark P.; Nixon, Susan J.; Lo, Harriet P.; Abankwa, Daniel; Luetterforst, Robert; Fernandez-Rojo, Manuel; Breen, Michael R.; Gygi, Steven P.; Vinten, Jorgen; Walser, Piers J.; North, Kathryn N.; Hancock, John F.; Pilch, Paul F.

2009-01-01

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein. PMID:19546242

WAVE2 Forms a Complex with PKA and Is Involved in PKA Enhancement of Membrane Protrusions*

PubMed Central

Yamashita, Hiroshi; Ueda, Kazumitsu; Kioka, Noriyuki

2011-01-01

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation. PMID:21119216

Recording information on protein complexes in an information management system

PubMed Central

Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

2011-01-01

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

N-cadherin in adult rat cardiomyocytes in culture. II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes.

PubMed

Hertig, C M; Butz, S; Koch, S; Eppenberger-Eberhardt, M; Kemler, R; Eppenberger, H M

1996-01-01

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The 'redifferentiation model' of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin which bind to either beta-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the following gap junction formation; a temporal sequence of the appearance of adherens junction proteins and of gap junctions forming connexin-43 is suggested.

Structure of human Fe-S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP-ISD11 interactions.

PubMed

Cory, Seth A; Van Vranken, Jonathan G; Brignole, Edward J; Patra, Shachin; Winge, Dennis R; Drennan, Catherine L; Rutter, Jared; Barondeau, David P

2017-07-03

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.

Spectral study and protein labeling of inclusion complex between dye and calixarene sulfonate.

PubMed

Fei, Xuening; Zhang, Yong; Zhu, Sen; Liu, Lijuan; Yu, Lu

2013-05-01

The host-guest inclusion complex of calix[6]arene sulfonate (SCA6) with thiazole orange (TO) formed in aqueous solution was studied. Absorption and fluorescence techniques were used for the analysis of this inclusion complex. The addition of calixarene sulfonate leads to a decrease in both absorption and fluorescence intensity of the dye, indicating that the inclusion complex was formed. Simultaneously, the inclusion phenomenon of another cyanine dye, Cy3, with calixarene sulfonate was investigated. The stability constant of the two complexes was determined, and the results were compared. The water solubility of TO dye was increased in the presence of calixarene sulfonate, and further protein labeling experiments suggested that this TO-SCA6 complex can act as a fluorescent probe for labeling of biomolecules.

A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion

DOE PAGES

Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko

2014-11-06

Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components thatmore » form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.« less

MFIB: a repository of protein complexes with mutual folding induced by binding.

PubMed

Fichó, Erzsébet; Reményi, István; Simon, István; Mészáros, Bálint

2017-11-15

It is commonplace that intrinsically disordered proteins (IDPs) are involved in crucial interactions in the living cell. However, the study of protein complexes formed exclusively by IDPs is hindered by the lack of data and such analyses remain sporadic. Systematic studies benefited other types of protein-protein interactions paving a way from basic science to therapeutics; yet these efforts require reliable datasets that are currently lacking for synergistically folding complexes of IDPs. Here we present the Mutual Folding Induced by Binding (MFIB) database, the first systematic collection of complexes formed exclusively by IDPs. MFIB contains an order of magnitude more data than any dataset used in corresponding studies and offers a wide coverage of known IDP complexes in terms of flexibility, oligomeric composition and protein function from all domains of life. The included complexes are grouped using a hierarchical classification and are complemented with structural and functional annotations. MFIB is backed by a firm development team and infrastructure, and together with possible future community collaboration it will provide the cornerstone for structural and functional studies of IDP complexes. MFIB is freely accessible at http://mfib.enzim.ttk.mta.hu/. The MFIB application is hosted by Apache web server and was implemented in PHP. To enrich querying features and to enhance backend performance a MySQL database was also created. simon.istvan@ttk.mta.hu, meszaros.balint@ttk.mta.hu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

Genetics Home Reference: celiac disease

MedlinePlus

... HLA-DQB1 genes belong to a family of genes called the human leukocyte antigen (HLA) complex . The HLA complex helps the immune system distinguish the body's own proteins from proteins made by foreign invaders such as ... and HLA-DQB1 genes attach (bind) to each other to form a ...

Unraveling the CHIP:Hsp70 complex as an information processor for protein quality control.

PubMed

VanPelt, Jamie; Page, Richard C

2017-02-01

The CHIP:Hsp70 complex stands at the crossroads of the cellular protein quality control system. Hsp70 facilitates active refolding of misfolded client proteins, while CHIP directs ubiquitination of misfolded client proteins bound to Hsp70. The direct competition between CHIP and Hsp70 for the fate of misfolded proteins leads to the question: how does the CHIP:Hsp70 complex execute triage decisions that direct misfolded proteins for either refolding or degradation? The current body of literature points toward action of the CHIP:Hsp70 complex as an information processor that takes inputs in the form of client folding state, dynamics, and posttranslational modifications, then outputs either refolded or ubiquitinated client proteins. Herein we examine the CHIP:Hsp70 complex beginning with the structure and function of CHIP and Hsp70, followed by an examination of recent studies of the interactions and dynamics of the CHIP:Hsp70 complex. Copyright © 2016 Elsevier B.V. All rights reserved.

Identifying protein complexes in PPI network using non-cooperative sequential game.

PubMed

Maulik, Ujjwal; Basu, Srinka; Ray, Sumanta

2017-08-21

Identifying protein complexes from protein-protein interaction (PPI) network is an important and challenging task in computational biology as it helps in better understanding of cellular mechanisms in various organisms. In this paper we propose a noncooperative sequential game based model for protein complex detection from PPI network. The key hypothesis is that protein complex formation is driven by mechanism that eventually optimizes the number of interactions within the complex leading to dense subgraph. The hypothesis is drawn from the observed network property named small world. The proposed multi-player game model translates the hypothesis into the game strategies. The Nash equilibrium of the game corresponds to a network partition where each protein either belong to a complex or form a singleton cluster. We further propose an algorithm to find the Nash equilibrium of the sequential game. The exhaustive experiment on synthetic benchmark and real life yeast networks evaluates the structural as well as biological significance of the network partitions.

Comparative analysis of activator-Eσ54 complexes formed with nucleotide-metal fluoride analogues

PubMed Central

Burrows, Patricia C.; Joly, Nicolas; Nixon, B. Tracy; Buck, Martin

2009-01-01

Bacterial RNA polymerase (RNAP) containing the major variant σ54 factor forms open promoter complexes in a reaction in which specialized activator proteins hydrolyse ATP. Here we probe binding interactions between σ54-RNAP (Eσ54) and the ATPases associated with various cellular activities (AAA+) domain of the Escherichia coli activator protein, PspF, using nucleotide-metal fluoride (BeF and AlF) analogues representing ground and transition states of ATP, which allow complexes (that are otherwise too transient with ATP) to be captured. We show that the organization and functionality of the ADP–BeF- and ADP–AlF-dependent complexes greatly overlap. Our data support an activation pathway in which the initial ATP-dependent binding of the activator to the Eσ54 closed complex results in the re-organization of Eσ54 with respect to the transcription start-site. However, the nucleotide-dependent binding interactions between the activator and the Eσ54 closed complex are in themselves insufficient for forming open promoter complexes when linear double-stranded DNA is present in the initial closed complex. PMID:19553192

Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles

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

Coleman, Matthew A.; Cappuccio, Jenny A.; Blanchette, Craig D.

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteinsmore » as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. Ultimately, these studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.« less

Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles

DOE PAGES

Coleman, Matthew A.; Cappuccio, Jenny A.; Blanchette, Craig D.; ...

2016-03-25

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteinsmore » as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. Ultimately, these studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.« less

Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals

DOEpatents

Kostic, Nenad M.; Chen, Jian

1991-03-05

Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme.

Applications of site-specific labeling to study HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.

PubMed

Mercer, Natalia; Ramakrishnan, Boopathy; Boeggeman, Elizabeth; Qasba, Pradman K

2011-01-01

Alpha-lactalbumin (α-LA) is a calcium-bound mammary gland-specific protein that is found in milk. This protein is a modulator of β1,4-galactosyltransferase enzyme, changing its acceptor specificity from N-acetyl-glucosamine to glucose, to produce lactose, milk's main carbohydrate. When calcium is removed from α-LA, it adopts a molten globule form, and this form, interestingly, when complexed with oleic acid (OA) acquires tumoricidal activity. Such a complex made from human α-LA (hLA) is known as HAMLET (Human A-lactalbumin Made Lethal to Tumor cells), and its tumoricidal activity has been well established. In the present work, we have used site-specific labeling, a technique previously developed in our laboratory, to label HAMLET with biotin, or a fluoroprobe for confocal microscopy studies. In addition to full length hLA, the α-domain of hLA (αD-hLA) alone is also included in the present study. We have engineered these proteins with a 17-amino acid C-terminal extension (hLA-ext and αD-hLA-ext). A single Thr residue in this extension is glycosylated with 2-acetonyl-galactose (C2-keto-galactose) using polypeptide-α-N-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules. We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity.

Integration of G protein α (Gα) signaling by the regulator of G protein signaling 14 (RGS14).

PubMed

Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose; Ramineni, Suneela; Ortlund, Eric A; Griffin, Patrick R; Hepler, John R

2015-04-03

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of amino acids that promote specific and rigid TAR RNA-tat protein complex formation.

PubMed

Edwards, Thomas E; Robinson, Bruce H; Sigurdsson, Snorri Th

2005-03-01

The Tat protein and the transactivation responsive (TAR) RNA form an essential complex in the HIV lifecycle, and mutations in the basic region of the Tat protein alter this RNA-protein molecular recognition. Here, EPR spectroscopy was used to identify amino acids, flanking an essential arginine of the Tat protein, which contribute to specific and rigid TAR-Tat complex formation by monitoring changes in the mobility of nitroxide spin-labeled TAR RNA nucleotides upon binding. Arginine to lysine N-terminal mutations did not affect TAR RNA interfacial dynamics. In contrast, C-terminal point mutations, R56 in particular, affected the mobility of nucleotides U23 and U38, which are involved in a base-triple interaction in the complex. This report highlights the role of dynamics in specific molecular complex formation and demonstrates the ability of EPR spectroscopy to study interfacial dynamics of macromolecular complexes.

HIV-1 Tat protein promotes formation of more-processive elongation complexes.

PubMed Central

Marciniak, R A; Sharp, P A

1991-01-01

The Tat protein of HIV-1 trans-activates transcription in vitro in a cell-free extract of HeLa nuclei. Quantitative analysis of the efficiency of elongation revealed that a majority of the elongation complexes generated by the HIV-1 promoter were not highly processive and terminated within the first 500 nucleotides. Tat trans-activation of transcription from the HIV-1 promoter resulted from an increase in processive character of the elongation complexes. More specifically, the analysis suggests that there exist two classes of elongation complexes initiating from the HIV promoter: a less-processive form and a more-processive form. Addition of purified Tat protein was found to increase the abundance of the more-processive class of elongation complex. The purine nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits transcription in this reaction by decreasing the efficiency of elongation. Surprisingly, stimulation of transcription elongation by Tat was preferentially inhibited by the addition of DRB. Images PMID:1756726

The LGI1–ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function

PubMed Central

Lovero, Kathryn L.; Fukata, Yuko; Granger, Adam J.; Fukata, Masaki; Nicoll, Roger A.

2015-01-01

Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95. PMID:26178195

A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

NASA Technical Reports Server (NTRS)

Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

1999-01-01

To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

Free-Energy Landscape of Protein-Ligand Interactions Coupled with Protein Structural Changes.

PubMed

Moritsugu, Kei; Terada, Tohru; Kidera, Akinori

2017-02-02

Protein-ligand interactions are frequently coupled with protein structural changes. Focusing on the coupling, we present the free-energy surface (FES) of the ligand-binding process for glutamine-binding protein (GlnBP) and its ligand, glutamine, in which glutamine binding accompanies large-scale domain closure. All-atom simulations were performed in explicit solvents by multiscale enhanced sampling (MSES), which adopts a multicopy and multiscale scheme to achieve enhanced sampling of systems with a large number of degrees of freedom. The structural ensemble derived from the MSES simulation yielded the FES of the coupling, described in terms of both the ligand's and protein's degrees of freedom at atomic resolution, and revealed the tight coupling between the two degrees of freedom. The derived FES led to the determination of definite structural states, which suggested the dominant pathways of glutamine binding to GlnBP: first, glutamine migrates via diffusion to form a dominant encounter complex with Arg75 on the large domain of GlnBP, through strong polar interactions. Subsequently, the closing motion of GlnBP occurs to form ligand interactions with the small domain, finally completing the native-specific complex structure. The formation of hydrogen bonds between glutamine and the small domain is considered to be a rate-limiting step, inducing desolvation of the protein-ligand interface to form the specific native complex. The key interactions to attain high specificity for glutamine, the "door keeper" existing between the two domains (Asp10-Lys115) and the "hydrophobic sandwich" formed between the ligand glutamine and Phe13/Phe50, have been successfully mapped on the pathway derived from the FES.

Stoichiometry for binding and transport by the twin arginine translocation system.

PubMed

Celedon, Jose M; Cline, Kenneth

2012-05-14

Twin arginine translocation (Tat) systems transport large folded proteins across sealed membranes. Tat systems accomplish this feat with three membrane components organized in two complexes. In thylakoid membranes, cpTatC and Hcf106 comprise a large receptor complex containing an estimated eight cpTatC-Hcf106 pairs. Protein transport occurs when Tha4 joins the receptor complex as an oligomer of uncertain size that is thought to form the protein-conducting structure. Here, binding analyses with intact membranes or purified complexes indicate that each receptor complex could bind eight precursor proteins. Kinetic analysis of translocation showed that each precursor-bound site was independently functional for transport, and, with sufficient Tha4, all sites were concurrently active for transport. Tha4 titration determined that ∼26 Tha4 protomers were required for transport of each OE17 (oxygen-evolving complex subunit of 17 kD) precursor protein. Our results suggest that, when fully saturated with precursor proteins and Tha4, the Tat translocase is an ∼2.2-megadalton complex that can individually transport eight precursor proteins or cooperatively transport multimeric precursors.

Structure of TatA Paralog, TatE, Suggests a Structurally Homogeneous Form of Tat Protein Translocase That Transports Folded Proteins of Differing Diameter

PubMed Central

Baglieri, Jacopo; Beck, Daniel; Vasisht, Nishi; Smith, Corinne J.; Robinson, Colin

2012-01-01

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ∼50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70–90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6–8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed. PMID:22190680

Molecular Investigations of the Structure and Function of the Protein Phosphatase 1:Spinophilin:Inhibitor-2 Heterotrimeric Complex

PubMed Central

Dancheck, Barbara; Ragusa, Michael J.; Allaire, Marc; Nairn, Angus C.; Page, Rebecca; Peti, Wolfgang

2011-01-01

Regulation of the major ser/thr phosphatase Protein Phosphatase 1 (PP1) is controlled by a diverse array of targeting and inhibitor proteins. Though many PP1 regulatory proteins share at least one PP1 binding motif, usually the RVxF motif, it was recently discovered that certain pairs of targeting and inhibitor proteins bind PP1 simultaneously to form PP1 heterotrimeric complexes. To date, structural information for these heterotrimeric complexes, and, in turn, how they direct PP1 activity is entirely lacking. Using a combination of NMR spectroscopy, biochemistry and small angle X-ray scattering (SAXS), we show that major structural rearrangements in both spinophilin (targeting) and Inhibitor-2 (I-2, inhibitor) are essential for the formation of the heterotrimeric PP1:spinophilin:I-2 (PSI) complex. The RVxF motif of I-2 is released from PP1 during the formation of PSI, making the less prevalent SILK motif of I-2 essential for complex stability. The release of the I-2 RVxF motif allows for enhanced flexibility of both I-2 and spinophilin in the heterotrimeric complex. In addition, we used inductively coupled plasma atomic emission spectroscopy to show that PP1 contains two metals in both heterodimeric complexes (PP1:spinophilin and PP1:I2) and PSI, demonstrating that PSI retains the biochemical characteristics of the PP1:I2 holoenzyme. Finally, we combined the NMR and biochemical data with SAXS and molecular dynamics simulations to generate a structural model of the full heterotrimeric PSI complex. Collectively, these data reveal the molecular events that enable PP1 heterotrimeric complexes to exploit both the targeting and inhibitory features of the PP1-regulatory proteins to form multi-functional PP1 holoenzymes. PMID:21218781

Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals

DOEpatents

Kostic, N.M.; Chen, J.

1991-03-05

Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme. No Drawings

Amyloid-like assembly of the low complexity domain of yeast Nab3.

PubMed

O'Rourke, Thomas W; Loya, Travis J; Head, PamelaSara E; Horton, John R; Reines, Daniel

2015-01-01

Termination of transcription of short non-coding RNAs is carried out in yeast by the Nab3-Nrd1-Sen1 complex. Nab3 and Nrd1 are hnRNP-like proteins that dimerize and bind RNA with sequence specificity. We show here that an essential region of Nab3 that is predicted to be prion-like based upon its sequence bias, formed amyloid-like filaments. A similar region from Nrd1 also assembled into filaments in vitro. The purified Nab3 domain formed a macroscopic gel whose lattice organization was observed by X-ray fiber diffraction. Filaments were resistant to dissociation in anionic detergent, bound the fluorescent dye thioflavin T, and showed a β-sheet rich structure by circular dichroism spectroscopy, similar to human amyloid β which served as a reference amyloid. A version of the Nab3 domain with a mutation that impairs its termination function, also formed fibers as observed by electron microscopy. Using a protein fragment interaction assay, the purified Nab3 domain was seen to interact with itself in living yeast. A similar observation was made for full length Nab3. These results suggest that the Nab3 and Nrd1 RNA-binding proteins can attain a complex polymeric form and raise the possibility that this property is important for organizing their functional state during termination. These findings are congruent with recent work showing that RNA binding proteins with low complexity domains form a dynamic subcellular matrix in which RNA metabolism takes place but can also aberrantly yield pathological aggregated particles.

Loss of Tsc1 in vivo impairs hippocampal mGluR-LTD and increases excitatory synaptic function.

PubMed

Bateup, Helen S; Takasaki, Kevin T; Saulnier, Jessica L; Denefrio, Cassandra L; Sabatini, Bernardo L

2011-06-15

The autism spectrum disorder tuberous sclerosis complex (TSC) is caused by mutations in the Tsc1 or Tsc2 genes, whose protein products form a heterodimeric complex that negatively regulates mammalian target of rapamycin-dependent protein translation. Although several forms of synaptic plasticity, including metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD), depend on protein translation at the time of induction, it is unknown whether these forms of plasticity require signaling through the Tsc1/2 complex. To examine this possibility, we postnatally deleted Tsc1 in vivo in a subset of hippocampal CA1 neurons using viral delivery of Cre recombinase in mice. We found that hippocampal mGluR-LTD was abolished by loss of Tsc1, whereas a protein synthesis-independent form of NMDA receptor-dependent LTD was preserved. Additionally, AMPA and NMDA receptor-mediated EPSCs and miniature spontaneous EPSC frequency were enhanced in Tsc1 KO neurons. These changes in synaptic function occurred in the absence of alterations in spine density, morphology, or presynaptic release probability. Our findings indicate that signaling through Tsc1/2 is required for the expression of specific forms of hippocampal synaptic plasticity as well as the maintenance of normal excitatory synaptic strength. Furthermore, these data suggest that perturbations of synaptic signaling may contribute to the pathogenesis of TSC.

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

PubMed

Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

2002-12-01

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

PubMed Central

Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

2002-01-01

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit. PMID:12515387

Structural and functional features of formate hydrogen lyase, an enzyme of mixed-acid fermentation from Escherichia coli.

PubMed

Bagramyan, K; Trchounian, A

2003-11-01

Formate hydrogen lyase from Escherichia coli is a membrane-bound complex that oxidizes formic acid to carbon dioxide and molecular hydrogen. Under anaerobic growth conditions and fermentation of sugars (glucose), it exists in two forms. One form is constituted by formate dehydrogenase H and hydrogenase 3, and the other one is the same formate dehydrogenase and hydrogenase 4; the presence of small protein subunits, carriers of electrons, is also probable. Other proteins may also be involved in formation of the enzyme complex, which requires the presence of metal (nickel-cobalt). Its formation also depends on the external pH and the presence of formate. Activity of both forms requires F(0)F(1)-ATPase; this explains dependence of the complex functioning on proton-motive force. It is also possible that the formate hydrogen lyase complex will exhibit its own proton-translocating function.

Molecular basis and quantitative assessment of TRF1 and TRF2 protein interactions with TIN2 and Apollo peptides.

PubMed

Kalathiya, Umesh; Padariya, Monikaben; Baginski, Maciej

2017-03-01

Shelterin is a six-protein complex (TRF1, TRF2, POT1, RAP1, TIN2, and TPP1) that also functions in smaller subsets in regulation and protection of human telomeres. Two closely related proteins, TRF1 and TRF2, make high-affinity contact directly with double-stranded telomeric DNA and serve as a molecular platform. Protein TIN2 binds to TRF1 and TRF2 dimer-forming domains, whereas Apollo makes interaction only with TRF2. To elucidate the molecular basis of these interactions, we employed molecular dynamics (MD) simulations of TRF1 TRFH -TIN2 TBM and TRF2 TRFH -TIN2 TBM /Apollo TBM complexes and of the isolated proteins. MD enabled a structural and dynamical comparison of protein-peptide complexes including H-bond interactions and interfacial residues that may regulate TRF protein binding to the given peptides, especially focusing on interactions described in crystallographic data. Residues with a selective function in both TRF1 TRFH and TRF2 TRFH and forming a stable hydrogen bond network with TIN2 TBM or Apollo TBM peptides were traced. Our study revealed that TIN2 TBM forms a well-defined binding mode with TRF1 TRFH as compared to TRF2 TRFH , and that the binding pocket of TIN2 TBM is deeper for TRF2 TRFH protein than Apollo TBM . The MD data provide a basis for the reinterpretation of mutational data obtained in crystallographic work for the TRF proteins. Together, the previously determined X-ray structure and our MD provide a detailed view of the TRF-peptide binding mode and the structure of TRF1/2 binding pockets. Particular TRF-peptide interactions are very specific for the formation of each protein-peptide complex, identifying TRF proteins as potential targets for the design of inhibitors/drugs modulating telomere machinery for anticancer therapy.

Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact.

PubMed

Heyduk, E; Baichoo, N; Heyduk, T

2001-11-30

The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence. To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization. The alpha dimer and DNA formed a 1:1 complex in solution. Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed. The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero. Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed.

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA

PubMed Central

Mori, Tetsuya; Saveliev, Sergei V.; Xu, Yao; Stafford, Walter F.; Cox, Michael M.; Inman, Ross B.; Johnson, Carl H.

2002-01-01

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecA/DnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecA/DnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns. PMID:12477935

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

PubMed

Mori, Tetsuya; Saveliev, Sergei V; Xu, Yao; Stafford, Walter F; Cox, Michael M; Inman, Ross B; Johnson, Carl H

2002-12-24

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

PubMed Central

Lloyd, Julie C.; Raines, Christine A.

2011-01-01

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein–protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the ‘non-regulatory’ A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed. PMID:21498632

Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex.

PubMed

Ortega-Roldan, Jose Luis; Jensen, Malene Ringkjøbing; Brutscher, Bernhard; Azuaga, Ana I; Blackledge, Martin; van Nuland, Nico A J

2009-05-01

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 muM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (K(d) = 132 +/- 13 muM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein-protein complexes.

New procyanidin B3-human salivary protein complexes by mass spectrometry. Effect of salivary protein profile, tannin concentration, and time stability.

PubMed

Perez-Gregorio, Maria Rosa; Mateus, Nuno; De Freitas, Victor

2014-10-15

Several factors could influence the tannin-protein interaction such as the human salivary protein profile, the tannin tested, and the tannin/protein ratio. The goal of this study aims to study the effect of different salivas (A, B, and C) and different tannin concentrations (0.5 and 1 mg/mL) on the interaction process as well as the complex's stability over time. This study is focused on the identification of new procyanidin B3-human salivary protein complexes. Thus, 48 major B3-human salivary protein aggregates were identified regardless of the saliva and tannin concentration tested. A higher number of aggregates was found at lower tannin concentration. Moreover, the number of protein moieties involved in the aggregation process was higher when the tannin concentration was also higher. The selectivity of the different groups of proteins to bind tannin was also confirmed. It was also verified that the B3-human salivary protein complexes formed evolved over time.

Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

PubMed

Shen, Xianjun; Yi, Li; Jiang, Xingpeng; He, Tingting; Yang, Jincai; Xie, Wei; Hu, Po; Hu, Xiaohua

2017-01-01

How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI) data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment). It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte Complex Micelle.

PubMed

Wu, Fu-Gen; Jiang, Yao-Wen; Chen, Zhan; Yu, Zhi-Wu

2016-04-19

The folding/unfolding behavior of proteins (enzymes) in confined space is important for their properties and functions, but such a behavior remains largely unexplored. In this article, we reported our finding that lysozyme and a double hydrophilic block copolymer, methoxypoly(ethylene glycol)5K-block-poly(l-aspartic acid sodium salt)10 (mPEG(5K)-b-PLD10), can form a polyelectrolyte complex micelle with a particle size of ∼30 nm, as verified by dynamic light scattering and transmission electron microscopy. The unfolding and refolding behaviors of lysozyme molecules in the presence of the copolymer were studied by microcalorimetry and circular dichroism spectroscopy. Upon complex formation with mPEG(5K)-b-PLD10, lysozyme changed from its initial native state to a new partially unfolded state. Compared with its native state, this copolymer-complexed new folding state of lysozyme has different secondary and tertiary structures, a decreased thermostability, and significantly altered unfolding/refolding behaviors. It was found that the native lysozyme exhibited reversible unfolding and refolding upon heating and subsequent cooling, while lysozyme in the new folding state (complexed with the oppositely charged PLD segments of the polymer) could unfold upon heating but could not refold upon subsequent cooling. By employing the heating-cooling-reheating procedure, the prevention of complex formation between lysozyme and polymer due to the salt screening effect was observed, and the resulting uncomplexed lysozyme regained its proper unfolding and refolding abilities upon heating and subsequent cooling. Besides, we also pointed out the important role the length of the PLD segment played during the formation of micelles and the monodispersity of the formed micelles. Furthermore, the lysozyme-mPEG(5K)-b-PLD10 mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates in solution as frequently observed in other protein-polyelectrolyte systems. Hence, the present protein-PEGylated poly(amino acid) mixture provides an ideal water-soluble model system to study the important role of electrostatic interaction in the complexation between proteins and polymers, leading to important new knowledge on the protein-polymer interactions. Moreover, the polyelectrolyte complex micelle formed between protein and PEGylated polymer may provide a good drug delivery vehicle for therapeutic proteins.

Structural Study of the RIPoptosome Core Reveals a Helical Assembly for Kinase Recruitment

PubMed Central

2015-01-01

Receptor interaction protein kinase 1 (RIP1) is a molecular cell-fate switch. RIP1, together with Fas-associated protein with death domain (FADD) and caspase-8, forms the RIPoptosome that activates apoptosis. RIP1 also associates with RIP3 to form the necrosome that triggers necroptosis. The RIPoptosome assembles through interactions between the death domains (DDs) of RIP1 and FADD and between death effector domains (DEDs) of FADD and caspase-8. In this study, we analyzed the overall structure of the RIP1 DD/FADD DD complex, the core of the RIPoptosome, by negative-stain electron microscopy and modeling. The results show that RIP1 DD and FADD DD form a stable complex in vitro similar to the previously described Fas DD/FADD DD complex, suggesting that the RIPoptosome and the Fas death-inducing signaling complex share a common assembly mechanism. Both complexes adopt a helical conformation that requires type I, II, and III interactions between the death domains. PMID:25119434

Psp Stress Response Proteins Form a Complex with Mislocalized Secretins in the Yersinia enterocolitica Cytoplasmic Membrane.

PubMed

Srivastava, Disha; Moumene, Amal; Flores-Kim, Josué; Darwin, Andrew J

2017-09-12

The bacterial phage shock protein system (Psp) is a conserved extracytoplasmic stress response that is essential for the virulence of some pathogens, including Yersinia enterocolitica It is induced by events that can compromise inner membrane (IM) integrity, including the mislocalization of outer membrane pore-forming proteins called secretins. In the absence of the Psp system, secretin mislocalization permeabilizes the IM and causes rapid cell death. The Psp proteins PspB and PspC form an integral IM complex with two independent roles. First, the PspBC complex is required to activate the Psp response in response to some inducing triggers, including a mislocalized secretin. Second, PspBC are sufficient to counteract mislocalized secretin toxicity. Remarkably, secretin mislocalization into the IM induces psp gene expression without significantly affecting the expression of any other genes. Furthermore, psp null strains are killed by mislocalized secretins, whereas no other null mutants have been found to share this specific secretin sensitivity. This suggests an exquisitely specific relationship between secretins and the Psp system, but there has been no mechanism described to explain this. In this study, we addressed this deficiency by using a coimmunoprecipitation approach to show that the Psp proteins form a specific complex with mislocalized secretins in the Y. enterocolitica IM. Importantly, analysis of different secretin mutant proteins also revealed that this interaction is absolutely dependent on a secretin adopting a multimeric state. Therefore, the Psp system has evolved with the ability to detect and detoxify dangerous secretin multimers while ignoring the presence of innocuous monomers. IMPORTANCE The phage shock protein (Psp) response has been linked to important phenotypes in diverse bacteria, including those related to antibiotic resistance, biofilm formation, and virulence. This has generated widespread interest in understanding various aspects of its function. Outer membrane secretin proteins are essential components of export systems required for the virulence of many bacterial pathogens. However, secretins can mislocalize into the inner membrane, and this induces the Psp response in a highly specific manner and kills Psp-defective strains with similar specificity. There has been no mechanism described to explain this exquisitely specific relationship between secretins and the Psp system. Therefore, this study provides a critical advance by discovering that Psp effector proteins form a complex with secretins in the Yersinia enterocolitica inner membrane. Remarkably, this interaction is absolutely dependent on a secretin adopting its multimeric state. Therefore, the Psp system detects and detoxifies dangerous secretin multimers, while ignoring the presence of innocuous secretin monomers. Copyright © 2017 Srivastava et al.

Crystal Structure of an L-Carnitine Complex with Pyrogallol[4]arene

NASA Astrophysics Data System (ADS)

Fujisawa, I.; Takeuchi, D.; Kitamura, Y.; Okamoto, R.; Aoki, K.

2012-03-01

L-Carnitine is essential for the transport of long-chain fatty acids from cytosol into mitochondria for generating metabolic energy. The survey of crystal structures of carnitine-containing proteins in the Protein Data Bank reveals that carnitine can take several conformations with the quarternary trimethylammonium terminal being always bound to aromatic residues through cation-π interactions in acyltransferases or carnitine-binding proteins. In order to demonstrate the importance of cation-π interaction as a carnitine recognition mechanism in the artificial receptor-ligand system that mimics the carnitine-binding sites, we have determined the crystal structure of a complex formed between L-carnitine and pyrogallol[4]arene (pyrogallol cyclic tetramer: PCT) as a carnitine receptor, 2PCT·2(L-carnitine)·4EtOH. There form two crystallographically independent monomeric [PCT·L-carnitine] substructures, which further form an obliquely arranged capsule-like dimeric [PCT·L-carnitine]2 structure through a pair of O-H (PCT)···O (L-carnitine) hydrogen bonds. This is the first report of PCT complex with chiral molecules. In each of the two monomeric [PCT·L-carnitine] substructures, the L-carnitine molecule takes the elongated form with an intramolecular hydrogen bond between the hydroxyl group and the carboxylate oxygen, and the cationic trimethylammonium moiety is incorporated into the cavity of the bowl-shaped PCT molecule through cation-π interactions. These features are similar to those at the D-carnitine-binding site in the crystal structure of the glycine betaine/carnitine/choline-binding protein complex.

Sample preparation for SFM imaging of DNA, proteins, and DNA-protein complexes.

PubMed

Ristic, Dejan; Sanchez, Humberto; Wyman, Claire

2011-01-01

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate, and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nanometer resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA-bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA, and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

Trypanosoma brucei RNA Editing Complex

PubMed Central

O'Hearn, Sean F.; Huang, Catherine E.; Hemann, Mike; Zhelonkina, Alevtina; Sollner-Webb, Barbara

2003-01-01

Maturation of Trypanosoma brucei mitochondrial mRNA involves massive posttranscriptional insertion and deletion of uridine residues. This RNA editing utilizes an enzymatic complex with seven major proteins, band I through band VII. We here use RNA interference (RNAi) to examine the band II and band V proteins. Band II is found essential for viability; it is needed to maintain the normal structure of the editing complex and to retain the band V ligase protein. Previously, band III was found essential for certain activities, including maintenance of the editing complex and retention of the band IV ligase protein. Thus, band II and band V form a protein pair with features analogous to the band III/band IV ligase pair. Since band V is specific for U insertion and since band IV is needed for U deletion, their parallel organization suggests that the editing complex has a pseudosymmetry. However, unlike the essential band IV ligase, RNAi to band V has only a morphological but no growth rate effect, suggesting that it is stimulatory but nonessential. Indeed, in vitro analysis of band V RNAi cell extract demonstrates that band IV can seal U insertion when band V is lacking. Thus, band IV ligase is the first activity of the basic editing complex shown able to serve in both forms of editing. Our studies also indicate that the U insertional portion may be less central in the editing complex than the corresponding U deletional portion. PMID:14560033

The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response

PubMed Central

Cicconi, Alessandro; Micheli, Emanuela; Vernì, Fiammetta; Jackson, Alison; Gradilla, Ana Citlali; Cipressa, Francesca; Raimondo, Domenico; Bosso, Giuseppe; Wakefield, James G.; Ciapponi, Laura; Cenci, Giovanni; Gatti, Maurizio

2017-01-01

Abstract Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin. PMID:27940556

Were protein internal repeats formed by "bricolage"?

PubMed

Lavorgna, G; Patthy, L; Boncinelli, E

2001-03-01

Is evolution an engineer, or is it a tinkerer--a "bricoleur"--building up complex molecules in organisms by increasing and adapting the materials at hand? An analysis of completely sequenced genomes suggests the latter, showing that increasing repetition of modules within the proteins encoded by these genomes is correlated with increasing complexity of the organism.

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis

PubMed Central

2018-01-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that ‘leftover’ proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles. PMID:29518071

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis.

PubMed

Holland, David O; Johnson, Margaret E

2018-03-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that 'leftover' proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles.

Short-Chain 3-Hydroxyacyl-Coenzyme A Dehydrogenase Associates with a Protein Super-Complex Integrating Multiple Metabolic Pathways

PubMed Central

Narayan, Srinivas B.; Master, Stephen R.; Sireci, Anthony N.; Bierl, Charlene; Stanley, Paige E.; Li, Changhong; Stanley, Charles A.; Bennett, Michael J.

2012-01-01

Proteins involved in mitochondrial metabolic pathways engage in functionally relevant multi-enzyme complexes. We previously described an interaction between short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) and glutamate dehydrogenase (GDH) explaining the clinical phenotype of hyperinsulinism in SCHAD-deficient patients and adding SCHAD to the list of mitochondrial proteins capable of forming functional, multi-pathway complexes. In this work, we provide evidence of SCHAD's involvement in additional interactions forming tissue-specific metabolic super complexes involving both membrane-associated and matrix-dwelling enzymes and spanning multiple metabolic pathways. As an example, in murine liver, we find SCHAD interaction with aspartate transaminase (AST) and GDH from amino acid metabolic pathways, carbamoyl phosphate synthase I (CPS-1) from ureagenesis, other fatty acid oxidation and ketogenesis enzymes and fructose-bisphosphate aldolase, an extra-mitochondrial enzyme of the glycolytic pathway. Most of the interactions appear to be independent of SCHAD's role in the penultimate step of fatty acid oxidation suggesting an organizational, structural or non-enzymatic role for the SCHAD protein. PMID:22496890

A Method for Visualization of Incoming Adenovirus Chromatin Complexes in Fixed and Living Cells

PubMed Central

Komatsu, Tetsuro; Dacheux, Denis; Kreppel, Florian; Nagata, Kyosuke; Wodrich, Harald

2015-01-01

Inside the adenovirus virion, the genome forms a chromatin-like structure with viral basic core proteins. Core protein VII is the major DNA binding protein and was shown to remain associated with viral genomes upon virus entry even after nuclear delivery. It has been suggested that protein VII plays a regulatory role in viral gene expression and is a functional component of viral chromatin complexes in host cells. As such, protein VII could be used as a maker to track adenoviral chromatin complexes in vivo. In this study, we characterize a new monoclonal antibody against protein VII that stains incoming viral chromatin complexes following nuclear import. Furthermore, we describe the development of a novel imaging system that uses Template Activating Factor-I (TAF-I/SET), a cellular chromatin protein tightly bound to protein VII upon infection. This setup allows us not only to rapidly visualize protein VII foci in fixed cells but also to monitor their movement in living cells. These powerful tools can provide novel insights into the spatio-temporal regulation of incoming adenoviral chromatin complexes. PMID:26332038

Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative analysis by electron microscopy.

PubMed

Giss, Dominic; Kemmerling, Simon; Dandey, Venkata; Stahlberg, Henning; Braun, Thomas

2014-05-20

Multimolecular protein complexes are important for many cellular processes. However, the stochastic nature of the cellular interactome makes the experimental detection of complex protein assemblies difficult and quantitative analysis at the single molecule level essential. Here, we present a fast and simple microfluidic method for (i) the quantitative isolation of endogenous levels of untagged protein complexes from minute volumes of cell lysates under close to physiological conditions and (ii) the labeling of specific components constituting these complexes. The method presented uses specific antibodies that are conjugated via a photocleavable linker to magnetic beads that are trapped in microcapillaries to immobilize the target proteins. Proteins are released by photocleavage, eluted, and subsequently analyzed by quantitative transmission electron microscopy at the single molecule level. Additionally, before photocleavage, immunogold can be employed to label proteins that interact with the primary target protein. Thus, the presented method provides a new way to study the interactome and, in combination with single molecule transmission electron microscopy, to structurally characterize the large, dynamic, heterogeneous multimolecular protein complexes formed.

Modifications in structure and interaction of nanoparticle-protein-surfactant complexes in electrolyte solution

NASA Astrophysics Data System (ADS)

Mehan, Sumit; Kumar, S.; Aswal, V. K.; Schweins, R.

2016-05-01

SANS experiments of three-component system of anionic silica nanoparticles, anionic BSA protein and anionic SDS surfactants have been carried out without and with electrolyte in aqueous solution. In both the cases, the interaction of surfactant with protein results in formation of bead-necklace structure of protein-surfactant complexes in solution. These protein-surfactant complexes interact very differently with nanoparticles in absence and presence of electrolyte. In absence of electrolyte, nanoparticles remain in dispersed phase in solution, whereas with the addition of electrolyte the nanoparticles fractal aggregates are formed. SANS describes the phase behavior to be governed by competition of electrostatic and depletion interactions among the components solution.

Reducing the allergenic capacity of peanut extracts and liquid peanut butter by phenolic compounds.

USDA-ARS?s Scientific Manuscript database

Phenolic compounds are known to form soluble and insoluble complexes with proteins. The objective of this study was to determine if phenolics, such as, caffeic, chlorogenic, and ferulic acids form insoluble and irreversible complexes with major peanut allergens. We also tested whether such complexat...

Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3

PubMed Central

Meng, He; Zhang, Xiaojie; Yu, Genggeng; Lee, Soo Jung; Chen, Y. Eugene; Prudovsky, Igor; Wang, Michael M.

2012-01-01

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the best understood cause of dominantly inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein accumulation and selective arterial smooth muscle degeneration. Previous studies show that NOTCH3 protein forms multimers. Here, we investigate protein interactions between NOTCH3 and other vascular Notch isoforms and characterize the effects of elevated NOTCH3 on smooth muscle gene regulation. We demonstrate that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y mutant NOTCH3 form complexes which are more resistant to detergents than wild type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we found significant inhibition of mutant NOTCH3 clearance. In coculture assays of NOTCH function, overexpressed wild type and mutant NOTCH3 significantly repressed NOTCH-regulated smooth muscle transcripts and potently impaired the activity of three independent smooth muscle promoters. Wildtype and R90C recombinant NOTCH3 proteins applied to cell cultures also blocked canonical Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle cells. PMID:23028706

The fanconi anemia proteins FANCA and FANCG stabilize each other and promote the nuclear accumulation of the Fanconi anemia complex.

PubMed

Garcia-Higuera, I; Kuang, Y; Denham, J; D'Andrea, A D

2000-11-01

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with 8 complementation groups. Four of the FA genes have been cloned, and at least 3 of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a multisubunit protein complex. The FANCG protein binds directly to the amino terminal nuclear localization sequence (NLS) of FANCA, suggesting that FANCG plays a role in regulating FANCA nuclear accumulation. In the current study the functional consequences of FANCG/FANCA binding were examined. Correction of an FA-G cell line with the FANCG complementary DNA (cDNA) resulted in FANCA/FANCG binding, prolongation of the cellular half-life of FANCA, and an increase in the nuclear accumulation of the FA protein complex. Similar results were obtained upon correction of an FA-A cell line, with a reciprocal increase in the half-life of FANCG. Patient-derived mutant forms of FANCA, containing an intact NLS sequence but point mutations in the carboxy-terminal leucine zipper region, bound FANCG in the cytoplasm. The mutant forms failed to translocate to the nucleus of transduced cells, thereby suggesting a model of coordinated binding and nuclear translocation. These results demonstrate that the FANCA/FANCG interaction is required to maintain the cellular levels of both proteins. Moreover, at least one function of FANCG and FANCA is to regulate the nuclear accumulation of the FA protein complex. Failure to accumulate the nuclear FA protein complex results in the characteristic spectrum of clinical and cellular abnormalities observed in FA.

High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

PubMed Central

2012-01-01

Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

Protein-Protein Interactions in the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL from Azotobacter vinelandii

PubMed Central

Money, Tracy; Barrett, Jason; Dixon, Ray; Austin, Sara

2001-01-01

The enhancer binding protein NIFA and the sensor protein NIFL from Azotobacter vinelandii comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. Using limited proteolysis of the purified proteins, we show here that changes in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred when the complex was formed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protected by NIFL. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are not detectable by cochromatography. PMID:11157949

Biological and Pathological Implications of an Alternative ATP-Powered Proteasomal Assembly With Cdc48 and the 20S Peptidase.

PubMed

Esaki, Masatoshi; Johjima-Murata, Ai; Islam, Md Tanvir; Ogura, Teru

2018-01-01

The ATP-powered protein degradation machinery plays essential roles in maintaining protein homeostasis in all organisms. Robust proteolytic activities are typically sequestered within protein complexes to avoid the fatal removal of essential proteins. Because the openings of proteolytic chambers are narrow, substrate proteins must undergo unfolding. AAA superfamily proteins (ATPases associated with diverse cellular activities) are mostly located at these openings and regulate protein degradation appropriately. The 26S proteasome, comprising 20S peptidase and 19S regulatory particles, is the major ATP-powered protein degradation machinery in eukaryotes. The 19S particles are composed of six AAA proteins and 13 regulatory proteins, and bind to both ends of a barrel-shaped proteolytic chamber formed by the 20S peptidase. Several recent studies have reported that another AAA protein, Cdc48, can replace the 19S particles to form an alternative ATP-powered proteasomal complex, i.e., the Cdc48-20S proteasome. This review focuses on our current knowledge of this alternative proteasome and its possible linkage to amyotrophic lateral sclerosis.

Solution structure of the phosphoryl transfer complex between the cytoplasmic A domain of the mannitol transporter IIMannitol and HPr of the Escherichia coli phosphotransferase system.

PubMed

Cornilescu, Gabriel; Lee, Byeong Ryong; Cornilescu, Claudia C; Wang, Guangshun; Peterkofsky, Alan; Clore, G Marius

2002-11-01

The solution structure of the complex between the cytoplasmic A domain (IIA(Mtl)) of the mannitol transporter II(Mannitol) and the histidine-containing phosphocarrier protein (HPr) of the Escherichia coli phosphotransferase system has been solved by NMR, including the use of conjoined rigid body/torsion angle dynamics, and residual dipolar couplings, coupled with cross-validation, to permit accurate orientation of the two proteins. A convex surface on HPr, formed by helices 1 and 2, interacts with a complementary concave depression on the surface of IIA(Mtl) formed by helix 3, portions of helices 2 and 4, and beta-strands 2 and 3. The majority of intermolecular contacts are hydrophobic, with a small number of electrostatic interactions at the periphery of the interface. The active site histidines, His-15 of HPr and His-65 of IIA(Mtl), are in close spatial proximity, and a pentacoordinate phosphoryl transition state can be readily accommodated with no change in protein-protein orientation and only minimal perturbations of the backbone immediately adjacent to the histidines. Comparison with two previously solved structures of complexes of HPr with partner proteins of the phosphotransferase system, the N-terminal domain of enzyme I (EIN) and enzyme IIA(Glucose) (IIA(Glc)), reveals a number of common features despite the fact that EIN, IIA(Glc), and IIA(Mtl) bear no structural resemblance to one another. Thus, entirely different underlying structural elements can form binding surfaces for HPr that are similar in terms of both shape and residue composition. These structural comparisons illustrate the roles of surface and residue complementarity, redundancy, incremental build-up of specificity and conformational side chain plasticity in the formation of transient specific protein-protein complexes in signal transduction pathways.

Surfactant titration of nanoparticle-protein corona.

PubMed

Maiolo, Daniele; Bergese, Paolo; Mahon, Eugene; Dawson, Kenneth A; Monopoli, Marco P

2014-12-16

Nanoparticles (NP), when exposed to biological fluids, are coated by specific proteins that form the so-called protein corona. While some adsorbing proteins exchange with the surroundings on a short time scale, described as a "dynamic" corona, others with higher affinity and long-lived interaction with the NP surface form a "hard" corona (HC), which is believed to mediate NP interaction with cellular machineries. In-depth NP protein corona characterization is therefore a necessary step in understanding the relationship between surface layer structure and biological outcomes. In the present work, we evaluate the protein composition and stability over time and we systematically challenge the formed complexes with surfactants. Each challenge is characterized through different physicochemical measurements (dynamic light scattering, ζ-potential, and differential centrifugal sedimentation) alongside proteomic evaluation in titration type experiments (surfactant titration). 100 nm silicon oxide (Si) and 100 nm carboxylated polystyrene (PS-COOH) NPs cloaked by human plasma HC were titrated with 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, zwitterionic), Triton X-100 (nonionic), sodium dodecyl sulfate (SDS, anionic), and dodecyltrimethylammonium bromide (DTAB, cationic) surfactants. Composition and density of HC together with size and ζ-potential of NP-HC complexes were tracked at each step after surfactant titration. Results on Si NP-HC complexes showed that SDS removes most of the HC, while DTAB induces NP agglomeration. Analogous results were obtained for PS NP-HC complexes. Interestingly, CHAPS and Triton X-100, thanks to similar surface binding preferences, enable selective extraction of apolipoprotein AI (ApoAI) from Si NP hard coronas, leaving unaltered the dispersion physicochemical properties. These findings indicate that surfactant titration can enable the study of NP-HC stability through surfactant variation and also selective separation of certain proteins from the HC. This approach thus has an immediate analytical value as well as potential applications in HC engineering.

Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence.

PubMed Central

Slama-Schwok, A; Zakrzewska, K; Léger, G; Leroux, Y; Takahashi, M; Käs, E; Debey, P

2000-01-01

Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation. PMID:10777751

A nacre protein forms mesoscale hydrogels that “hijack” the biomineralization process within a seawater environment

DOE PAGES

Pendola, Martin; Jain, Gaurav; Davidyants, Anastasia; ...

2016-09-26

We examined the mineralization performance of a nacre protein, AP7, within seawater mineralization assays that form aragonite and magnesium calcite. Under these conditions AP7 forms hydrogel particles that vary in size and complexity depending upon ionic conditions. These hydrogels “hijack” the mineralization process by limiting nucleation in bulk solution and promoting nucleation within the hydrogels.

The nanoparticle protein corona formed in human blood or human blood fractions.

PubMed

Lundqvist, Martin; Augustsson, Cecilia; Lilja, Malin; Lundkvist, Kristoffer; Dahlbäck, Björn; Linse, Sara; Cedervall, Tommy

2017-01-01

The protein corona formed around nanoparticles in protein-rich fluids plays an important role for nanoparticle biocompatibility, as found in several studies during the last decade. Biological fluids have complex compositions and the molecular components interact and function together in intricate networks. Therefore, the process to isolate blood or the preparation of blood derivatives may lead to differences in the composition of the identified protein corona around nanoparticles. Here, we show distinct differences in the protein corona formed in whole blood, whole blood with EDTA, plasma, or serum. Furthermore, the ratio between particle surface area to protein concentration influences the detected corona. We also show that the nanoparticle size per se influences the formed protein corona due to curvature effects. These results emphasize the need of investigating the formation and biological importance of the protein corona in the same environment as the nanoparticles are intended for or released into.

Concerted formation of macromolecular Suppressor–mutator transposition complexes

PubMed Central

Raina, Ramesh; Schläppi, Michael; Karunanandaa, Balasulojini; Elhofy, Adam; Fedoroff, Nina

1998-01-01

Transposition of the maize Suppressor–mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA–DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein–protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range. PMID:9671711

Contribution of Human Oral Cells to Astringency by Binding Salivary Protein/Tannin Complexes.

PubMed

Soares, Susana; Ferrer-Galego, Raúl; Brandão, Elsa; Silva, Mafalda; Mateus, Nuno; Freitas, Victor de

2016-10-10

The most widely accepted mechanism to explain astringency is the interaction and precipitation of salivary proteins by food tannins, in particular proline-rich proteins. However, other mechanisms have been arising to explain astringency, such as binding of tannins to oral cells. In this work, an experimental method was adapted to study the possible contribution of both salivary proteins and oral cells to astringency induced by grape seed procyanidin fractions. Overall, in the absence of salivary proteins, the extent of procyanidin complexation with oral cells increased with increasing procyanidin degree of polymerization (mDP). Procyanidin fractions rich in monomers were the ones with the lowest ability to bind to oral cells. In the presence of salivary proteins and for procyanidins with mDP 2 the highest concentrations (1.5 and 2.0 mM) resulted in an increased binding of procyanidins to oral cells. This was even more evident for fractions III and IV at 1.0 mM and upper concentrations. Regarding the salivary proteins affected, it was possible to observe a decrease of P-B peptide and aPRP proteins for fractions II and III. This decrease is greater as the procyanidins' mDP increases. In fact, for fraction IV an almost total depletion of all salivary proteins was observed. This decrease is due to the formation of insoluble salivary protein/procyanidin complexes. Altogether, these data suggest that some procyanidins are able to bind to oral cells and that the salivary proteins interact with procyanidins forming salivary protein/procyanidin complexes that are also able to link to oral cells. The procyanidins that remain unbound to oral cells are able to bind to salivary proteins forming a large network of salivary protein/procyanidin complexes. Overall, the results presented herein provide one more step to understand food oral astringency onset.

Crystallization of Mitochondrial Respiratory Complex II from Chicken Heart: a Membrane Protein Complex Diffracting to 2.0 Å.

PubMed Central

Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen; Berry, Edward

2006-01-01

Synopsis A multi-subunit mitochondrial membrane protein complex involved in the Krebs Cycle and respiratory chain has been crystallized in a form suitable for near-atomic resolution structure determination. A procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Å with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites. PMID:15805592

Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs.

PubMed

Navarro, Gemma; Cordomí, Arnau; Zelman-Femiak, Monika; Brugarolas, Marc; Moreno, Estefania; Aguinaga, David; Perez-Benito, Laura; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Pardo, Leonardo; García-Sáez, Ana J; McCormick, Peter J; Franco, Rafael

2016-04-05

G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.

A novel method for identifying disease associated protein complexes based on functional similarity protein complex networks.

PubMed

Le, Duc-Hau

2015-01-01

Protein complexes formed by non-covalent interaction among proteins play important roles in cellular functions. Computational and purification methods have been used to identify many protein complexes and their cellular functions. However, their roles in terms of causing disease have not been well discovered yet. There exist only a few studies for the identification of disease-associated protein complexes. However, they mostly utilize complicated heterogeneous networks which are constructed based on an out-of-date database of phenotype similarity network collected from literature. In addition, they only apply for diseases for which tissue-specific data exist. In this study, we propose a method to identify novel disease-protein complex associations. First, we introduce a framework to construct functional similarity protein complex networks where two protein complexes are functionally connected by either shared protein elements, shared annotating GO terms or based on protein interactions between elements in each protein complex. Second, we propose a simple but effective neighborhood-based algorithm, which yields a local similarity measure, to rank disease candidate protein complexes. Comparing the predictive performance of our proposed algorithm with that of two state-of-the-art network propagation algorithms including one we used in our previous study, we found that it performed statistically significantly better than that of these two algorithms for all the constructed functional similarity protein complex networks. In addition, it ran about 32 times faster than these two algorithms. Moreover, our proposed method always achieved high performance in terms of AUC values irrespective of the ways to construct the functional similarity protein complex networks and the used algorithms. The performance of our method was also higher than that reported in some existing methods which were based on complicated heterogeneous networks. Finally, we also tested our method with prostate cancer and selected the top 100 highly ranked candidate protein complexes. Interestingly, 69 of them were evidenced since at least one of their protein elements are known to be associated with prostate cancer. Our proposed method, including the framework to construct functional similarity protein complex networks and the neighborhood-based algorithm on these networks, could be used for identification of novel disease-protein complex associations.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

PubMed Central

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

2015-01-01

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

Interaction of acute-phase-inducible and liver-enriched nuclear factors with the promoter region of the mouse alpha sub 1 -acid glycoprotein gene-1

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

Alam, T.; Papaconstantinou, J.

1992-02-25

The synthesis and secretion of several acute-phase proteins increases markedly following physiological stress. {alpha}{sub 1}-Acid glycoprotein (AGP), a major acute-phase reactant made by the liver, is strongly induced by inflammatory agents such as lipopolysaccharide (LPS). Nuclear run-on assay showed a 17-fold increase in the rate of AGP transcription 4 h following LPS injection. DNase I footprinting assays revealed multiple protein binding domains in the mouse AGP-1 promoter region. Region B ({minus}104 to {minus}91) is protected by a liver-enriched transcription factor that is heat labile and in limiting quantity. An adjacent region, C ({minus}125 to {minus}104), is well-protected by nuclear extractsmore » from hepatocytes. Electrophoretic mobility shift assays indicated that only one DNA-protein complex can form with an oligonucleotide corresponding to region B. However, nuclear proteins from untreated mouse liver can form three strong complexes (C1, C2, and C3) and a weak one (C4) with oligonucleotide C. An acute-phase-inducible DNA-binding protein (AP-DBP) forms complex 4. A dramatic increase (over 11-fold) in AP-DBP binding activity is seen with nuclear proteins from LPS-stimulated animals. Interestingly, AP-DBP, a heat-stable factor, can form heterodimers with the transcription factor CCAAT/enhancer binding protein (C/EBP). Furthermore, purified C/EBP also binds avidly to region C. The studies indicate that several liver-enriched nuclear factors can interact with AGP-1 promoter and that AP-DBP binds to the AGP-1 promoter with high affinity only during the acute-phase induction.« less

Discovering functional interdependence relationship in PPI networks for protein complex identification.

PubMed

Lam, Winnie W M; Chan, Keith C C

2012-04-01

Protein molecules interact with each other in protein complexes to perform many vital functions, and different computational techniques have been developed to identify protein complexes in protein-protein interaction (PPI) networks. These techniques are developed to search for subgraphs of high connectivity in PPI networks under the assumption that the proteins in a protein complex are highly interconnected. While these techniques have been shown to be quite effective, it is also possible that the matching rate between the protein complexes they discover and those that are previously determined experimentally be relatively low and the "false-alarm" rate can be relatively high. This is especially the case when the assumption of proteins in protein complexes being more highly interconnected be relatively invalid. To increase the matching rate and reduce the false-alarm rate, we have developed a technique that can work effectively without having to make this assumption. The name of the technique called protein complex identification by discovering functional interdependence (PCIFI) searches for protein complexes in PPI networks by taking into consideration both the functional interdependence relationship between protein molecules and the network topology of the network. The PCIFI works in several steps. The first step is to construct a multiple-function protein network graph by labeling each vertex with one or more of the molecular functions it performs. The second step is to filter out protein interactions between protein pairs that are not functionally interdependent of each other in the statistical sense. The third step is to make use of an information-theoretic measure to determine the strength of the functional interdependence between all remaining interacting protein pairs. Finally, the last step is to try to form protein complexes based on the measure of the strength of functional interdependence and the connectivity between proteins. For performance evaluation, PCIFI was used to identify protein complexes in real PPI network data and the protein complexes it found were matched against those that were previously known in MIPS. The results show that PCIFI can be an effective technique for the identification of protein complexes. The protein complexes it found can match more known protein complexes with a smaller false-alarm rate and can provide useful insights into the understanding of the functional interdependence relationships between proteins in protein complexes.

Identification of increased amounts of eppin protein complex components in sperm cells of diabetic and obese individuals by difference gel electrophoresis.

PubMed

Paasch, Uwe; Heidenreich, Falk; Pursche, Theresia; Kuhlisch, Eberhard; Kettner, Karina; Grunewald, Sonja; Kratzsch, Jürgen; Dittmar, Gunnar; Glander, Hans-Jürgen; Hoflack, Bernard; Kriegel, Thomas M

2011-08-01

Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ≤ 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, β-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress.

Recent advances in inflammasome biology.

PubMed

Place, David E; Kanneganti, Thirumala-Devi

2018-02-01

The inflammasome is a complex of proteins that through the activity of caspase-1 and the downstream substrates gasdermin D, IL-1β, and IL-18 execute an inflammatory form of cell death termed pyroptosis. Activation of this complex often involves the adaptor protein ASC and upstream sensors including NLRP1, NLRP3, NLRC4, AIM2, and pyrin, which are activated by different stimuli including infectious agents and changes in cell homeostasis. Here we discuss new regulatory mechanisms that have been identified for the canonical inflammasomes, the most recently identified NLRP9b inflammasome, and the new gasdermin family of proteins that mediate pyroptosis and other forms of regulated cell death. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tob38, a novel essential component in the biogenesis of β-barrel proteins of mitochondria

PubMed Central

Waizenegger, Thomas; Habib, Shukry J; Lech, Maciej; Mokranjac, Dejana; Paschen, Stefan A; Hell, Kai; Neupert, Walter; Rapaport, Doron

2004-01-01

Insertion of β-barrel proteins into the outer membrane of mitochondria is mediated by the TOB complex. Known constituents of this complex are Tob55 and Mas37. We identified a novel component, Tob38. It is essential for viability of yeast and the function of the TOB complex. Tob38 is exposed on the surface of the mitochondrial outer membrane. It interacts with Mas37 and Tob55 and is associated with Tob55 even in the absence of Mas37. The Tob38–Tob55 core complex binds precursors of β-barrel proteins and facilitates their insertion into the outer membrane. Depletion of Tob38 results in strongly reduced levels of Tob55 and Mas37 and the residual proteins no longer form a complex. Tob38-depleted mitochondria are deficient in the import of β-barrel precursor proteins, but not of other outer membrane proteins or proteins of other mitochondrial subcompartments. We conclude that Tob38 has a crucial function in the biogenesis of β-barrel proteins of mitochondria. PMID:15205677

Identifying protein complexes based on brainstorming strategy.

PubMed

Shen, Xianjun; Zhou, Jin; Yi, Li; Hu, Xiaohua; He, Tingting; Yang, Jincai

2016-11-01

Protein complexes comprising of interacting proteins in protein-protein interaction network (PPI network) play a central role in driving biological processes within cells. Recently, more and more swarm intelligence based algorithms to detect protein complexes have been emerging, which have become the research hotspot in proteomics field. In this paper, we propose a novel algorithm for identifying protein complexes based on brainstorming strategy (IPC-BSS), which is integrated into the main idea of swarm intelligence optimization and the improved K-means algorithm. Distance between the nodes in PPI network is defined by combining the network topology and gene ontology (GO) information. Inspired by human brainstorming process, IPC-BSS algorithm firstly selects the clustering center nodes, and then they are separately consolidated with the other nodes with short distance to form initial clusters. Finally, we put forward two ways of updating the initial clusters to search optimal results. Experimental results show that our IPC-BSS algorithm outperforms the other classic algorithms on yeast and human PPI networks, and it obtains many predicted protein complexes with biological significance. Copyright © 2016 Elsevier Inc. All rights reserved.

Who is Mr. HAMLET? Interaction of human alpha-lactalbumin with monomeric oleic acid.

PubMed

Knyazeva, Ekaterina L; Grishchenko, Valery M; Fadeev, Roman S; Akatov, Vladimir S; Permyakov, Sergei E; Permyakov, Eugene A

2008-12-09

A specific state of the human milk Ca(2+) binding protein alpha-lactalbumin (hLA) complexed with oleic acid (OA) prepared using an OA-pretreated ion-exchange column (HAMLET) triggers several cell death pathways in various tumor cells. The possibility of preparing a hLA-OA complex with structural and cytotoxic properties similar to those of the HAMLET but under solution conditions has been explored. The complex was formed by titration of hLA by OA at pH 8.3 up to OA critical micelle concentration. We have shown that complex formation strongly depends on calcium, ionic strength, and temperature; the optimal conditions were established. The spectrofluorimetrically estimated number of OA molecules irreversibly bound per hLA molecule (after dialysis of the OA-loaded preparation against water followed by lyophilization) depends upon temperature: 2.9 at 17 degrees C (native apo-hLA; resulting complex referred to as LA-OA-17 state) and 9 at 45 degrees C (thermally unfolded apo-hLA; LA-OA-45). Intrinsic tryptophan fluorescence measurements revealed substantially decreased thermal stability of Ca(2+)-free forms of HAMLET, LA-OA-45, and OA-saturated protein. The irreversibly bound OA does not affect the Ca(2+) association constant of the protein. Phase plot analysis of fluorimetric and CD data indicates that the OA binding process involves several hLA intermediates. The effective pseudoequilibrium OA association constants for Ca(2+)-free hLA were estimated. The far-UV CD spectra of Ca(2+)-free hLA show that all OA-bound forms of the protein are characterized by elevated content of alpha-helical structure. The various hLA-OA complexes possess similar cytotoxic activities against human epidermoid larynx carcinoma cells. Overall, the LA-OA-45 complex possesses physicochemical, structural, and cytotoxic properties closely resembling those of HAMLET. The fact that the HAMLET-like complex can be formed in aqueous solution makes the process of its preparation more transparent and controllable, opening up opportunities for formation of active complexes with specific properties.

Evolution of an ancient protein function involved in organized multicellularity in animals.

PubMed

Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

2016-01-07

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which - the evolution of GKPID's capacity to bind the cortical marker protein - can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals.

Structure of MyTH4-FERM domains in myosin VIIa tail bound to cargo.

PubMed

Wu, Lin; Pan, Lifeng; Wei, Zhiyi; Zhang, Mingjie

2011-02-11

The unconventional myosin VIIa (MYO7A) is one of the five proteins that form a network of complexes involved in formation of stereocilia. Defects in these proteins cause syndromic deaf-blindness in humans [Usher syndrome I (USH1)]. Many disease-causing mutations occur in myosin tail homology 4-protein 4.1, ezrin, radixin, moesin (MyTH4-FERM) domains in the myosin tail that binds to another USH1 protein, Sans. We report the crystal structure of MYO7A MyTH4-FERM domains in complex with the central domain (CEN) of Sans at 2.8 angstrom resolution. The MyTH4 and FERM domains form an integral structural and functional supramodule binding to two highly conserved segments (CEN1 and 2) of Sans. The MyTH4-FERM/CEN complex structure provides mechanistic explanations for known deafness-causing mutations in MYO7A MyTH4-FERM. The structure will also facilitate mechanistic and functional studies of MyTH4-FERM domains in other myosins.

Interdependence of free zinc changes and protein complex assembly - insights into zinc signal regulation.

PubMed

Kocyła, Anna; Adamczyk, Justyna; Krężel, Artur

2018-01-24

Cellular zinc (Zn(ii)) is bound with proteins that are part of the proteomes of all domains of life. It is mostly utilized as a catalytic or structural protein cofactor, which results in a vast number of binding architectures. The Zn(ii) ion is also important for the formation of transient protein complexes with a Zn(ii)-dependent quaternary structure that is formed upon cellular zinc signals. The mechanisms by which proteins associate with and dissociate from Zn(ii) and the connection with cellular Zn(ii) changes remain incompletely understood. In this study, we aimed to examine how zinc protein domains with various Zn(ii)-binding architectures are formed under free Zn(ii) concentration changes and how formation of the Zn(ii)-dependent assemblies is related to the protein concentration and reactivity. To accomplish these goals we chose four zinc domains with different Zn(ii)-to-protein binding stoichiometries: classical zinc finger (ZnP), LIM domain (Zn 2 P), zinc hook (ZnP 2 ) and zinc clasp (ZnP 1 P 2 ) folds. Our research demonstrated a lack of changes in the saturation level of intraprotein zinc binding sites, despite various peptide concentrations, while homo- and heterodimers indicated a concentration-dependent tendency. In other words, at a certain free Zn(ii) concentration, the fraction of a formed dimeric complex increases or decreases with subunit concentration changes. Secondly, even small or local changes in free Zn(ii) may significantly affect protein saturation depending on its architecture, function and subcellular concentration. In our paper, we indicate the importance of interdependence of free Zn(ii) availability and protein subunit concentrations for cellular zinc signal regulation.

The ubiquitin conjugating enzyme UbcH7, controls cell migration

USDA-ARS?s Scientific Manuscript database

Post translational modification by ubiquitination can target proteins for degradation, allow the interaction of proteins to form complexes or direct relocalization of proteins to different subcellular compartments. As such, ubiquitin controls a variety of essential cellular processes. Previously we ...

Identification of a small TAF complex and its role in the assembly of TAF-containing complexes.

PubMed

Demény, Màté A; Soutoglou, Evi; Nagy, Zita; Scheer, Elisabeth; Jànoshàzi, Agnes; Richardot, Magalie; Argentini, Manuela; Kessler, Pascal; Tora, Laszlo

2007-03-21

TFIID plays a role in nucleating RNA polymerase II preinitiation complex assembly on protein-coding genes. TFIID is a multisubunit complex comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). Another class of multiprotein transcriptional regulatory complexes having histone acetyl transferase (HAT) activity, and containing TAFs, includes TFTC, STAGA and the PCAF/GCN5 complex. Looking for as yet undiscovered subunits by a proteomic approach, we had identified TAF8 and SPT7L in human TFTC preparations. Subsequently, however, we demonstrated that TAF8 was not a stable component of TFTC, but that it is present in a small TAF complex (SMAT), containing TAF8, TAF10 and SPT7L, that co-purified with TFTC. Thus, TAF8 is a subunit of both TFIID and SMAT. The latter has to be involved in a pathway of complex formation distinct from the other known TAF complexes, since these three histone fold (HF)-containing proteins (TAF8, TAF10 and SPT7L) can never be found together either in TFIID or in STAGA/TFTC HAT complexes. Here we show that TAF8 is absolutely necessary for the integration of TAF10 in a higher order TFIID core complex containing seven TAFs. TAF8 forms a heterodimer with TAF10 through its HF and proline rich domains, and also interacts with SPT7L through its C-terminal region, and the three proteins form a complex in vitro and in vivo. Thus, the TAF8-TAF10 and TAF10-SPT7L HF pairs, and also the SMAT complex, seem to be important regulators of the composition of different TFIID and/or STAGA/TFTC complexes in the nucleus and consequently may play a role in gene regulation.

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

PubMed

Hallée, Stéphanie; Thériault, Catherine; Gagnon, Dominic; Kehrer, Jessica; Frischknecht, Friedrich; Mair, Gunnar R; Richard, Dave

2018-03-26

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages. © 2018 John Wiley & Sons Ltd.

Complementarity of stability patches at the interfaces of protein complexes: Implication for the structural organization of energetic hot spots.

PubMed

Kuttner, Yosef Y; Engel, Stanislav

2018-02-01

A rational design of protein complexes with defined functionalities and of drugs aimed at disrupting protein-protein interactions requires fundamental understanding of the mechanisms underlying the formation of specific protein complexes. Efforts to develop efficient small-molecule or protein-based binders often exploit energetic hot spots on protein surfaces, namely, the interfacial residues that provide most of the binding free energy in the complex. The molecular basis underlying the unusually high energy contribution of the hot spots remains obscure, and its elucidation would facilitate the design of interface-targeted drugs. To study the nature of the energetic hot spots, we analyzed the backbone dynamic properties of contact surfaces in several protein complexes. We demonstrate that, in most complexes, the backbone dynamic landscapes of interacting surfaces form complementary "stability patches," in which static areas from the opposing surfaces superimpose, and that these areas are predominantly located near the geometric center of the interface. We propose that a diminished enthalpy-entropy compensation effect augments the degree to which residues positioned within the complementary stability patches contribute to complex affinity, thereby giving rise to the energetic hot spots. These findings offer new insights into the nature of energetic hot spots and the role that backbone dynamics play in facilitating intermolecular recognition. Mapping the interfacial stability patches may provide guidance for protein engineering approaches aimed at improving the stability of protein complexes and could facilitate the design of ligands that target complex interfaces. © 2017 Wiley Periodicals, Inc.

XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes.

PubMed Central

van der Spek, P J; Eker, A; Rademakers, S; Visser, C; Sugasawa, K; Masutani, C; Hanaoka, F; Bootsma, D; Hoeijmakers, J H

1996-01-01

The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER). The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al. (1994) EMBO J. 8, 1831-1843). Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC. In contrast, we cannot detect any bound HHR23A. Thus the HHR23 proteins may have an additional function independent of XPC. The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction. Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation. Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex. However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex. Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus. PMID:8692695

Solution and Gas-Phase H/D Exchange of Protein-Small-Molecule Complexes: Cex and Its Inhibitors

NASA Astrophysics Data System (ADS)

Kang, Yang; Terrier, Peran; Ding, Chuanfan; Douglas, D. J.

2012-01-01

The properties of noncovalent complexes of the enzyme exo-1,4-β-D-glycanase ("Cex") with three aza-sugar inhibitors, deoxynojirimycin (X2DNJ), isofagomine lactam (X2IL), and isofagomine (X2IF), have been studied with solution and gas-phase hydrogen deuterium exchange (H/Dx) and measurements of collision cross sections of gas-phase ions. In solution, complexes have lower H/Dx levels than free Cex because binding the inhibitors blocks some sites from H/Dx and reduces fluctuations of the protein. In mass spectra of complexes, abundant Cex ions are seen, which mostly are formed by dissociation of complexes in the ion sampling interface. Both complex ions and Cex ions formed from a solution containing complexes have lower cross sections than Cex ions from a solution of Cex alone. This suggests the Cex ions formed by dissociation "remember" their solution conformations. For a given charge, ions of the complexes have greater gas-phase H/Dx levels than ions of Cex. Unlike cross sections, H/Dx levels of the complexes do not correlate with the relative gas-phase binding strengths measured by MS/MS. Cex ions from solutions with or without inhibitors, which have different cross sections, show the same H/Dx level after 15 s, indicating the ions may fold or unfold on the seconds time scale of the H/Dx experiment. Thus, cross sections show that complexes have more compact conformations than free protein ions on the time scale of ca. 1 ms. The gas-phase H/Dx measurements show that at least some complexes retain different conformations from the Cex ions on a time scale of seconds.

Complex lasso: new entangled motifs in proteins

NASA Astrophysics Data System (ADS)

Niemyska, Wanda; Dabrowski-Tumanski, Pawel; Kadlof, Michal; Haglund, Ellinor; Sułkowski, Piotr; Sulkowska, Joanna I.

2016-11-01

We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules.

Template-Based Modeling of Protein-RNA Interactions.

PubMed

Zheng, Jinfang; Kundrotas, Petras J; Vakser, Ilya A; Liu, Shiyong

2016-09-01

Protein-RNA complexes formed by specific recognition between RNA and RNA-binding proteins play an important role in biological processes. More than a thousand of such proteins in human are curated and many novel RNA-binding proteins are to be discovered. Due to limitations of experimental approaches, computational techniques are needed for characterization of protein-RNA interactions. Although much progress has been made, adequate methodologies reliably providing atomic resolution structural details are still lacking. Although protein-RNA free docking approaches proved to be useful, in general, the template-based approaches provide higher quality of predictions. Templates are key to building a high quality model. Sequence/structure relationships were studied based on a representative set of binary protein-RNA complexes from PDB. Several approaches were tested for pairwise target/template alignment. The analysis revealed a transition point between random and correct binding modes. The results showed that structural alignment is better than sequence alignment in identifying good templates, suitable for generating protein-RNA complexes close to the native structure, and outperforms free docking, successfully predicting complexes where the free docking fails, including cases of significant conformational change upon binding. A template-based protein-RNA interaction modeling protocol PRIME was developed and benchmarked on a representative set of complexes.

The Fanconi anemia protein FANCF forms a nuclear complex with FANCA, FANCC and FANCG.

PubMed

de Winter, J P; van der Weel, L; de Groot, J; Stone, S; Waisfisz, Q; Arwert, F; Scheper, R J; Kruyt, F A; Hoatlin, M E; Joenje, H

2000-11-01

Fanconi anemia (FA) is a chromosomal instability syndrome associated with a strong predisposition to cancer, particularly acute myeloid leukemia and squamous cell carcinoma. At the cellular level, FA is characterized by spontaneous chromosomal breakage and a unique hypersensitivity to DNA cross-linking agents. Complementation analysis has indicated that at least seven distinct genes are involved in the pathogenesis of FA. Despite the identification of four of these genes (FANCA, FANCC, FANCF and FANCG), the nature of the 'FA pathway' has remained enigmatic, as the FA proteins lack sequence homologies or motifs that could point to a molecular function. To further define this pathway, we studied the subcellular localizations and mutual interactions of the FA proteins, including the recently identified FANCF protein, in human lymphoblasts. FANCF was found predominantly in the nucleus, where it complexes with FANCA, FANCC and FANCG. These interactions were detected in wild-type and FA-D lymphoblasts, but not in lymphoblasts of other FA complementation groups. This implies that each of the FA proteins, except FANCD, is required for these complexes to form. Similarly, we show that the interaction between FANCA and FANCC is restricted to wild-type and FA-D cells. Furthermore, we document the subcellular localization of FANCA and the FANCA/FANCG complex in all FA complementation groups. Our results, along with published data, culminate in a model in which a multi-protein FA complex serves a nuclear function to maintain genomic integrity.

Tor forms a dimer through an N-terminal helical solenoid with a complex topology

NASA Astrophysics Data System (ADS)

Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M.; Williams, Roger L.

2016-04-01

The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended `railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.

Encapsulation of a rhodamine dye within a bile acid binding protein: toward water processable functional bio host-guest materials.

PubMed

Tomaselli, Simona; Giovanella, Umberto; Pagano, Katiuscia; Leone, Giuseppe; Zanzoni, Serena; Assfalg, Michael; Meinardi, Francesco; Molinari, Henriette; Botta, Chiara; Ragona, Laura

2013-10-14

New strategies are requested for the preparation of bioinspired host-guest complexes to be employed in technologically relevant applications, as sensors and optoelectronic devices. We report here a new approach employing a single monomeric protein as host for the strongly fluorescent rhodamine dye. The selected protein, belonging to the intracellular lipid binding protein family, fully encapsulates one rhodamine molecule inside its cavity forming a host-guest complex stabilized by H and π-hydrogen bonds, a salt bridge, and favorable hydrophobic contacts, as revealed by the NMR derived structural model. The protein-dye solutions are easily processable and form homogeneous thin films exhibiting excellent photophysical and morphological properties, as derived from photoluminescence and AFM data. The obtained results represent the proof of concept of the viability of this bio host-guest system for the development of bioinspired optoelectronic devices.

Application of virtual screening and molecular dynamics for the analysis of selectivity of inhibitors of HU proteins targeted to the DNA-recognition site

NASA Astrophysics Data System (ADS)

Talyzina, A. A.; Agapova, Yu. K.; Podshivalov, D. D.; Timofeev, V. I.; Sidorov-Biryukov, D. D.; Rakitina, T. V.

2017-11-01

DNA-Binding HU proteins are essential for the maintenance of genomic DNA supercoiling and compaction in prokaryotic cells and are promising pharmacological targets for the design of new antibacterial agents. The virtual screening for low-molecular-weight compounds capable of specifically interacting with the DNA-recognition loop of the HU protein from the mycoplasma Spiroplasma melliferum was performed. The ability of the initially selected ligands to form stable complexes with the protein target was assessed by molecular dynamics simulation. One compound, which forms an unstable complex, was eliminated by means of a combination of computational methods, resulting in a decrease in the number of compounds that will pass to the experimental test phase. This approach can be used to solve a wide range of problems related to the search for and validation of low-molecular-weight inhibitors specific for a particular protein target.

Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks.

PubMed

Chiva, M; Saperas, N; Ribes, E

2011-12-01

In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and conclude that the appearance of precursor (intermediate) molecules facilitated the development of complex patterns of condensation and, as a consequence, a great diversity of forms in the sperm cell nuclei Copyright © 2011 Elsevier Ltd. All rights reserved.

The New Kid on the Block: A Specialized Secretion System during Bacterial Sporulation.

PubMed

Morlot, Cécile; Rodrigues, Christopher D A

2018-02-02

The transport of proteins across the bacterial cell envelope is mediated by protein complexes called specialized secretion systems. These nanomachines exist in both Gram-positive and Gram-negative bacteria and have been categorized into different types based on their structural components and function. Interestingly, multiple studies suggest the existence of a protein complex in endospore-forming bacteria that appears to be a new type of specialized secretion system. This protein complex is called the SpoIIIA-SpoIIQ complex and is an exception to the categorical norm since it appears to be a hybrid composed of different parts from well-defined specialized secretion systems. Here we summarize and discuss the current understanding of this complex and its potential role as a specialized secretion system. Copyright © 2018 Elsevier Ltd. All rights reserved.

Analysis of the 3H8 antigen of Candida albicans reveals new aspects of the organization of fungal cell wall proteins.

PubMed

Sentandreu, Rafael; Caminero, Antonio; Rentería, Itzel; León-Ramirez, Claudia; González-de-la-Vara, Luis; Valentin-Gomez, Eulogio; Ruiz-Herrera, José

2018-06-01

The walls of both, yeast and mycelial cells of Candida albicans possess a species-specific antigen that is recognized by a monoclonal antibody (MAb 3H8). This antigen can be extracted in the form of a very high Mr complex, close or over 106 Da, by treatment, with β-1,3-glucanase, β mercaptoethanol or dithothreitol, or mild alkali, but not by saturated hydrogen fluoride (HF) in pyridine, suggesting that the complex is bound to wall β-1,3 glucans, and to proteins by disulfide bonds, but not to β-1,6 glucans. Through its sensitivity to trypsin and different deglycosylation procedures, it was concluded that the epitope is associated to a glycoprotein containing N-glycosidic, but not O-glycosidic mannan moieties. By means of electrophoresis in polycrylamide gradient gels, followed by mass spectrometric analysis, the epitope was pinpointed to a very high MW complex containing Agglutinin-Like Sequence (ALS) family proteins, and other cytoplasmic, membrane and secreted proteins. The components of this complex are bound by unknown covalent bonds. The material extracted with β mercaptoethanol or dilute alkali appeared under the electron microscope as large aggregates in the form of spheroidal and mostly web-like structures of large sizes. These, and additional data, suggest that this protein complex may constitute an important part of the basic glycoprotein structure of C. albicans. The possibility that similar complexes exist in the wall of other fungi is an attractive, although yet untested possibility.

Rice black streaked dwarf virus P7-2 forms a SCF complex through binding to Oryza sativa SKP1-like proteins, and interacts with GID2 involved in the gibberellin pathway.

PubMed

Tao, Tao; Zhou, Cui-Ji; Wang, Qian; Chen, Xiang-Ru; Sun, Qian; Zhao, Tian-Yu; Ye, Jian-Chun; Wang, Ying; Zhang, Zong-Ying; Zhang, Yong-Liang; Guo, Ze-Jian; Wang, Xian-Bing; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2017-01-01

As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1) plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA), jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV), a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK) proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2) from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection.

Rice black streaked dwarf virus P7-2 forms a SCF complex through binding to Oryza sativa SKP1-like proteins, and interacts with GID2 involved in the gibberellin pathway

PubMed Central

Wang, Qian; Chen, Xiang-Ru; Sun, Qian; Zhao, Tian-Yu; Ye, Jian-Chun; Wang, Ying; Zhang, Zong-Ying; Zhang, Yong-Liang; Guo, Ze-Jian; Wang, Xian-Bing; Li, Da-Wei; Yu, Jia-Lin

2017-01-01

As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1) plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA), jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV), a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK) proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2) from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection. PMID:28494021

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

Guo, Zheng; Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Dadao Bei, Guangzhou 510515; Zhou, Yuning

Highlights: Black-Right-Pointing-Pointer Rictor associates with FBXW7 to form an E3 complex. Black-Right-Pointing-Pointer Knockdown of rictor decreases ubiquitination of c-Myc and cylin E. Black-Right-Pointing-Pointer Knockdown of rictor increases protein levels of c-Myc and cylin E. Black-Right-Pointing-Pointer Overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Black-Right-Pointing-Pointer Rictor regulation of c-Myc and cyclin E requires FBXW7. -- Abstract: Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation.more » Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.« less

Description of the topographical changes associated to the different stages of the DsbA catalytic cycle

PubMed Central

Vinci, Floriana; Couprie, Joël; Pucci, Piero; Quéméneur, Eric; Moutiez, Mireille

2002-01-01

This paper provides a description of the surface topography of DsbA, the bacterial disulfide-bond forming enzyme, in the different phases of its catalytic cycle. Three representative states, that is, oxidized and reduced protein and a covalent complex mimicking the DsbA-substrate disulfide intermediate, have been investigated by a combination of limited proteolysis experiments and mass spectrometry methodologies. Protease-accessible sites are largely distributed in the oxidized form with a small predominance inside the thioredoxin domain. Proteolysis occurs even in secondary structure elements, revealing a significant mobility of the protein. Many cleavage sites disappear in the reduced form and most of the remaining ones appear with strongly reduced kinetics. The protein within the complex shows an intermediate behavior. This variation of flexibility in DsbA is probably the determining factor for the course of its catalytic cycle. In particular, the great mobility of the oxidized protein might facilitate the accommodation of its various substrates, whereas the increasing rigidity from the complexed to the reduced form could help the release of oxidized products. The formation of the complex between PID peptide and DsbA does not significantly protect the enzyme against proteolysis, reinforcing the results previously obtained by calorimetry concerning the weakness of their interaction. The few cleavage sites observed, however, are in favor of the presence of the peptide in the binding site postulated from crystallographic studies. As for the peptide itself, the proteolytic pattern and the protection effect exerted by DsbA could be explained by a preferential orientation within the binding site. PMID:12070313

Solution and electron microscopy characterization of lactococcal phage baseplates expressed in Escherichia coli.

PubMed

Campanacci, Valérie; Veesler, David; Lichière, Julie; Blangy, Stéphanie; Sciara, Giuliano; Moineau, Sylvain; van Sinderen, Douwe; Bron, Patrick; Cambillau, Christian

2010-10-01

We report here the characterization of several large structural protein complexes forming the baseplates (or part of them) of Siphoviridae phages infecting Lactococcus lactis: TP901-1, Tuc2009 and p2. We revisited a "block cloning" expression strategy and extended this approach to genomic fragments encoding proteins whose interacting partners have not yet been clearly identified. Biophysical characterization of some of these complexes using circular dichroism and size exclusion chromatography, coupled with on-line light scattering and refractometry, demonstrated that the over-produced recombinant proteins interact with each other to form large (up to 1.9MDa) and stable baseplate assemblies. Some of these complexes were characterized by electron microscopy confirming their structural homogeneity as well as providing a picture of their overall molecular shapes and symmetry. Finally, using these results, we were able to highlight similarities and differences with the well characterized much larger baseplate of the myophage T4.

3D Complex: A Structural Classification of Protein Complexes

PubMed Central

Levy, Emmanuel D; Pereira-Leal, Jose B; Chothia, Cyrus; Teichmann, Sarah A

2006-01-01

Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes. PMID:17112313

[Fanconi anemia: genes and function(s) revisited].

PubMed

Papadopoulo, Dora; Moustacchi, Ethel

2005-01-01

Fanconi anemia (FA), a rare inherited disorder, exhibits a complex phenotype including progressive bone marrow failure, congenital malformations and increased risk of cancers, mainly acute myeloid leukaemia. At the cellular level, FA is characterized by hypersensitivity to DNA cross-linking agents and by high frequencies of induced chromosomal aberrations, a property used for diagnosis. FA results from mutations in one of the eleven FANC (FANCA to FANCJ) genes. Nine of them have been identified. In addition, FANCD1 gene has been shown to be identical to BRCA2, one of the two breast cancer susceptibility genes. Seven of the FANC proteins form a complex, which exists in four different forms depending of its subcellular localisation. Four FANC proteins (D1(BRCA2), D2, I and J) are not associated to the complex. The presence of the nuclear form of the FA core complex is necessary for the mono-ubiquitinylation of FANCD2 protein, a modification required for its re-localization to nuclear foci, likely to be sites of DNA repair. A clue towards understanding the molecular function of the FANC genes comes from the recently identified connection of FANC to the BRCA1, ATM, NBS1 and ATR genes. Two of the FANC proteins (A and D2) directly interact with BRCA1, which in turn interacts with the MRE11/RAD50/NBS1 complex, which is one of the key components in the mechanisms involved in the cellular response to DNA double strand breaks (DSB). Moreover, ATM, a protein kinase that plays a central role in the network of DSB signalling, phosphorylates in vitro and in vivo FANCD2 in response to ionising radiations. Moreover, the NBS1 protein and the monoubiquitinated form of FANCD2 seem to act together in response to DNA crosslinking agents. Taken together with the previously reported impaired DSB and DNA interstrand crosslinks repair in FA cells, the connection of FANC genes to the ATM, ATR, NBS1 and BRCA1 links the FANC genes function to the finely orchestrated network involved in the sensing, signalling and repair of DNA replication-blocking lesions.

Augmenting β-augmentation: structural basis of how BamB binds BamA and may support folding of outer membrane proteins.

PubMed

Heuck, Alexander; Schleiffer, Alexander; Clausen, Tim

2011-03-11

β-Barrel proteins are frequently found in the outer membrane of mitochondria, chloroplasts and Gram-negative bacteria. In Escherichia coli, these proteins are inserted in the outer membrane by the Bam (β-barrel assembly machinery) complex, a multiprotein machinery formed by the β-barrel protein BamA and the four peripheral membrane proteins BamB, BamC, BamD and BamE. The periplasmic part of BamA binds prefolded β-barrel proteins by a β-augmentation mechanism, thereby stabilizing the precursors prior to their membrane insertion. However, the role of the associated proteins within the Bam complex remains unknown. Here, we describe the crystal structure of BamB, a nonessential component of the Bam complex. The structure shows a typical eight-bladed β-propeller fold. Two sequence stretches of BamB were previously identified to be important for interaction with BamA. In our structure, both motifs are located in close proximity to each other and contribute to a conserved region forming a narrow groove on the top of the propeller. Moreover, crystal contacts reveal two interaction modes of how BamB might bind unfolded β-barrel proteins. In the crystal lattice, BamB binds to exposed β-strands by β-augmentation, whereas peptide stretches rich in aromatic residues can be accommodated in hydrophobic pockets located at the bottom of the propeller. Thus, BamB could simultaneously bind to BamA and prefolded β-barrel proteins, thereby enhancing the folding and membrane insertion capability of the Bam complex. Copyright © 2011 Elsevier Ltd. All rights reserved.

CUB domain-containing protein 1 and the epidermal growth factor receptor cooperate to induce cell detachment.

PubMed

Law, Mary E; Ferreira, Renan B; Davis, Bradley J; Higgins, Paul J; Kim, Jae-Sung; Castellano, Ronald K; Chen, Sixue; Luesch, Hendrik; Law, Brian K

2016-08-05

While localized malignancies often respond to available therapies, most disseminated cancers are refractory. Novel approaches, therefore, are needed for the treatment of metastatic disease. CUB domain-containing protein1 (CDCP1) plays an important role in metastasis and drug resistance; the mechanism however, is poorly understood. Breast cancer cell lines were engineered to stably express EGFR, CDCP1 or phosphorylation site mutants of CDCP1. These cell lines were used for immunoblot analysis or affinity purification followed by immunoblot analysis to assess protein phosphorylation and/or protein complex formation with CDCP1. Kinase activity was evaluated using phosphorylation site-specific antibodies and immunoblot analysis in in vitro kinase assays. Protein band excision and mass spectrometry was utilized to further identify proteins complexed with CDCP1 or ΔCDCP1, which is a mimetic of the cleaved form of CDCP1. Cell detachment was assessed using cell counting. This paper reports that CDCP1 forms ternary protein complexes with Src and EGFR, facilitating Src activation and Src-dependent EGFR transactivation. Importantly, we have discovered that a class of compounds termed Disulfide bond Disrupting Agents (DDAs) blocks CDCP1/EGFR/Src ternary complex formation and downstream signaling. CDCP1 and EGFR cooperate to induce detachment of breast cancer cells from the substratum and to disrupt adherens junctions. Analysis of CDCP1-containing complexes using proteomics techniques reveals that CDCP1 associates with several proteins involved in cell adhesion, including adherens junction and desmosomal cadherins, and cytoskeletal elements. Together, these results suggest that CDCP1 may facilitate loss of adhesion by promoting activation of EGFR and Src at sites of cell-cell and cell-substratum contact.

Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction.

PubMed

Papadakos, Grigorios; Sharma, Amit; Lancaster, Lorna E; Bowen, Rebecca; Kaminska, Renata; Leech, Andrew P; Walker, Daniel; Redfield, Christina; Kleanthous, Colin

2015-04-29

The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost.

Simulation-Based Validation of the p53 Transcriptional Activity with Hybrid Functional Petri Net.

PubMed

Doi, Atsushi; Nagasaki, Masao; Matsuno, Hiroshi; Miyano, Satoru

2011-01-01

MDM2 and p19ARF are essential proteins in cancer pathways forming a complex with protein p53 to control the transcriptional activity of protein p53. It is confirmed that protein p53 loses its transcriptional activity by forming the functional dimer with protein MDM2. However, it is still unclear that protein p53 keeps its transcriptional activity when it forms the trimer with proteins MDM2 and p19ARF. We have observed mutual behaviors among genes p53, MDM2, p19ARF and their products on a computational model with hybrid functional Petri net (HFPN) which is constructed based on information described in the literature. The simulation results suggested that protein p53 should have the transcriptional activity in the forms of the trimer of proteins p53, MDM2, and p19ARF. This paper also discusses the advantages of HFPN based modeling method in terms of pathway description for simulations.

Signaling complexes of voltage-gated calcium channels

PubMed Central

Turner, Ray W; Anderson, Dustin

2011-01-01

Voltage-gated calcium channels are key mediators of depolarization induced calcium entry into electrically excitable cells. There is increasing evidence that voltage-gated calcium channels, like many other types of ionic channels, do not operate in isolation, but instead form complexes with signaling molecules, G protein coupled receptors, and other types of ion channels. Furthermore, there appears to be bidirectional signaling within these protein complexes, thus allowing not only for efficient translation of calcium signals into cellular responses, but also for tight control of calcium entry per se. In this review, we will focus predominantly on signaling complexes between G protein-coupled receptors and high voltage activated calcium channels, and on complexes of voltage-gated calcium channels and members of the potassium channel superfamily. PMID:21832880

Changes in mouse whole saliva soluble proteome induced by tannin-enriched diet

PubMed Central

2010-01-01

Background Previous studies suggested that dietary tannin ingestion may induce changes in mouse salivary proteins in addition to the primarily studied proline-rich proteins (PRPs). The aim of the present study was to determine the protein expression changes induced by condensed tannin intake on the fraction of mouse whole salivary proteins that are unable to form insoluble tannin-protein complexes. Two-dimensional polyacrylamide gel electrophoresis protein separation was used, followed by protein identification by mass spectrometry. Results Fifty-seven protein spots were excised from control group gels, and 21 different proteins were identified. With tannin consumption, the expression levels of one α-amylase isoform and one unidentified protein increased, whereas acidic mammalian chitinase and Muc10 decreased. Additionally, two basic spots that stained pink with Coomassie Brilliant Blue R-250 were newly observed, suggesting that some induced PRPs may remain uncomplexed or form soluble complexes with tannins. Conclusion This proteomic analysis provides evidence that other salivary proteins, in addition to tannin-precipitating proteins, are affected by tannin ingestion. Changes in the expression levels of the acidic mammalian chitinase precursor and in one of the 14 salivary α-amylase isoforms underscores the need to further investigate their role in tannin ingestion. PMID:21159160

Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex.

PubMed

Tuttle, Lisa M; Pacheco, Derek; Warfield, Linda; Luo, Jie; Ranish, Jeff; Hahn, Steven; Klevit, Rachel E

2018-03-20

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs.

PubMed

Moreno, Renata; Hernández-Arranz, Sofía; La Rosa, Ruggero; Yuste, Luis; Madhushani, Anjana; Shingler, Victoria; Rojo, Fernando

2015-01-01

The Crc protein is a global regulator that has a key role in catabolite repression and optimization of metabolism in Pseudomonads. Crc inhibits gene expression post-transcriptionally, preventing translation of mRNAs bearing an AAnAAnAA motif [the catabolite activity (CA) motif] close to the translation start site. Although Crc was initially believed to bind RNA by itself, this idea was recently challenged by results suggesting that a protein co-purifying with Crc, presumably the Hfq protein, could account for the detected RNA-binding activity. Hfq is an abundant protein that has a central role in post-transcriptional gene regulation. Herein, we show that the Pseudomonas putida Hfq protein can recognize the CA motifs of RNAs through its distal face and that Crc facilitates formation of a more stable complex at these targets. Crc was unable to bind RNA in the absence of Hfq. However, pull-down assays showed that Crc and Hfq can form a co-complex with RNA containing a CA motif in vitro. Inactivation of the hfq or the crc gene impaired catabolite repression to a similar extent. We propose that Crc and Hfq cooperate in catabolite repression, probably through forming a stable co-complex with RNAs containing CA motifs to result in inhibition of translation initiation. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

SAS6-like protein in Plasmodium indicates that conoid-associated apical complex proteins persist in invasive stages within the mosquito vector.

PubMed

Wall, Richard J; Roques, Magali; Katris, Nicholas J; Koreny, Ludek; Stanway, Rebecca R; Brady, Declan; Waller, Ross F; Tewari, Rita

2016-06-24

The SAS6-like (SAS6L) protein, a truncated paralogue of the ubiquitous basal body/centriole protein SAS6, has been characterised recently as a flagellum protein in trypanosomatids, but associated with the conoid in apicomplexan Toxoplasma. The conoid has been suggested to derive from flagella parts, but is thought to have been lost from some apicomplexans including the malaria-causing genus Plasmodium. Presence of SAS6L in Plasmodium, therefore, suggested a possible role in flagella assembly in male gametes, the only flagellated stage. Here, we have studied the expression and role of SAS6L throughout the Plasmodium life cycle using the rodent malaria model P. berghei. Contrary to a hypothesised role in flagella, SAS6L was absent during gamete flagellum formation. Instead, SAS6L was restricted to the apical complex in ookinetes and sporozoites, the extracellular invasive stages that develop within the mosquito vector. In these stages SAS6L forms an apical ring, as we show is also the case in Toxoplasma tachyzoites. The SAS6L ring was not apparent in blood-stage invasive merozoites, indicating that the apical complex is differentiated between the different invasive forms. Overall this study indicates that a conoid-associated apical complex protein and ring structure is persistent in Plasmodium in a stage-specific manner.

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ.

PubMed

Dharmaiah, Srisathiyanarayanan; Bindu, Lakshman; Tran, Timothy H; Gillette, William K; Frank, Peter H; Ghirlando, Rodolfo; Nissley, Dwight V; Esposito, Dominic; McCormick, Frank; Stephen, Andrew G; Simanshu, Dhirendra K

2016-11-01

Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated-methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated-methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated-methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated-methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein-protein interaction interface in KRAS4b-PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.

Cryo-EM of dynamic protein complexes in eukaryotic DNA replication.

PubMed

Sun, Jingchuan; Yuan, Zuanning; Bai, Lin; Li, Huilin

2017-01-01

DNA replication in Eukaryotes is a highly dynamic process that involves several dozens of proteins. Some of these proteins form stable complexes that are amenable to high-resolution structure determination by cryo-EM, thanks to the recent advent of the direct electron detector and powerful image analysis algorithm. But many of these proteins associate only transiently and flexibly, precluding traditional biochemical purification. We found that direct mixing of the component proteins followed by 2D and 3D image sorting can capture some very weakly interacting complexes. Even at 2D average level and at low resolution, EM images of these flexible complexes can provide important biological insights. It is often necessary to positively identify the feature-of-interest in a low resolution EM structure. We found that systematically fusing or inserting maltose binding protein (MBP) to selected proteins is highly effective in these situations. In this chapter, we describe the EM studies of several protein complexes involved in the eukaryotic DNA replication over the past decade or so. We suggest that some of the approaches used in these studies may be applicable to structural analysis of other biological systems. © 2016 The Protein Society.

Predicting protein complex geometries with a neural network.

PubMed

Chae, Myong-Ho; Krull, Florian; Lorenzen, Stephan; Knapp, Ernst-Walter

2010-03-01

A major challenge of the protein docking problem is to define scoring functions that can distinguish near-native protein complex geometries from a large number of non-native geometries (decoys) generated with noncomplexed protein structures (unbound docking). In this study, we have constructed a neural network that employs the information from atom-pair distance distributions of a large number of decoys to predict protein complex geometries. We found that docking prediction can be significantly improved using two different types of polar hydrogen atoms. To train the neural network, 2000 near-native decoys of even distance distribution were used for each of the 185 considered protein complexes. The neural network normalizes the information from different protein complexes using an additional protein complex identity input neuron for each complex. The parameters of the neural network were determined such that they mimic a scoring funnel in the neighborhood of the native complex structure. The neural network approach avoids the reference state problem, which occurs in deriving knowledge-based energy functions for scoring. We show that a distance-dependent atom pair potential performs much better than a simple atom-pair contact potential. We have compared the performance of our scoring function with other empirical and knowledge-based scoring functions such as ZDOCK 3.0, ZRANK, ITScore-PP, EMPIRE, and RosettaDock. In spite of the simplicity of the method and its functional form, our neural network-based scoring function achieves a reasonable performance in rigid-body unbound docking of proteins. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Toll-like receptor signaling in vertebrates: testing the integration of protein, complex, and pathway data in the protein ontology framework.

PubMed

Arighi, Cecilia; Shamovsky, Veronica; Masci, Anna Maria; Ruttenberg, Alan; Smith, Barry; Natale, Darren A; Wu, Cathy; D'Eustachio, Peter

2015-01-01

The Protein Ontology (PRO) provides terms for and supports annotation of species-specific protein complexes in an ontology framework that relates them both to their components and to species-independent families of complexes. Comprehensive curation of experimentally known forms and annotations thereof is expected to expose discrepancies, differences, and gaps in our knowledge. We have annotated the early events of innate immune signaling mediated by Toll-Like Receptor 3 and 4 complexes in human, mouse, and chicken. The resulting ontology and annotation data set has allowed us to identify species-specific gaps in experimental data and possible functional differences between species, and to employ inferred structural and functional relationships to suggest plausible resolutions of these discrepancies and gaps.

Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and Its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

NASA Astrophysics Data System (ADS)

Farrugia, Mark A.; Han, Linjie; Zhong, Yueyang; Boer, Jodi L.; Ruotolo, Brandon T.; Hausinger, Robert P.

2013-09-01

Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)2 quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)2 and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)2 complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation.

Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex

PubMed Central

Ortega-Roldan, Jose Luis; Jensen, Malene Ringkjøbing; Brutscher, Bernhard; Azuaga, Ana I.; Blackledge, Martin; van Nuland, Nico A. J.

2009-01-01

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 μM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (Kd = 132 ± 13 μM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein–protein complexes. PMID:19359362

Role of Glutamine 17 of the Bovine Papillomavirus E5 Protein in Platelet-Derived Growth Factor β Receptor Activation and Cell Transformation

PubMed Central

Klein, Ophir; Polack, Glenda W.; Surti, Toral; Kegler-Ebo, Deena; Smith, Steven O.; DiMaio, Daniel

1998-01-01

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) β receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF β receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF β receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF β receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF β receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF β receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF β receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF β receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF β receptor. PMID:9765437

Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growth factor beta receptor activation and cell transformation.

PubMed

Klein, O; Polack, G W; Surti, T; Kegler-Ebo, D; Smith, S O; DiMaio, D

1998-11-01

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF beta receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF beta receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF beta receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF beta receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF beta receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF beta receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF beta receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF beta receptor.

Conformational heterogeneity in the Hsp70 chaperone-substrate ensemble identified from analysis of NMR-detected titration data.

PubMed

Sekhar, Ashok; Nagesh, Jayashree; Rosenzweig, Rina; Kay, Lewis E

2017-11-01

The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl-TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR-based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1-DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes. © 2017 The Protein Society.

Identification of SFBB-containing canonical and noncanonical SCF complexes in pollen of apple (Malus × domestica).

PubMed

Minamikawa, Mai F; Koyano, Ruriko; Kikuchi, Shinji; Koba, Takato; Sassa, Hidenori

2014-01-01

Gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a single polymorphic S locus. The S locus contains at least two genes, S-RNase and F-box protein encoding gene SLF/SFB/SFBB that control pistil and pollen specificity, respectively. Generally, the F-box protein forms an E3 ligase complex, SCF complex with Skp1, Cullin1 (CUL1) and Rbx1, however, in Petunia inflata, SBP1 (S-RNase binding protein1) was reported to play the role of Skp1 and Rbx1, and form an SCFSLF-like complex for ubiquitination of non-self S-RNases. On the other hand, in Petunia hybrida and Petunia inflata of Solanaceae, Prunus avium and Pyrus bretschneideri of Rosaceae, SSK1 (SLF-interacting Skp1-like protein1) is considered to form the SCFSLF/SFB complex. Here, we isolated pollen-expressed apple homologs of SSK1 and CUL1, and named MdSSK1, MdCUL1A and MdCUL1B. MdSSK1 was preferentially expressed in pollen, but weakly in other organs analyzed, while, MdCUL1A and MdCUL1B were almost equally expressed in all the organs analyzed. MdSSK1 transcript abundance was significantly (>100 times) higher than that of MdSBP1. In vitro binding assays showed that MdSSK1 and MdSBP1 interacted with MdSFBB1-S9 and MdCUL1, and MdSFBB1-S9 interacted more strongly with MdSSK1 than with MdSBP1. The results suggest that both MdSSK1-containing SCFSFBB1 and MdSBP1-containing SCFSFBB1-like complexes function in pollen of apple, and the former plays a major role.

Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova*

PubMed Central

Buczek, Pawel; Horvath, Martin P.

2009-01-01

In Sterkiella nova, α and β telomere proteins bind cooperatively with single-stranded DNA to form a ternary α·β·DNA complex. Association of telomere protein subunits is DNA-dependent, and α-β association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, α and DNA first form a stable α·DNA complex followed by addition of β in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different with ΔCp = −305 ± 3 cal mol−1 K−1 for α binding with DNA and ΔCp = −2010 ± 20 cal mol−1 K−1 for addition of β to complete the α·β·DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed α·β complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of β. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length β or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of β. PMID:17082188

ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo.

PubMed

Beilschmidt, Lena Kristina; Ollagnier de Choudens, Sandrine; Fournier, Marjorie; Sanakis, Ioannis; Hograindleur, Marc-André; Clémancey, Martin; Blondin, Geneviève; Schmucker, Stéphane; Eisenmann, Aurélie; Weiss, Amélie; Koebel, Pascale; Messaddeq, Nadia; Puccio, Hélène; Martelli, Alain

2017-05-11

Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron-sulfur cluster (Fe-S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial Fe 4 S 4 proteins. Here we report that mouse ISCA1 and ISCA2 are Fe 2 S 2 -containing proteins that combine all features of Fe-S carrier proteins. We use biochemical, spectroscopic and in vivo approaches to demonstrate that despite forming a complex, ISCA1 and ISCA2 establish discrete interactions with components of the late Fe-S machinery. Surprisingly, knockdown experiments in mouse skeletal muscle and in primary cultures of neurons suggest that ISCA1, but not ISCA2, is required for mitochondrial Fe 4 S 4 proteins biogenesis. Collectively, our data suggest that cellular processes with different requirements for ISCA1, ISCA2 and ISCA1-ISCA2 complex seem to exist.

ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo

PubMed Central

Beilschmidt, Lena Kristina; Ollagnier de Choudens, Sandrine; Fournier, Marjorie; Sanakis, Ioannis; Hograindleur, Marc-André; Clémancey, Martin; Blondin, Geneviève; Schmucker, Stéphane; Eisenmann, Aurélie; Weiss, Amélie; Koebel, Pascale; Messaddeq, Nadia; Puccio, Hélène; Martelli, Alain

2017-01-01

Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron–sulfur cluster (Fe–S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial Fe4S4 proteins. Here we report that mouse ISCA1 and ISCA2 are Fe2S2-containing proteins that combine all features of Fe–S carrier proteins. We use biochemical, spectroscopic and in vivo approaches to demonstrate that despite forming a complex, ISCA1 and ISCA2 establish discrete interactions with components of the late Fe–S machinery. Surprisingly, knockdown experiments in mouse skeletal muscle and in primary cultures of neurons suggest that ISCA1, but not ISCA2, is required for mitochondrial Fe4S4 proteins biogenesis. Collectively, our data suggest that cellular processes with different requirements for ISCA1, ISCA2 and ISCA1–ISCA2 complex seem to exist. PMID:28492233

Imaging of DNA and Protein by SFM and Combined SFM-TIRF Microscopy.

PubMed

Grosbart, Małgorzata; Ristić, Dejan; Sánchez, Humberto; Wyman, Claire

2018-01-01

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nm resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

Phycomyces MADB interacts with MADA to form the primary photoreceptor complex for fungal phototropism.

PubMed

Sanz, Catalina; Rodríguez-Romero, Julio; Idnurm, Alexander; Christie, John M; Heitman, Joseph; Corrochano, Luis M; Eslava, Arturo P

2009-04-28

The fungus Phycomyces blakesleeanus reacts to environmental signals, including light, gravity, touch, and the presence of nearby objects, by changing the speed and direction of growth of its fruiting body (sporangiophore). Phototropism, growth toward light, shares many features in fungi and plants but the molecular mechanisms remain to be fully elucidated. Phycomyces mutants with altered phototropism were isolated approximately 40 years ago and found to have mutations in the mad genes. All of the responses to light in Phycomyces require the products of the madA and madB genes. We showed that madA encodes a protein similar to the Neurospora blue-light photoreceptor, zinc-finger protein WC-1. We show here that madB encodes a protein similar to the Neurospora zinc-finger protein WC-2. MADA and MADB interact to form a complex in yeast 2-hybrid assays and when coexpressed in E. coli, providing evidence that phototropism and other responses to light are mediated by a photoresponsive transcription factor complex. The Phycomyces genome contains 3 genes similar to wc-1, and 4 genes similar to wc-2, many of which are regulated by light in a madA or madB dependent manner. We did not detect any interactions between additional WC proteins in yeast 2-hybrid assays, which suggest that MADA and MADB form the major photoreceptor complex in Phycomyces. However, the presence of multiple wc genes in Phycomyces may enable perception across a broad range of light intensities, and may provide specialized photoreceptors for distinct photoresponses.

Phycomyces MADB interacts with MADA to form the primary photoreceptor complex for fungal phototropism

PubMed Central

Sanz, Catalina; Rodríguez-Romero, Julio; Idnurm, Alexander; Christie, John M.; Heitman, Joseph; Corrochano, Luis M.; Eslava, Arturo P.

2009-01-01

The fungus Phycomyces blakesleeanus reacts to environmental signals, including light, gravity, touch, and the presence of nearby objects, by changing the speed and direction of growth of its fruiting body (sporangiophore). Phototropism, growth toward light, shares many features in fungi and plants but the molecular mechanisms remain to be fully elucidated. Phycomyces mutants with altered phototropism were isolated ≈40 years ago and found to have mutations in the mad genes. All of the responses to light in Phycomyces require the products of the madA and madB genes. We showed that madA encodes a protein similar to the Neurospora blue-light photoreceptor, zinc-finger protein WC-1. We show here that madB encodes a protein similar to the Neurospora zinc-finger protein WC-2. MADA and MADB interact to form a complex in yeast 2-hybrid assays and when coexpressed in E. coli, providing evidence that phototropism and other responses to light are mediated by a photoresponsive transcription factor complex. The Phycomyces genome contains 3 genes similar to wc-1, and 4 genes similar to wc-2, many of which are regulated by light in a madA or madB dependent manner. We did not detect any interactions between additional WC proteins in yeast 2-hybrid assays, which suggest that MADA and MADB form the major photoreceptor complex in Phycomyces. However, the presence of multiple wc genes in Phycomyces may enable perception across a broad range of light intensities, and may provide specialized photoreceptors for distinct photoresponses. PMID:19380729

α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

PubMed Central

Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M.; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

2014-01-01

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. PMID:24778182

Understanding the nanoparticle-protein corona complexes using computational and experimental methods.

PubMed

Kharazian, B; Hadipour, N L; Ejtehadi, M R

2016-06-01

Nanoparticles (NP) have capability to adsorb proteins from biological fluids and form protein layer, which is called protein corona. As the cell sees corona coated NPs, the protein corona can dictate biological response to NPs. The composition of protein corona is varied by physicochemical properties of NPs including size, shape, surface chemistry. Processing of protein adsorption is dynamic phenomena; to that end, a protein may desorb or leave a surface vacancy that is rapidly filled by another protein and cause changes in the corona composition mainly by the Vroman effect. In this review, we discuss the interaction between NP and proteins and the available techniques for identification of NP-bound proteins. Also we review current developed computational methods for understanding the NP-protein complex interactions. Copyright © 2016. Published by Elsevier Ltd.

PucC and LhaA direct efficient assembly of the light‐harvesting complexes in Rhodobacter sphaeroides

PubMed Central

Mothersole, David J.; Jackson, Philip J.; Vasilev, Cvetelin; Tucker, Jaimey D.; Brindley, Amanda A.; Dickman, Mark J.

2015-01-01

Summary The mature architecture of the photosynthetic membrane of the purple phototroph R hodobacter sphaeroides has been characterised to a level where an atomic‐level membrane model is available, but the roles of the putative assembly proteins LhaA and PucC in establishing this architecture are unknown. Here we investigate the assembly of light‐harvesting LH2 and reaction centre‐light‐harvesting1‐PufX (RC‐LH1‐PufX) photosystem complexes using spectroscopy, pull‐downs, native gel electrophoresis, quantitative mass spectrometry and fluorescence lifetime microscopy to characterise a series of lha A and puc C mutants. LhaA and PucC are important for specific assembly of LH1 or LH2 complexes, respectively, but they are not essential; the few LH1 subunits found in Δlha A mutants assemble to form normal RC‐LH1‐PufX core complexes showing that, once initiated, LH1 assembly round the RC is cooperative and proceeds to completion. LhaA and PucC form oligomers at sites of initiation of membrane invagination; LhaA associates with RCs, bacteriochlorophyll synthase (BchG), the protein translocase subunit YajC and the YidC membrane protein insertase. These associations within membrane nanodomains likely maximise interactions between pigments newly arriving from BchG and nascent proteins within the SecYEG‐SecDF‐YajC‐YidC assembly machinery, thereby co‐ordinating pigment delivery, the co‐translational insertion of LH polypeptides and their folding and assembly to form photosynthetic complexes. PMID:26419219

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

PubMed Central

Castillo, Virginia; Ventura, Salvador

2009-01-01

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins. PMID:19696882

The Legionella IcmS-IcmW protein complex is important for Dot/Icm-mediated protein translocation.

PubMed

Ninio, Shira; Zuckman-Cholon, Deborah M; Cambronne, Eric D; Roy, Craig R

2005-02-01

The intracellular pathogen Legionella pneumophila can infect and replicate within macrophages of a human host. To establish infection, Legionella require the Dot/Icm secretion system to inject protein substrates directly into the host cell cytoplasm. The mechanism by which substrate proteins are engaged and translocated by the Dot/Icm system is not well understood. Here we show that two cytosolic components of the Dot/Icm secretion machinery, the proteins IcmS and IcmW, play an important role in substrate translocation. Biochemical analysis indicates that IcmS and IcmW form a stable protein complex. In Legionella, the IcmW protein is rapidly degraded in the absence of the IcmS protein. Substrate proteins translocated into mammalian host cells by the Dot/Icm system were identified using the IcmW protein as bait in a yeast two-hybrid screen. It was determined that the IcmS-IcmW complex interacts with these substrates and plays an important role in translocation of these proteins into mammalian cells. These data are consistent with the IcmS-IcmW complex being involved in the recognition and Dot/Icm-dependent translocation of substrate proteins during Legionella infection of host cells.

Uncoordinated (UNC)119: coordinating the trafficking of myristoylated proteins.

PubMed

Constantine, Ryan; Zhang, Houbin; Gerstner, Cecilia D; Frederick, Jeanne M; Baehr, Wolfgang

2012-12-15

The mechanism by which myristoylated proteins are targeted to specific subcellular membrane compartments is poorly understood. Two novel acyl-binding proteins, UNC119A and UNC119B, have been shown recently to function as chaperones/co-factors in the transport of myristoylated G protein α-subunits and src-type tyrosine kinases. UNC119 polypeptides feature an immunoglobulin-like β-sandwich fold that forms a hydrophobic pocket capable of binding lauroyl (C12) and myristoyl (C14) side chains. UNC119A in rod photoreceptors facilitates the transfer of transducin α subunits (Tα) from inner segment to outer segment membranes by forming an intermediate diffusible UNC119-Tα complex. Similar complexes are formed in other sensory neurons, as the G proteins ODR-3 and GPA-13 in Caenorhabditis elegans unc-119 mutants traffic inappropriately. UNC119B knockdown in IMCD3 cells prevents trafficking ofmyristoylated nephrocystin-3 (NPHP3), a protein associated with nephronophthisis, to cilia. Further, UNC119A was shown to transport myristoylated src-type tyrosine kinases to cell membranes and to affect T-cell receptor (TCR) and interleukin-5 receptor (IL-5R) activities. These interactions establish UNC119 polypeptides as novel lipid-binding chaperones with specificity for a diverse subset of myristoylated proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

Uncoordinated (UNC)119: Coordinating the Trafficking of Myristoylated Proteins

PubMed Central

Constantine, Ryan; Zhang, Houbin; Gerstner, Cecilia D.; Frederick, Jeanne M.; Baehr, Wolfgang

2012-01-01

The mechanism by which myristoylated proteins are targeted to specific subcellular membrane compartments is poorly understood. Two novel acyl-binding proteins, UNC119A and UNC119B, have been shown recently to function as chaperones/co-factors in the transport of myristoylated G protein α-subunits and src-type tyrosine kinases. UNC119 polypeptides feature an immunoglobulin-like β-sandwich fold that forms a hydrophobic pocket capable of binding lauroyl (C12) and myristoyl (C14) side chains. UNC119A in rod photoreceptors facilitates the transfer of transducin α subunits (Tα) from inner segment to outer segment membranes by forming an intermediate diffusible UNC119-Tα complex. Similar complexes are formed in other sensory neurons, as the G proteins ODR-3 and GPA-13 in C. elegans unc-119 mutants traffic inappropriately. UNC119B knockdown in IMCD3 cells prevents trafficking of myristoylated nephrocystin-3 (NPHP3), a protein associated with nephronophthisis, to cilia. Further, UNC119A was shown to transpot myristoylated src-type tyrosine kinases to cell membranes and to affect T-cell receptor (TCR) and interleukin-5 receptor (IL-5R) activities. These interactions establish UNC119 polypeptides as novel lipid-binding chaperones with specificity for a diverse subset of myristoylated proteins. PMID:23000199

Protein Corona Influences Cell-Biomaterial Interactions in Nanostructured Tissue Engineering Scaffolds.

PubMed

Serpooshan, Vahid; Mahmoudi, Morteza; Zhao, Mingming; Wei, Ke; Sivanesan, Senthilkumar; Motamedchaboki, Khatereh; Malkovskiy, Andrey V; Gladstone, Andrew B; Cohen, Jeffrey E; Yang, Phillip C; Rajadas, Jayakumar; Bernstein, Daniel; Woo, Y Joseph; Ruiz-Lozano, Pilar

2015-07-22

Biomaterials are extensively used to restore damaged tissues, in the forms of implants (e.g. tissue engineered scaffolds) or biomedical devices (e.g. pacemakers). Once in contact with the physiological environment, nanostructured biomaterials undergo modifications as a result of endogenous proteins binding to their surface. The formation of this macromolecular coating complex, known as 'protein corona', onto the surface of nanoparticles and its effect on cell-particle interactions are currently under intense investigation. In striking contrast, protein corona constructs within nanostructured porous tissue engineering scaffolds remain poorly characterized. As organismal systems are highly dynamic, it is conceivable that the formation of distinct protein corona on implanted scaffolds might itself modulate cell-extracellular matrix interactions. Here, we report that corona complexes formed onto the fibrils of engineered collagen scaffolds display specific, distinct, and reproducible compositions that are a signature of the tissue microenvironment as well as being indicative of the subject's health condition. Protein corona formed on collagen matrices modulated cellular secretome in a context-specific manner ex-vivo , demonstrating their role in regulating scaffold-cellular interactions. Together, these findings underscore the importance of custom-designing personalized nanostructured biomaterials, according to the biological milieu and disease state. We propose the use of protein corona as in situ biosensor of temporal and local biomarkers.

Protein associations in DnaA-ATP hydrolysis mediated by the Hda-replicase clamp complex.

PubMed

Su'etsugu, Masayuki; Shimuta, Toh-Ru; Ishida, Takuma; Kawakami, Hironori; Katayama, Tsutomu

2005-02-25

In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the beta-sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified mutant Hda proteins were used in a staged in vitro RIDA system followed by a pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arg(168) in the AAA(+) Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain approximately 50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA(+) proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA(+) domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.

A novel germ cell-specific protein, SHIP1, forms a complex with chromatin remodeling activity during spermatogenesis.

PubMed

Choi, Eunyoung; Han, Cecil; Park, Inju; Lee, Boyeon; Jin, Sora; Choi, Heejin; Kim, Do Han; Park, Zee Yong; Eddy, Edward M; Cho, Chunghee

2008-12-12

To determine the mechanisms of spermatogenesis, it is essential to identify and characterize germ cell-specific genes. Here we describe a protein encoded by a novel germ cell-specific gene, Mm.290718/ZFP541, identified from the mouse spermatocyte UniGene library. The protein contains specific motifs and domains potentially involved in DNA binding and chromatin reorganization. An antibody against Mm.290718/ZFP541 revealed the existence of the protein in testicular spermatogenic cells (159 kDa) but not testicular and mature sperm. Immunostaining analysis of cells at various stages of spermatogenesis consistently showed that the protein is present in spermatocytes and round spermatids only. Transfection assays and immunofluorescence studies indicate that the protein is localized specifically in the nucleus. Proteomic analyses performed to explore the functional characteristics of Mm.290718/ZFP541 showed that the protein forms a unique complex. Other major components of the complex included histone deacetylase 1 (HDAC1) and heat-shock protein A2. Disappearance of Mm.290718/ZFP541 was highly correlated with hyperacetylation in spermatids during spermatogenesis, and specific domains of the protein were involved in the regulation of interactions and nuclear localization of HDAC1. Furthermore, we found that premature hyperacetylation, induced by an HDAC inhibitor, is associated with an alteration in the integrity of Mm.290718/ZFP541 in spermatogenic cells. Our results collectively suggest that the Mm.290718/ZFP541 complex is implicated in chromatin remodeling during spermatogenesis, and we provide further information on the previously unknown molecular mechanism. Consequently, we re-designate Mm.290718/ZFP541 as "SHIP1" representing spermatogenic cell HDAC-interacting protein 1.

Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

PubMed Central

Urata, Shuzo; Yokosawa, Hideyoshi; Yasuda, Jiro

2007-01-01

Background HTLV-1 Gag protein is a matrix protein that contains the PTAP and PPPY sequences as L-domain motifs and which can be released from mammalian cells in the form of virus-like particles (VLPs). The cellular factors Tsg101 and Nedd4.1 interact with PTAP and PPPY, respectively, within the HTLV-1 Gag polyprotein. Tsg101 forms a complex with Vps28 and Vps37 (ESCRT-I complex) and plays an important role in the class E Vps pathway, which mediates protein sorting and invagination of vesicles into multivesicular bodies. Nedd4.1 is an E3 ubiquitin ligase that binds to the PPPY motif through its WW motif, but its function is still unknown. In the present study, to investigate the mechanism of HTLV-1 budding in detail, we analyzed HTLV-1 budding using dominant negative (DN) forms of the class E proteins. Results Here, we report that DN forms of Vps4A, Vps4B, and AIP1 inhibit HTLV-1 budding. Conclusion These findings suggest that HTLV-1 budding utilizes the MVB pathway and that these class E proteins may be targets for prevention of mother-to-infant vertical transmission of the virus. PMID:17601348

A Glutaredoxin·BolA Complex Serves as an Iron-Sulfur Cluster Chaperone for the Cytosolic Cluster Assembly Machinery*♦

PubMed Central

Frey, Avery G.; Palenchar, Daniel J.; Wildemann, Justin D.; Philpott, Caroline C.

2016-01-01

Cells contain hundreds of proteins that require iron cofactors for activity. Iron cofactors are synthesized in the cell, but the pathways involved in distributing heme, iron-sulfur clusters, and ferrous/ferric ions to apoproteins remain incompletely defined. In particular, cytosolic monothiol glutaredoxins and BolA-like proteins have been identified as [2Fe-2S]-coordinating complexes in vitro and iron-regulatory proteins in fungi, but it is not clear how these proteins function in mammalian systems or how this complex might affect Fe-S proteins or the cytosolic Fe-S assembly machinery. To explore these questions, we use quantitative immunoprecipitation and live cell proximity-dependent biotinylation to monitor interactions between Glrx3, BolA2, and components of the cytosolic iron-sulfur cluster assembly system. We characterize cytosolic Glrx3·BolA2 as a [2Fe-2S] chaperone complex in human cells. Unlike complexes formed by fungal orthologs, human Glrx3-BolA2 interaction required the coordination of Fe-S clusters, whereas Glrx3 homodimer formation did not. Cellular Glrx3·BolA2 complexes increased 6–8-fold in response to increasing iron, forming a rapidly expandable pool of Fe-S clusters. Fe-S coordination by Glrx3·BolA2 did not depend on Ciapin1 or Ciao1, proteins that bind Glrx3 and are involved in cytosolic Fe-S cluster assembly and distribution. Instead, Glrx3 and BolA2 bound and facilitated Fe-S incorporation into Ciapin1, a [2Fe-2S] protein functioning early in the cytosolic Fe-S assembly pathway. Thus, Glrx3·BolA is a [2Fe-2S] chaperone complex capable of transferring [2Fe-2S] clusters to apoproteins in human cells. PMID:27519415

Domain-Swapped Dimers of Intracellular Lipid-Binding Proteins: Evidence for Ordered Folding Intermediates.

PubMed

Assar, Zahra; Nossoni, Zahra; Wang, Wenjing; Santos, Elizabeth M; Kramer, Kevin; McCornack, Colin; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H

2016-09-06

Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form. Copyright © 2016 Elsevier Ltd. All rights reserved.

Crystal structure of the Csm3-Csm4 subcomplex in the type III-A CRISPR-Cas interference complex.

PubMed

Numata, Tomoyuki; Inanaga, Hideko; Sato, Chikara; Osawa, Takuo

2015-01-30

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci play a pivotal role in the prokaryotic host defense system against invading genetic materials. The CRISPR loci are transcribed to produce CRISPR RNAs (crRNAs), which form interference complexes with CRISPR-associated (Cas) proteins to target the invading nucleic acid for degradation. The interference complex of the type III-A CRISPR-Cas system is composed of five Cas proteins (Csm1-Csm5) and a crRNA, and targets invading DNA. Here, we show that the Csm1, Csm3, and Csm4 proteins from Methanocaldococcus jannaschii form a stable subcomplex. We also report the crystal structure of the M. jannaschii Csm3-Csm4 subcomplex at 3.1Å resolution. The complex structure revealed the presence of a basic concave surface around their interface, suggesting the RNA and/or DNA binding ability of the complex. A gel retardation analysis showed that the Csm3-Csm4 complex binds single-stranded RNA in a non-sequence-specific manner. Csm4 structurally resembles Cmr3, a component of the type III-B CRISPR-Cas interference complex. Based on bioinformatics, we constructed a model structure of the Csm1-Csm4-Csm3 ternary complex, which provides insights into its role in the Csm interference complex. Copyright © 2014 Elsevier Ltd. All rights reserved.

Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein.

PubMed

Tayou, Junior; Wang, Qiang; Jang, Geeng-Fu; Pronin, Alexey N; Orlandi, Cesare; Martemyanov, Kirill A; Crabb, John W; Slepak, Vladlen Z

2016-04-22

RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gβ5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gβ5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular architecture of polycomb repressive complexes

PubMed Central

Chittock, Emily C.; Latwiel, Sebastian; Miller, Thomas C.R.

2017-01-01

The polycomb group (PcG) proteins are a large and diverse family that epigenetically repress the transcription of key developmental genes. They form three broad groups of polycomb repressive complexes (PRCs) known as PRC1, PRC2 and Polycomb Repressive DeUBiquitinase, each of which modifies and/or remodels chromatin by distinct mechanisms that are tuned by having variable compositions of core and accessory subunits. Until recently, relatively little was known about how the various PcG proteins assemble to form the PRCs; however, studies by several groups have now allowed us to start piecing together the PcG puzzle. Here, we discuss some highlights of recent PcG structures and the insights they have given us into how these complexes regulate transcription through chromatin. PMID:28202673

Structural characterization of more potent alternatives to HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.

PubMed

Nemashkalova, Ekaterina L; Kazakov, Alexei S; Khasanova, Leysan M; Permyakov, Eugene A; Permyakov, Sergei E

2013-09-10

HAMLET is a complex of human α-lactalbumin (hLA) with oleic acid (OA) that kills various tumor cells and strains of Streptococcus pneumoniae. More potent protein-OA complexes were previously reported for bovine α-lactalbumin (bLA) and β-lactoglobulin (bLG), and pike parvalbumin (pPA), and here we explore their structural features. The concentration dependencies of the tryptophan fluorescence of hLA, bLA, and bLG complexes with OA reveal their disintegration at protein concentrations below the micromolar level. Chemical cross-linking experiments provide evidence that association with OA shifts the distribution of oligomeric forms of hLA, bLA, bLG, and pPA toward higher-order oligomers. This effect is confirmed for bLA and bLG using the dynamic light scattering method, while pPA is shown to associate with OA vesicles. Like hLA binding, OA binding increases the affinity of bLG for small unilamellar dipalmitoylphosphatidylcholine vesicles, while pPA efficiently binds to the vesicles irrespective of OA binding. The association of OA with bLG and pPA increases their α-helix and cross-β-sheet content and resistance to enzymatic proteolysis, which is indicative of OA-induced protein structuring. The lack of excess heat sorption during melting of bLG and pPA in complex with OA and the presence of a cooperative thermal transition at the level of their secondary structure suggest that the OA-bound forms of bLG and pPA lack a fixed tertiary structure but exhibit a continuous thermal transition. Overall, despite marked differences, the HAMLET-like complexes that were studied exhibit a common feature: a tendency toward protein oligomerization. Because OA-induced oligomerization has been reported for other proteins, this phenomenon is inherent to many proteins.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

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

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding sitemore » are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.« less

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

PubMed

Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

2015-06-05

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

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

Bienz, K.; Egger, D.; Troxler, M.

1990-03-01

Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but didmore » not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed.« less

Proteins required for lipopolysaccharide assembly in Escherichia coli form a trans-envelope complex†

PubMed Central

Chng, Shu-Sin; Gronenberg, Luisa S.; Kahne, Daniel

2010-01-01

The viability of Gram-negative organisms is dependent on the proper placement of lipopolysaccharide (LPS) in the outer leaflet of its outer membrane. LPS is synthesized inside the cell and transported to the surface by seven essential Lpt proteins. How these proteins cooperate to transport LPS is unknown. We show that these Lpt proteins can be found in a membrane fraction that contains inner and outer membranes, and that they co-purify. This constitutes the first evidence that the Lpt proteins form a trans-envelope complex. We suggest that this protein bridge provides a route for LPS transport across the cell envelope. PMID:20446753

Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb.

PubMed

Neuveut, C; Low, K G; Maldarelli, F; Schmitt, I; Majone, F; Grassmann, R; Jeang, K T

1998-06-01

Human T-cell leukemia virus type 1 is etiologically linked to the development of adult T-cell leukemia and various human neuropathies. The Tax protein of human T-cell leukemia virus type I has been implicated in cellular transformation. Like other oncoproteins, such as Myc, Jun, and Fos, Tax is a transcriptional activator. How it mechanistically dysregulates the cell cycle is unclear. Previously, it was suggested that Tax affects cell-phase transition by forming a direct protein-protein complex with p16(INK4a), thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16(INK4a), Tax can compel an egress of cells from G0/G1 into S despite the absence of serum. We also show that in undifferentiated myocytes, expression of Tax represses cellular differentiation. In both settings, Tax expression was found to increase cyclin D-cdk activity and to enhance pRb phosphorylation. In T cells, a Tax-associated increase in steady-state E2F2 protein was also documented. In searching for a molecular explanation for these observations, we found that Tax forms a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein. Taken together, these findings suggest that Tax might directly influence cyclin D-cdk activity and function, perhaps by a route independent of cdk inhibitors such as p16(INK4a).

The trehalose/maltose-binding protein as the sensitive element of a glucose biosensor

NASA Astrophysics Data System (ADS)

Fonin, A. V.; Povarova, O. I.; Staiano, M.; D'Auria, S.; Turoverov, K. K.; Kuznetsova, I. M.

2014-08-01

The promising direction of the development of a modern glucometer is the construction of sensing element on the basis of stained (dyed) protein which changes its fluorescence upon glucose binding. One of the proteins that can be used for this purpose is the D-trehalose/D-maltose-binding protein (TMBP) from the thermophilic bacteria Thermococcus litoralis. We investigated the physical-chemical properties of the protein and evaluated its stability to the denaturing action of GdnHCl and heating. It was confirmed that TMBP is an extremely stable protein. In vivo, the intrinsic ligands of TMBP are trehalose and maltose, but TMBP can also bind glucose. The dissociation constant of the TMBP-glucose complex is in the range of 3-8 mM. The binding of glucose does not noticeably change the intrinsic fluorescence of the TMBP. To register protein-glucose binding, we used the fluorescence of the thiol-reactive dye BADAN attached to TMBP. Because the fluorescence of BADAN attached to the cysteine Cys182 of TMBP does not change upon glucose binding, the mutant forms ТМВР/C182S/X_Cys were created. In these mutant proteins, Cys182 is replaced by Ser, removing intrinsic binding site of BADAN and a new dye binding sites were introduced. The largest increase (by 1.4 times) in the intensity of the dye fluorescence was observed upon TMBP/C182S/A14C-BADAN-Glc complex formation. The dissociation constant of this complex is 3.4 ± 0.1 mM. We consider TMBP/C182S/A14C mutant form with attached fluorescent dye BADAN as a good basis for further research aimed to develop of series of TMBP mutant forms with different affinities to glucose labeled with fluorescent dyes.

AAA-ATPases in Protein Degradation

PubMed Central

Yedidi, Ravikiran S.; Wendler, Petra; Enenkel, Cordula

2017-01-01

Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea. PMID:28676851

AAA-ATPases in Protein Degradation.

PubMed

Yedidi, Ravikiran S; Wendler, Petra; Enenkel, Cordula

2017-01-01

Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

Function of multiple Lis-Homology domain/WD-40 repeat-containing proteins in feed-forward transcriptional repression by silencing mediator for retinoic and thyroid receptor/nuclear receptor corepressor complexes.

PubMed

Choi, Hyo-Kyoung; Choi, Kyung-Chul; Kang, Hee-Bum; Kim, Han-Cheon; Lee, Yoo-Hyun; Haam, Seungjoo; Park, Hyoung-Gi; Yoon, Ho-Geun

2008-05-01

Lis-homology (LisH) motifs are involved in protein dimerization, and the discovery of the conserved N-terminal LisH domain in transducin beta-like protein 1 and its receptor (TBL1 and TBLR1) led us to examine the role of this domain in transcriptional repression. Here we show that multiple beta-transducin (WD-40) repeat-containing proteins interact to form oligomers in solution and that oligomerization depends on the presence of the LisH domain in each protein. Repression of transcription, as assayed using Gal4 fusion proteins, also depended on the presence of the LisH domain, suggesting that oligomerization is a prerequisite for efficient transcriptional repression. Furthermore, we show that the LisH domain is responsible for the binding to the hypoacetylated histone H4 tail and for stable chromatin targeting by the nuclear receptor corepressor complex. Mutations in conserved residues in the LisH motif of TBL1 and TBLR1 block histone binding, oligomerization, and transcriptional repression, supporting the functional importance of the LisH motif in transcriptional repression. Our results indicate that another WD-40 protein, TBL3, also preferentially binds to the N-terminal domain of TBL1 and TBLR1, and forms oligomers with other WD-40 proteins. Finally, we observed that the WD-40 proteins RbAp46 and RbAp48 of the sin3A corepressor complex failed to dimerize. We also found the specific interaction UbcH/E2 with TBL1, but not RbAp46/48. Altogether, our results thus indicate that the presence of multiple LisH/WD-40 repeat containing proteins is exclusive to nuclear receptor corepressor/ silencing mediator for retinoic and thyroid receptor complexes compared with other class 1 histone deacetylase-containing corepessor complexes.

Nuclear Pore Complexes: Global Conservation and Local Variation.

PubMed

Holzer, Guillaume; Antonin, Wolfram

2018-06-04

Nuclear pore complexes are the transport gates to the nucleus. Most proteins forming these huge complexes are evolutionarily conserved, as is the eightfold symmetry of these complexes. A new study reporting the structure of the yeast nuclear pore complex now shows striking differences from its human counterpart. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biochemical indicators of nephrotoxicity in blood serum of rats treated with novel 4-thiazolidinone derivatives or their complexes with polyethylene glycol-containing nanoscale polymeric carrier

PubMed

Kоbyli nska, L I; Havrylyuk, D Ya; Mitina, N E; Zaichenko, A S; Lesyk, R B; Zіme nkovsky, B S; Stoika, R S

2016-01-01

The aim of this study was to compare the effect of new synthetic 4-thiazolidinone derivatives (potential anticancer compounds denoted as 3882, 3288 and 3833) and doxorubicin (positive control) in free form and in their complexes with synthetic polyethylene glycol-containing nanoscale polymeric carrier on the biochemical indicators of nephrotoxicity in blood serum of rats. The concentration of total protein, urea, creatinine, glucose, ions of sodium, potassium, calcium, iron and chloride was measured. It was found that after injection of the investigated compounds, the concentration of sodium cations and chloride anions in blood serum was increased compared with control (untreated animals). Doxorubicin’s injection was accompanied by a decrease in the concentration of iron cations. The concentration of total protein, urea and creatinine decreased under the influence of the studied compounds. Complexation of these аntineoplastic substances with a synthetic polymeric nanocarrier lowered the concentration of the investigated metabolites substantially compared to the effect of these compounds in free form. The normalization of concentration of total protein, urea and creatinine in blood serum of rats treated with complexes of the studied compounds with the polymeric carrier comparing with increased concentration of these indicators at the introduction of such compounds in free form was found.

Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis.

PubMed

Chen, Jia-Liang; Zhao, Yu; Gong, Yan-Jun; Pan, Bin-Bin; Wang, Xiao; Su, Xun-Cheng

2018-02-01

Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein-ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.

Triage of oxidation-prone proteins by Sqstm1/p62 within the mitochondria

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

Lee, Minjung; Shin, Jaekyoon, E-mail: jkshin@med.skku.ac.kr

2011-09-16

Highlights: {yields} The mitochondrion contains its own protein quality control system. {yields} p62 localizes within the mitochondria and forms mega-dalton sized complexes. {yields} p62 interacts with oxidation-prone proteins and the proteins of quality control. {yields} In vitro delivery of p62 improves mitochondrial functions. {yields} p62 is implicated as a participant in mitochondrial protein quality control. -- Abstract: As the mitochondrion is vulnerable to oxidative stress, cells have evolved several strategies to maintain mitochondrial integrity, including mitochondrial protein quality control mechanisms and autophagic removal of damaged mitochondria. Involvement of an autophagy adaptor, Sqstm1/p62, in the latter process has been recently described.more » In the present study, we provide evidence that a portion of p62 directly localizes within the mitochondria and supports stable electron transport by forming heterogeneous protein complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of mitochondrial proteins co-purified with p62 revealed that p62 interacts with several oxidation-prone proteins, including a few components of the electron transport chain complexes, as well as multiple chaperone molecules and redox regulatory enzymes. Accordingly, p62-deficient mitochondria exhibited compromised electron transport, and the compromised function was partially restored by in vitro delivery of p62. These results suggest that p62 plays an additional role in maintaining mitochondrial integrity at the vicinity of target machineries through its function in relation to protein quality control.« less

Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

PubMed Central

Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

2011-01-01

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

Probing the modulated formation of gold nanoparticles-beta-lactoglobulin corona complexes and their applications.

PubMed

Yang, Jiang; Wang, Bo; You, Youngsang; Chang, Woo-Jin; Tang, Ke; Wang, Yi-Cheng; Zhang, Wenzhao; Ding, Feng; Gunasekaran, Sundaram

2017-11-23

Understanding the interactions between proteins and nanoparticles (NPs) along with the underlying structural and dynamic information is of utmost importance to exploit nanotechnology for biomedical applications. Upon adsorption onto a NP surface, proteins form a well-organized layer, termed the corona, that dictates the identity of the NP-protein complex and governs its biological pathways. Given its high biological relevance, in-depth molecular investigations and applications of NPs-protein corona complexes are still scarce, especially since different proteins form unique corona patterns, making identification of the biomolecular motifs at the interface critical. In this work, we provide molecular insights and structural characterizations of the bio-nano interface of a popular food-based protein, namely bovine beta-lactoglobulin (β-LG), with gold nanoparticles (AuNPs) and report on our investigations of the formation of corona complexes by combined molecular simulations and complementary experiments. Two major binding sites in β-LG were identified as being driven by citrate-mediated electrostatic interactions, while the associated binding kinetics and conformational changes in the secondary structures were also characterized. More importantly, the superior stability of the corona led us to further explore its biomedical applications, such as in the smartphone-based point-of-care biosensing of Escherichia coli (E. coli) and in the computed tomography (CT) of the gastrointestinal (GI) tract through oral administration to probe GI tolerance and functions. Considering their biocompatibility, edible nature, and efficient excretion through defecation, AuNPs-β-LG corona complexes have shown promising perspectives for future in vitro and in vivo clinical settings.

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers.

PubMed

Veith, Paul D; Glew, Michelle D; Gorasia, Dhana G; Reynolds, Eric C

2017-10-01

The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum. Proteins secreted by the T9SS have an N-terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C-terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C-terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components. © 2017 John Wiley & Sons Ltd.

Template-Based Modeling of Protein-RNA Interactions

PubMed Central

Zheng, Jinfang; Kundrotas, Petras J.; Vakser, Ilya A.

2016-01-01

Protein-RNA complexes formed by specific recognition between RNA and RNA-binding proteins play an important role in biological processes. More than a thousand of such proteins in human are curated and many novel RNA-binding proteins are to be discovered. Due to limitations of experimental approaches, computational techniques are needed for characterization of protein-RNA interactions. Although much progress has been made, adequate methodologies reliably providing atomic resolution structural details are still lacking. Although protein-RNA free docking approaches proved to be useful, in general, the template-based approaches provide higher quality of predictions. Templates are key to building a high quality model. Sequence/structure relationships were studied based on a representative set of binary protein-RNA complexes from PDB. Several approaches were tested for pairwise target/template alignment. The analysis revealed a transition point between random and correct binding modes. The results showed that structural alignment is better than sequence alignment in identifying good templates, suitable for generating protein-RNA complexes close to the native structure, and outperforms free docking, successfully predicting complexes where the free docking fails, including cases of significant conformational change upon binding. A template-based protein-RNA interaction modeling protocol PRIME was developed and benchmarked on a representative set of complexes. PMID:27662342

Regulation of SMN Protein Stability▿ †

PubMed Central

Burnett, Barrington G.; Muñoz, Eric; Tandon, Animesh; Kwon, Deborah Y.; Sumner, Charlotte J.; Fischbeck, Kenneth H.

2009-01-01

Spinal muscular atrophy (SMA) is caused by mutations of the survival of motor neuron (SMN1) gene and deficiency of full-length SMN protein (FL-SMN). All SMA patients retain one or more copies of the SMN2 gene, but the principal protein product of SMN2 lacks exon 7 (SMNΔ7) and is unable to compensate for a deficiency of FL-SMN. SMN is known to oligomerize and form a multimeric protein complex; however, the mechanisms regulating stability and degradation of FL-SMN and SMNΔ7 proteins have been largely unexplored. Using pulse-chase analysis, we characterized SMN protein turnover and confirmed that SMN was ubiquitinated and degraded by the ubiquitin proteasome system (UPS). The SMNΔ7 protein had a twofold shorter half-life than FL-SMN in cells despite similar intrinsic rates of turnover by the UPS in a cell-free assay. Mutations that inhibited SMN oligomerization and complex formation reduced the FL-SMN half-life. Furthermore, recruitment of SMN into large macromolecular complexes as well as increased association with several Gemin proteins was regulated in part by protein kinase A. Together, our data indicate that SMN protein stability is modulated by complex formation. Promotion of the SMN complex formation may be an important novel therapeutic strategy for SMA. PMID:19103745

(PS)2: protein structure prediction server version 3.0.

PubMed

Huang, Tsun-Tsao; Hwang, Jenn-Kang; Chen, Chu-Huang; Chu, Chih-Sheng; Lee, Chi-Wen; Chen, Chih-Chieh

2015-07-01

Protein complexes are involved in many biological processes. Examining coupling between subunits of a complex would be useful to understand the molecular basis of protein function. Here, our updated (PS)(2) web server predicts the three-dimensional structures of protein complexes based on comparative modeling; furthermore, this server examines the coupling between subunits of the predicted complex by combining structural and evolutionary considerations. The predicted complex structure could be indicated and visualized by Java-based 3D graphics viewers and the structural and evolutionary profiles are shown and compared chain-by-chain. For each subunit, considerations with or without the packing contribution of other subunits cause the differences in similarities between structural and evolutionary profiles, and these differences imply which form, complex or monomeric, is preferred in the biological condition for the subunit. We believe that the (PS)(2) server would be a useful tool for biologists who are interested not only in the structures of protein complexes but also in the coupling between subunits of the complexes. The (PS)(2) is freely available at http://ps2v3.life.nctu.edu.tw/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure and Function of p97 and Pex1/6 Type II AAA+ Complexes.

PubMed

Saffert, Paul; Enenkel, Cordula; Wendler, Petra

2017-01-01

Protein complexes of the Type II AAA+ (ATPases associated with diverse cellular activities) family are typically hexamers of 80-150 kDa protomers that harbor two AAA+ ATPase domains. They form double ring assemblies flanked by associated domains, which can be N-terminal, intercalated or C-terminal to the ATPase domains. Most prominent members of this family include NSF (N-ethyl-maleimide sensitive factor), p97/VCP (valosin-containing protein), the Pex1/Pex6 complex and Hsp104 in eukaryotes and ClpB in bacteria. Tremendous efforts have been undertaken to understand the conformational dynamics of protein remodeling type II AAA+ complexes. A uniform mode of action has not been derived from these works. This review focuses on p97/VCP and the Pex1/6 complex, which both structurally remodel ubiquitinated substrate proteins. P97/VCP plays a role in many processes, including ER- associated protein degradation, and the Pex1/Pex6 complex dislocates and recycles the transport receptor Pex5 from the peroxisomal membrane during peroxisomal protein import. We give an introduction into existing knowledge about the biochemical and cellular activities of the complexes before discussing structural information. We particularly emphasize recent electron microscopy structures of the two AAA+ complexes and summarize their structural differences.

Complexes prepared from protein A and human serum, IgG, or Fc gamma fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion.

PubMed

Langone, J J; Das, C; Mainwaring, R; Shearer, W T

1985-01-01

Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of 125I-protein A and human 131I-IgG alone or in serum, and 131I-Fc gamma fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess 131I-IgG or 131I-Fc, all the 125I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess 125I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc gamma in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.

Protein-protein interactions in the RPS4/RRS1 immune receptor complex

PubMed Central

Sarris, Panagiotis F.

2017-01-01

Plant NLR (Nucleotide-binding domain and Leucine-rich Repeat) immune receptor proteins are encoded by Resistance (R) genes and confer specific resistance to pathogen races that carry the corresponding recognized effectors. Some NLR proteins function in pairs, forming receptor complexes for the perception of specific effectors. We show here that the Arabidopsis RPS4 and RRS1 NLR proteins are both required to make an authentic immune complex. Over-expression of RPS4 in tobacco or in Arabidopsis results in constitutive defense activation; this phenotype is suppressed in the presence of RRS1. RRS1 protein co-immunoprecipitates (co-IPs) with itself in the presence or absence of RPS4, but in contrast, RPS4 does not associate with itself in the absence of RRS1. In the presence of RRS1, RPS4 associates with defense signaling regulator EDS1 solely in the nucleus, in contrast to the extra-nuclear location found in the absence of RRS1. The AvrRps4 effector does not disrupt RPS4-EDS1 association in the presence of RRS1. In the absence of RRS1, AvrRps4 interacts with EDS1, forming nucleocytoplasmic aggregates, the formation of which is disturbed by the co-expression of PAD4 but not by SAG101. These data indicate that the study of an immune receptor protein complex in the absence of all components can result in misleading inferences, and reveals an NLR complex that dynamically interacts with the immune regulators EDS1/PAD4 or EDS1/SAG101, and with effectors, during the process by which effector recognition is converted to defense activation. PMID:28475615

Specific Binding of Protoporphyrin IX to a Membrane-Bound 63 Kilodalton Polypeptide in Cucumber Cotyledons Treated with Diphenyl Ether-Type Herbicides.

PubMed

Sato, R; Oshio, H; Koike, H; Inoue, Y; Yoshida, S; Takahashi, N

1991-06-01

Porphyrin accumulation in excised cucumber cotyledons (Cucumis sativus L.) treated with a N-phenylimide S-23142 (N-[4-chloro-2-fluoro-5-propargyloxyphenyl]-3,4,5,6- tetrahydrophthalimide) and a diphenylether acifluorfen-ethyl (ethyl-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitro benzoic acid) was studied. Most of the accumulated porphyrins were found in the membrane fractions of 6,000g and 30,000g pellets, forming a complex with a membrane polypeptide. The complex was solubilized with 1% n-dodecyl beta-d-maltoside and its molecular mass was estimated to be 63,000 and 66,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation high performance liquid chromatography (HPLC), respectively. The polypeptide also existed in untreated cotyledons but had little protoporphyrin IX. The complex was also formed in vitro by mixing the 30,000g pellets from untreated cotyledons and authentic protoporphyrin IX. However, protoporphyrin IX formed the complex specifically with the 63,000 dalton polypeptide and not with the other proteins both in vivo and in vitro. At least four fluorescent porphyrins, including protoporphyrin IX, were found in the acetone extract of the cotyledons by HPLC using a reversed phase column. Protoporphyrin IX was one of the two porphyrins that formed the complex. These results suggest that S-23142 and acifluorfenethyl enhance the accumulation of protoporphyrin IX, which forms the complex with the membrane protein.

Vitamin D in the avian egg. Its molecular identity and mechanism of incorporation into yolk.

PubMed Central

Fraser, D R; Emtage, J S

1976-01-01

The chemical identity of vitamin D in the egg of the domestic fowl was studied by analysing radioactivity in eggs from hens injected with [3H]cholecalciferol. Labelled molecules were found throughout the egg, but the concentration of total radioactivity in albumin was only 5-7% of that in yolk. In lipid extracts of yolk, more than 90% of the radioactivity was as unchanged cholecalciferol and 5% as 25-hydroxycholecalciferol. Only about 3% of the radioactivity in albumin was chloroform-soluble, and of this 40% was 25-hydroxycholecalciferol and 15% was cholecalciferol. Evidence is presented to support the idea that the specific transport of cholecalciferol into yolk is mediated by a cholecalciferol-binding protein in blood. This protein forms a complex with yolk proteins in transit from liver to ovary via the blood. A cholecalciferol-binding protein, chromatographically similar to that from blood, was found in egg yolk. It is postulated that cholecalciferol forms part of a complex with its specific binding protein, Ca2+ and the yolk phosphoprotein, phosvitin. This complex is then incorporated into yolk by the thecal cells of the ovarian follicle. PMID:189757

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin.

PubMed

Song, Tao; Fang, Liurong; Wang, Dang; Zhang, Ruoxi; Zeng, Songlin; An, Kang; Chen, Huanchun; Xiao, Shaobo

2016-06-16

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has heavily impacted the global swine industry. The PRRSV nonstructural protein 2 (nsp2) plays crucial roles in viral replication and host immune regulation, most likely by interacting with viral or cellular proteins that have not yet been identified. In this study, a quantitative interactome approach based on immunoprecipitation and stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify nsp2-interacting proteins in PRRSV-infected cells with an nsp2-specific monoclonal antibody. Nine viral proteins and 62 cellular proteins were identified as potential nsp2-interacting partners. Our data demonstrate that the PRRSV nsp1α, nsp1β, and nucleocapsid proteins all interact directly with nsp2. Nsp2-interacting cellular proteins were classified into different functional groups and an interactome network of nsp2 was generated. Interestingly, cellular vimentin, a known receptor for PRRSV, forms a complex with nsp2 by using viral nucleocapsid protein as an intermediate. Taken together, the nsp2 interactome under the condition of virus infection clarifies a role of nsp2 in PRRSV replication and immune evasion. Viral proteins must interact with other virus-encoded proteins and/or host cellular proteins to function, and interactome analysis is an ideal approach for identifying such interacting proteins. In this study, we used the quantitative interactome methodology to identify the viral and cellular proteins that potentially interact with the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) under virus infection conditions, thus providing a rich source of potential viral and cellular interaction partners for PRRSV nsp2. Based on the interactome data, we further demonstrated that PRRSV nsp2 and nucleocapsid protein together with cellular vimentin, form a complex that may be essential for viral attachment and replication, which partly explains the role of nsp2 in PRRSV replication and immune evasion. Copyright © 2016 Elsevier B.V. All rights reserved.

Purification of Transcript-Specific mRNP Complexes Formed In Vivo from Saccharomyces cerevisiae.

PubMed

Smith, Jenna E; Baker, Kristian E

2017-01-01

RNA binding proteins play critical roles in shaping the complex life cycle of cellular transcripts. For most RNAs, the association with a distinct complement of proteins serves to orchestrate its unique pattern of maturation, localization, translation, and stability. A key aspect to understanding how transcripts are differentially regulated lies, therefore, in the ability to identify the particular repertoire of protein binding partners associated with an individual transcript. We describe here an optimized experimental procedure for purifying a single mRNA population from yeast cells for the characterization of transcript-specific mRNA-protein complexes (mRNPs) as they exist in vivo. Chemical cross-linking is used to trap native mRNPs and facilitate the co-purification of protein complexes associated with an individual transcript population that is captured under stringent conditions from cell lysates through hybridization to complementary DNA oligonucleotides. The resulting mRNP is highly enriched and largely devoid of non-target transcripts, and can be used for a number of downstream analyses including protein identification by mass spectrometry.

Evolution of an ancient protein function involved in organized multicellularity in animals

PubMed Central

Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

2016-01-01

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which – the evolution of GKPID’s capacity to bind the cortical marker protein – can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals. DOI: http://dx.doi.org/10.7554/eLife.10147.001 PMID:26740169

Proteomic analysis of the crayfish gastrolith chitinous extracellular matrix reveals putative protein complexes and a central role for GAP 65.

PubMed

Glazer, Lilah; Roth, Ziv; Weil, Simy; Aflalo, Eliahu D; Khalaila, Isam; Sagi, Amir

2015-10-14

Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network that constitutes the scaffold upon which cuticles form. The chitin fibers that form this network are closely associated with specific structural proteins, while the cuticular matrix contains many additional structural, enzymatic and other proteins. We study the crayfish gastrolith as a simple model for the assembly of calcified cuticular structures, with particular focus on the proteins involved in this process. The present study integrates a gastrolith-forming epithelium transcriptomic library with data from mass spectrometry analysis of proteins extracted from the gastrolith matrix to obtain a near-complete picture of gastrolith protein content. Using native protein separation we identified 24 matrix proteins, of which 14 are novel. Further analysis led to discovery of three putative protein complexes, all containing GAP 65 the most abundant gastrolith structural protein. Using immunological methods we further studied the role of GAP 65 in the gastrolith matrix and forming epithelium, as well as in the newly identified protein complexes. We propose that gastrolith matrix construction is a sequential process in which protein complexes are dynamically assembled and disassembled around GAP 65, thus changing their functional properties to perform each step in the construction process. The scientific interest on which this study is based arises from three main features of gastroliths: (1) Gastroliths possess partial analogy to cuticles both in structural and molecular properties, and may be regarded, with the appropriate reservations (see Introduction), as simple models for cuticle assembly. At the same time, gastroliths are terminally assembled during a well-defined period, which can be controlled in the laboratory, making them significantly easier to study than cuticles. (2) Gastroliths, like the crayfish exoskeleton, contain stable amorphous calcium carbonate (ACC) rather than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification). Copyright © 2015. Published by Elsevier B.V.

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane

PubMed Central

Richardson, Lynn G. L.; Paila, Yamuna D.; Siman, Steven R.; Chen, Yi; Smith, Matthew D.; Schnell, Danny J.

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus. PMID:24966864

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

PubMed

Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

Hsp70 Forms Antiparallel Dimers Stabilized by Post-translational Modifications to Position Clients for Transfer to Hsp90

PubMed Central

Morgner, Nina; Schmidt, Carla; Beilsten-Edmands, Victoria; Ebong, Ima-obong; Patel, Nisha A.; Clerico, Eugenia M.; Kirschke, Elaine; Daturpalli, Soumya; Jackson, Sophie E.; Agard, David; Robinson, Carol V.

2015-01-01

Summary Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90. PMID:25921532

TALE factors poise promoters for activation by Hox proteins.

PubMed

Choe, Seong-Kyu; Ladam, Franck; Sagerström, Charles G

2014-01-27

Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription. Copyright © 2014 Elsevier Inc. All rights reserved.

Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates

PubMed Central

Chen, Miao-Hsueh; Li, Ya-Jun; Kawakami, Takatoshi; Xu, Shan-Mei; Chuang, Pao-Tien

2004-01-01

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue in Hh signaling is to elucidate the molecular mechanism by which a Hh protein morphogen gradient is formed despite its membrane association. In this study, we used a combination of genetic, cellular, and biochemical approaches to address the role of lipid modifications in long-range vertebrate Hh signaling. Our molecular analysis of knockout mice deficient in Skn, the murine homolog of the Drosophila ski gene, which catalyzes Hh palmitoylation, and gene-targeted mice producing a nonpalmitoylated form of Shh indicates that Hh palmitoylation is essential for its activity as well as the generation of a protein gradient in the developing embryos. Furthermore, our biochemical data show that Hh lipid modifications are required for producing a soluble multimeric protein complex, which constitutes the major active component for Hh signaling. These results suggest that soluble Hh multimeric complex travels in the morphogenetic field to activate Hh signaling in distant Hh-responsive cells. PMID:15075292

In vitro folding of inclusion body proteins.

PubMed

Rudolph, R; Lilie, H

1996-01-01

Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.

Nucleoplasmin-like domain of FKBP39 from Drosophila melanogaster forms a tetramer with partly disordered tentacle-like C-terminal segments

PubMed Central

Kozłowska, Małgorzata; Tarczewska, Aneta; Jakób, Michał; Bystranowska, Dominika; Taube, Michał; Kozak, Maciej; Czarnocki-Cieciura, Mariusz; Dziembowski, Andrzej; Orłowski, Marek; Tkocz, Katarzyna; Ożyhar, Andrzej

2017-01-01

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a β-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins. PMID:28074868

The Role of Shape Complementarity in the Protein-Protein Interactions

PubMed Central

Li, Ye; Zhang, Xianren; Cao, Dapeng

2013-01-01

We use a dissipative particle dynamic simulation to investigate the effects of shape complementarity on the protein-protein interactions. By monitoring different kinds of protein shape-complementarity modes, we gave a clear mechanism to reveal the role of the shape complementarity in the protein-protein interactions, i.e., when the two proteins with shape complementarity approach each other, the conformation of lipid chains between two proteins would be restricted significantly. The lipid molecules tend to leave the gap formed by two proteins to maximize the configuration entropy, and therefore yield an effective entropy-induced protein-protein attraction, which enhances the protein aggregation. In short, this work provides an insight into understanding the importance of the shape complementarity in the protein-protein interactions especially for protein aggregation and antibody–antigen complexes. Definitely, the shape complementarity is the third key factor affecting protein aggregation and complex, besides the electrostatic-complementarity and hydrophobic complementarity. PMID:24253561

The Role of Shape Complementarity in the Protein-Protein Interactions

NASA Astrophysics Data System (ADS)

Li, Ye; Zhang, Xianren; Cao, Dapeng

2013-11-01

We use a dissipative particle dynamic simulation to investigate the effects of shape complementarity on the protein-protein interactions. By monitoring different kinds of protein shape-complementarity modes, we gave a clear mechanism to reveal the role of the shape complementarity in the protein-protein interactions, i.e., when the two proteins with shape complementarity approach each other, the conformation of lipid chains between two proteins would be restricted significantly. The lipid molecules tend to leave the gap formed by two proteins to maximize the configuration entropy, and therefore yield an effective entropy-induced protein-protein attraction, which enhances the protein aggregation. In short, this work provides an insight into understanding the importance of the shape complementarity in the protein-protein interactions especially for protein aggregation and antibody-antigen complexes. Definitely, the shape complementarity is the third key factor affecting protein aggregation and complex, besides the electrostatic-complementarity and hydrophobic complementarity.

A Versatile Platform for Nanotechnology Based on Circular Permutation of a Chaperonin Protein

NASA Technical Reports Server (NTRS)

Paavola, Chad; McMillan, Andrew; Trent, Jonathan; Chan, Suzanne; Mazzarella, Kellen; Li, Yi-Fen

2004-01-01

A number of protein complexes have been developed as nanoscale templates. These templates can be functionalized using the peptide sequences that bind inorganic materials. However, it is difficult to integrate peptides into a specific position within a protein template. Integrating intact proteins with desirable binding or catalytic activities is an even greater challenge. We present a general method for modifying protein templates using circular permutation so that additional peptide sequence can be added in a wide variety of specific locations. Circular permutation is a reordering of the polypeptide chain such that the original termini are joined and new termini are created elsewhere in the protein. New sequence can be joined to the protein termini without perturbing the protein structure and with minimal limitation on the size and conformation of the added sequence. We have used this approach to modify a chaperonin protein template, placing termini at five different locations distributed across the surface of the protein complex. These permutants are competent to form the double-ring structures typical of chaperonin proteins. The permuted double-rings also form the same assemblies as the unmodified protein. We fused a fluorescent protein to two representative permutants and demonstrated that it assumes its active structure and does not interfere with assembly of chaperonin double-rings.

Quantitative analyses of bifunctional molecules.

PubMed

Braun, Patrick D; Wandless, Thomas J

2004-05-11

Small molecules can be discovered or engineered to bind tightly to biologically relevant proteins, and these molecules have proven to be powerful tools for both basic research and therapeutic applications. In many cases, detailed biophysical analyses of the intermolecular binding events are essential for improving the activity of the small molecules. These interactions can often be characterized as straightforward bimolecular binding events, and a variety of experimental and analytical techniques have been developed and refined to facilitate these analyses. Several investigators have recently synthesized heterodimeric molecules that are designed to bind simultaneously with two different proteins to form ternary complexes. These heterodimeric molecules often display compelling biological activity; however, they are difficult to characterize. The bimolecular interaction between one protein and the heterodimeric ligand (primary dissociation constant) can be determined by a number of methods. However, the interaction between that protein-ligand complex and the second protein (secondary dissociation constant) is more difficult to measure due to the noncovalent nature of the original protein-ligand complex. Consequently, these heterodimeric compounds are often characterized in terms of their activity, which is an experimentally dependent metric. We have developed a general quantitative mathematical model that can be used to measure both the primary (protein + ligand) and secondary (protein-ligand + protein) dissociation constants for heterodimeric small molecules. These values are largely independent of the experimental technique used and furthermore provide a direct measure of the thermodynamic stability of the ternary complexes that are formed. Fluorescence polarization and this model were used to characterize the heterodimeric molecule, SLFpYEEI, which binds to both FKBP12 and the Fyn SH2 domain, demonstrating that the model is useful for both predictive as well as ex post facto analytical applications.

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

PubMed Central

Lindin, Inger; Wuxiuer, Yimingjiang; Ravna, Aina Westrheim; Moens, Ugo; Sylte, Ingebrigt

2014-01-01

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding. PMID:24651460

Role of membrane contact sites in protein import into mitochondria

PubMed Central

Horvath, Susanne E; Rampelt, Heike; Oeljeklaus, Silke; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus

2015-01-01

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture. PMID:25514890

The influence of different cucumariosides on immunogenicity of OmpF porin from Yersinia pseudotuberulosis as a model protein antigen of tubular immunostimulating complex

NASA Astrophysics Data System (ADS)

Sanina, N. M.; Chopenko, N. S.; Davydova, L. A.; Mazeika, A. N.; Portnyagina, O. Yu.; Kim, N. Yu.; Golotin, V. A.; Kostetsky, E. Y.; Shnyrov, V. L.

2017-09-01

Nanoparticulate tubular immunostimulating complex (TI-complex) is a novel promising adjuvant carrier of antigens allowing to create safe and effective vaccines of new generation. The adjuvant activity of TI-complexes based on monogalactosyldyacylglycerol (MGDG) from the sea alga Ulva lactuca and different triterpene glycosides cucumariosides (CDs) from marine invertebrate Cucumaria japonica and their fractions was studied to assess effects of different CDs on the immunogenicity of porin OmpF from Yersinia pseudotuberculosis (YOmpF). TI-complexes with cucumarioside A2-2 (CDA2-2) maximally stimulated anti-porin antibody production. Studies of protein intrinsic fluorescence showed that all CDs had a relaxing effect on the conformation of YOmpF, loosening peripheral region of protein and promoting exposure of the protein antigenic determinants to the water environment. The greatest immunostimulating effect of TI-complexes comprising CDA2-2 was accompanied by mild effect of this CD on the tertiary structure of protein antigen YOmpF, whereas cucumarioside E (CDE) and cucumarioside A2-4 (CDA2-4) caused especially sharp redistribution of spectral form of the YOmpF corresponding to the emission of an intrinsic protein fluorophore tryptophan.

HcRed, a Genetically Encoded Fluorescent Binary Cross-Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

PubMed Central

Gong, Lan; Ramm, Georg; Devenish, Rodney J.; Prescott, Mark

2012-01-01

Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase. PMID:22496895

Different KChIPs compete for heteromultimeric assembly with pore-forming Kv4 subunits.

PubMed

Zhou, Jingheng; Tang, Yiquan; Zheng, Qin; Li, Meng; Yuan, Tianyi; Chen, Liangyi; Huang, Zhuo; Wang, KeWei

2015-06-02

Auxiliary Kv channel-interacting proteins 1-4 (KChIPs1-4) coassemble with pore-forming Kv4 α-subunits to form channel complexes underlying somatodendritic subthreshold A-type current that regulates neuronal excitability. It has been hypothesized that different KChIPs can competitively bind to Kv4 α-subunit to form variable channel complexes that can exhibit distinct biophysical properties for modulation of neural function. In this study, we use single-molecule subunit counting by total internal reflection fluorescence microscopy in combinations with electrophysiology and biochemistry to investigate whether different isoforms of auxiliary KChIPs, KChIP4a, and KChIP4bl, can compete for binding of Kv4.3 to coassemble heteromultimeric channel complexes for modulation of channel function. To count the number of photobleaching steps solely from cell membrane, we take advantage of a membrane tethered k-ras-CAAX peptide that anchors cytosolic KChIP4 proteins to the surface for reduction of background noise. Single-molecule subunit counting reveals that the number of KChIP4 isoforms in Kv4.3-KChIP4 complexes can vary depending on the KChIP4 expression level. Increasing the amount of KChIP4bl gradually reduces bleaching steps of KChIP4a isoform proteins, and vice versa. Further analysis of channel gating kinetics from different Kv4-KChIP4 subunit compositions confirms that both KChIP4a and KChIP4bl can modulate the channel complex function upon coassembly. Taken together, our findings show that auxiliary KChIPs can heteroassemble with Kv4 in a competitive manner to form heteromultimeric Kv4-KChIP4 channel complexes that are biophysically distinct and regulated under physiological or pathological conditions. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease

PubMed Central

Fong, Yu Hang; Wong, Ho Chun; Yuen, Man Hon; Lau, Pak Ho; Chen, Yu Wai; Wong, Kam-Bo

2013-01-01

Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease. PMID:24115911

Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I.

PubMed

Moparthi, Vamsi K; Kumar, Brijesh; Al-Eryani, Yusra; Sperling, Eva; Górecki, Kamil; Drakenberg, Torbjörn; Hägerhäll, Cecilia

2014-01-01

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na(+)/H(+) antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity. © 2013. Published by Elsevier B.V. All rights reserved.

Usher syndrome: molecular links of pathogenesis, proteins and pathways.

PubMed

Kremer, Hannie; van Wijk, Erwin; Märker, Tina; Wolfrum, Uwe; Roepman, Ronald

2006-10-15

Usher syndrome is the most common form of deaf-blindness. The syndrome is both clinically and genetically heterogeneous, and to date, eight causative genes have been identified. The proteins encoded by these genes are part of a dynamic protein complex that is present in hair cells of the inner ear and in photoreceptor cells of the retina. The localization of the Usher proteins and the phenotype in animal models indicate that the Usher protein complex is essential in the morphogenesis of the stereocilia bundle in hair cells and in the calycal processes of photoreceptor cells. In addition, the Usher proteins are important in the synaptic processes of both cell types. The association of other proteins with the complex indicates functional links to a number of basic cell-biological processes. Prominently present is the connection to the dynamics of the actin cytoskeleton, involved in cellular morphology, cell polarity and cell-cell interactions. The Usher protein complex can also be linked to the cadherins/catenins in the adherens junction-associated protein complexes, suggesting a role in cell polarity and tissue organization. A third link can be established to the integrin transmembrane signaling network. The Usher interactome, as outlined in this review, participates in pathways common in inner ear and retina that are disrupted in the Usher syndrome.

A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2.

PubMed

Rodgers, K K; Villey, I J; Ptaszek, L; Corbett, E; Schatz, D G; Coleman, J E

1999-07-15

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.

Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry

NASA Astrophysics Data System (ADS)

Belov, Arseniy M.; Viner, Rosa; Santos, Marcia R.; Horn, David M.; Bern, Marshall; Karger, Barry L.; Ivanov, Alexander R.

2017-12-01

Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). [Figure not available: see fulltext.

Identification of alpha-enolase as a nuclear DNA-binding protein in the zona fasciculata but not the zona reticularis of the human adrenal cortex.

PubMed

Wang, Weiye; Wang, Lishan; Endoh, Akira; Hummelke, Geoffrey; Hawks, Christina L; Hornsby, Peter J

2005-01-01

In order to establish whether there are differences in DNA-binding proteins between zona fasciculata (ZF) and zona reticularis (ZR) cells of the human adrenal cortex, we prepared nuclear extracts from separated ZF and ZR cells. The formation of DNA-protein complexes was studied using an element in the first intron of the type I and type II 3beta-hydroxysteroid dehydrogenase genes (HSD3B1 and HSD3B2). Using the element in the HSD3B2 gene as a probe, a complex (C1) was formed with extracts from ZF cells but was formed only at a low level with ZR cell extracts. Another pair of complexes (C2/C3) was formed with both ZF and ZR cell extracts. The ZF-specific protein forming C1 was enriched by column chromatography on DEAE-Sepharose and carboxymethyl-Sepharose. Oligonucleotide competition analysis on the enriched fraction gave results consistent with those obtained on the unfractionated material. A further enrichment was brought about by passing the protein over an oligonucleotide affinity column based on the HSD3B2 element. The protein bound to the column was identified as alpha-enolase by mass spectrometry. Although alpha-enolase is a glycolytic enzyme, it binds to specific DNA sequences and has been found to be present in nuclei of various cell types. We performed immunohistochemistry on sections of adult human adrenal cortex and found alpha-enolase to be located in nuclei of ZF cells but to be predominantly cytoplasmic in ZR cells. Transfection of an alpha-enolase expression vector into NCI-H295R human adrenocortical cells increased HSD3B2 promoter activity, suggesting a possible functional role for this protein in regulation of HSD3B2 expression.

HotRegion: a database of predicted hot spot clusters.

PubMed

Cukuroglu, Engin; Gursoy, Attila; Keskin, Ozlem

2012-01-01

Hot spots are energetically important residues at protein interfaces and they are not randomly distributed across the interface but rather clustered. These clustered hot spots form hot regions. Hot regions are important for the stability of protein complexes, as well as providing specificity to binding sites. We propose a database called HotRegion, which provides the hot region information of the interfaces by using predicted hot spot residues, and structural properties of these interface residues such as pair potentials of interface residues, accessible surface area (ASA) and relative ASA values of interface residues of both monomer and complex forms of proteins. Also, the 3D visualization of the interface and interactions among hot spot residues are provided. HotRegion is accessible at http://prism.ccbb.ku.edu.tr/hotregion.

A primary microcephaly protein complex forms a ring around parental centrioles.

PubMed

Sir, Joo-Hee; Barr, Alexis R; Nicholas, Adeline K; Carvalho, Ofelia P; Khurshid, Maryam; Sossick, Alex; Reichelt, Stefanie; D'Santos, Clive; Woods, C Geoffrey; Gergely, Fanni

2011-10-09

Autosomal recessive primary microcephaly (MCPH) is characterized by a substantial reduction in prenatal human brain growth without alteration of the cerebral architecture and is caused by biallelic mutations in genes coding for a subset of centrosomal proteins. Although at least three of these proteins have been implicated in centrosome duplication, the nature of the centrosome dysfunction that underlies the neurodevelopmental defect in MCPH is unclear. Here we report a homozygous MCPH-causing mutation in human CEP63. CEP63 forms a complex with another MCPH protein, CEP152, a conserved centrosome duplication factor. Together, these two proteins are essential for maintaining normal centrosome numbers in cells. Using super-resolution microscopy, we found that CEP63 and CEP152 co-localize in a discrete ring around the proximal end of the parental centriole, a pattern specifically disrupted in CEP63-deficient cells derived from patients with MCPH. This work suggests that the CEP152-CEP63 ring-like structure ensures normal neurodevelopment and that its impairment particularly affects human cerebral cortex growth.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

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

Neto, J.L. Siqueira; Instituto de Biologia, UNICAMP, Campinas, SP; Lira, C.B.B.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 ismore » a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.« less

A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

PubMed

Biedendieck, Rebekka

2016-01-01

For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

The structure and protein binding of amyloid-specific dye reagents.

PubMed

Stopa, Barbara; Piekarska, Barbara; Konieczny, Leszek; Rybarska, Janina; Spólnik, Paweł; Zemanek, Grzegorz; Roterman, Irena; Król, Marcin

2003-01-01

The self-assembling tendency and protein complexation capability of dyes related to Congo red and also some dyes of different structure were compared to explain the mechanism of Congo red binding and the reason for its specific affinity for beta-structure. Complexation with proteins was measured directly and expressed as the number of dye molecules bound to heat-aggregated IgG and to two light chains with different structural stability. Binding of dyes to rabbit antibodies was measured indirectly as the enhancement effect of the dye on immune complex formation. Self-assembling was tested using dynamic light scattering to measure the size of the supramolecular assemblies. In general the results show that the supramolecular form of a dye is the main factor determining its complexation capability. Dyes that in their compact supramolecular organization are ribbon-shaped may adhere to polypeptides of beta-conformation due to the architectural compatibility in this unique structural form. The optimal fit in complexation seems to depend on two contradictory factors involving, on the one hand, the compactness of the non-covalently stabilized supramolecular ligand, and the dynamic character producing its plasticity on the other. As a result, the highest protein binding capability is shown by dyes with a moderate self-assembling tendency, while those arranging into either very rigid or very unstable supramolecular entities are less able to bind.

Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation

PubMed Central

Roa-Espitia, Ana L.; Hernández-Rendón, Eva R.; Baltiérrez-Hoyos, Rafael; Muñoz-Gotera, Rafaela J.; Cote-Vélez, Antonieta; Jiménez, Irma; González-Márquez, Humberto

2016-01-01

ABSTRACT Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by β1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca2+ dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton. PMID:27402964

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

Scott, F.; Stec, B; Pop, C

The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis1, 2, 3. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation4, 5. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed andmore » isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FADD complex bridge governed by weak protein-protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.« less

Initiation of DNA replication: functional and evolutionary aspects

PubMed Central

Bryant, John A.; Aves, Stephen J.

2011-01-01

Background The initiation of DNA replication is a very important and highly regulated step in the cell division cycle. It is of interest to compare different groups of eukaryotic organisms (a) to identify the essential molecular events that occur in all eukaryotes, (b) to start to identify higher-level regulatory mechanisms that are specific to particular groups and (c) to gain insights into the evolution of initiation mechanisms. Scope This review features a wide-ranging literature survey covering replication origins, origin recognition and usage, modification of origin usage (especially in response to plant hormones), assembly of the pre-replication complex, loading of the replisome, genomics, and the likely origin of these mechanisms and proteins in Archaea. Conclusions In all eukaryotes, chromatin is organized for DNA replication as multiple replicons. In each replicon, replication is initiated at an origin. With the exception of those in budding yeast, replication origins, including the only one to be isolated so far from a plant, do not appear to embody a specific sequence; rather, they are AT-rich, with short tracts of locally bent DNA. The proteins involved in initiation are remarkably similar across the range of eukaryotes. Nevertheless, their activity may be modified by plant-specific mechanisms, including regulation by plant hormones. The molecular features of initiation are seen in a much simpler form in the Archaea. In particular, where eukaryotes possess a number of closely related proteins that form ‘hetero-complexes’ (such as the origin recognition complex and the MCM complex), archaeans typically possess one type of protein (e.g. one MCM) that forms a homo-complex. This suggests that several eukaryotic initiation proteins have evolved from archaeal ancestors by gene duplication and divergence. PMID:21508040

Export of a Toxoplasma gondii Rhoptry Neck Protein Complex at the Host Cell Membrane to Form the Moving Junction during Invasion

PubMed Central

Poncet, Joël; Dubremetz, Jean-François; Lebrun, Maryse

2009-01-01

One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes. PMID:19247437

Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection.

PubMed

Albecka, Anna; Owen, Danielle J; Ivanova, Lyudmila; Brun, Juliane; Liman, Rukayya; Davies, Laura; Ahmed, M Firoz; Colaco, Susanna; Hollinshead, Michael; Graham, Stephen C; Crump, Colin M

2017-01-15

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells. Copyright © 2017 Albecka et al.

Influence of pea protein aggregates on the structure and stability of pea protein/soybean polysaccharide complex emulsions.

PubMed

Yin, Baoru; Zhang, Rujing; Yao, Ping

2015-03-20

The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI) with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS), and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

PubMed Central

Farrugia, Mark A.; Han, Linjie; Zhong, Yueyang; Boer, Jodi L.; Ruotolo, Brandon T.; Hausinger, Robert P.

2013-01-01

Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)2 quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)2 and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)2 complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation. PMID:23797863

Mature DIABLO/Smac Is Produced by the IMP Protease Complex on the Mitochondrial Inner Membrane

PubMed Central

Burri, Lena; Strahm, Yvan; Hawkins, Christine J.; Gentle, Ian E.; Puryer, Michelle A.; Verhagen, Anne; Callus, Bernard; Vaux, David; Lithgow, Trevor

2005-01-01

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX5P in Imp1 and NX5S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space. PMID:15814844

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps

PubMed Central

Rakesh, Ramachandran; Joseph, Agnel Praveen; Bhaskara, Ramachandra M.; Srinivasan, Narayanaswamy

2016-01-01

ABSTRACT Pre-mRNA splicing in eukaryotes is performed by the spliceosome, a highly complex macromolecular machine. SF3b is a multi-protein complex which recognizes the branch point adenosine of pre-mRNA as part of a larger U2 snRNP or U11/U12 di-snRNP in the dynamic spliceosome machinery. Although a cryo-EM map is available for human SF3b complex, the structure and relative spatial arrangement of all components in the complex are not yet known. We have recognized folds of domains in various proteins in the assembly and generated comparative models. Using an integrative approach involving structural and other experimental data, guided by the available cryo-EM density map, we deciphered a pseudo-atomic model of the closed form of SF3b which is found to be a “fuzzy complex” with highly flexible components and multiplicity of folds. Further, the model provides structural information for 5 proteins (SF3b10, SF3b155, SF3b145, SF3b130 and SF3b14b) and localization information for 4 proteins (SF3b10, SF3b145, SF3b130 and SF3b14b) in the assembly for the first time. Integration of this model with the available U11/U12 di-snRNP cryo-EM map enabled elucidation of an open form. This now provides new insights on the mechanistic features involved in the transition between closed and open forms pivoted by a hinge region in the SF3b155 protein that also harbors cancer causing mutations. Moreover, the open form guided model of the 5′ end of U12 snRNA, which includes the branch point duplex, shows that the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation with potential implications for branch point adenosine recognition fidelity. PMID:27618338

Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide.

PubMed Central

Chiaruttini, C; Expert-Bezançon, A; Hayes, D; Ehresmann, B

1982-01-01

1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide. Images PMID:6760129

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

PubMed Central

Immink, Richard GH; Tonaco, Isabella AN; de Folter, Stefan; Shchennikova, Anna; van Dijk, Aalt DJ; Busscher-Lange, Jacqueline; Borst, Jan W; Angenent, Gerco C

2009-01-01

Background Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study. Results In total, 106 multimeric complexes were identified; in more than half of these at least one SEP protein was present. Besides the known complexes involved in determining floral organ identity, various complexes consisting of combinations of proteins known to play a role in floral organ identity specification, and flowering time determination were discovered. The capacity to form this latter type of complex suggests that homeotic factors play essential roles in down-regulation of the MADS box genes involved in floral timing in the flower via negative auto-regulatory loops. Furthermore, various novel complexes were identified that may be important for the direct regulation of the floral transition process. A subsequent detailed analysis of the APETALA3, PISTILLATA, and SEP3 proteins in living plant cells suggests the formation of a multimeric complex in vivo. Conclusions Overall, these results provide strong indications that higher-order complex formation is a general and essential molecular mechanism for plant MADS box protein functioning and attribute a pivotal role to the SEP3 'glue' protein in mediating multimerization. PMID:19243611

Biochemical characterization of native Usher protein complexes from a vesicular subfraction of tracheal epithelial cells.

PubMed

Zallocchi, Marisa; Sisson, Joseph H; Cosgrove, Dominic

2010-02-16

Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 x 10(6) Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

BIOCHEMICAL CHARACTERIZATION OF NATIVE USHER PROTEIN COMPLEXES FROM A VESICULAR SUBFRACTION OF TRACHEAL EPITHELIAL CELLS†

PubMed Central

Zallocchi, Marisa; Sisson, Joseph H.; Cosgrove, Dominic

2010-01-01

Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells, and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in non-detergent buffer, and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15 and VLGR-1, and a different one at the top of the gradient that included all the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100–200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins co-sediment into the gradient at a sedimentation coefficient of approximately 50S, correlating with a predicted molecular mass of 2 × 106 Daltons. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher proteins complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors. PMID:20058854

Predicting protein complexes from weighted protein-protein interaction graphs with a novel unsupervised methodology: Evolutionary enhanced Markov clustering.

PubMed

Theofilatos, Konstantinos; Pavlopoulou, Niki; Papasavvas, Christoforos; Likothanassis, Spiros; Dimitrakopoulos, Christos; Georgopoulos, Efstratios; Moschopoulos, Charalampos; Mavroudi, Seferina

2015-03-01

Proteins are considered to be the most important individual components of biological systems and they combine to form physical protein complexes which are responsible for certain molecular functions. Despite the large availability of protein-protein interaction (PPI) information, not much information is available about protein complexes. Experimental methods are limited in terms of time, efficiency, cost and performance constraints. Existing computational methods have provided encouraging preliminary results, but they phase certain disadvantages as they require parameter tuning, some of them cannot handle weighted PPI data and others do not allow a protein to participate in more than one protein complex. In the present paper, we propose a new fully unsupervised methodology for predicting protein complexes from weighted PPI graphs. The proposed methodology is called evolutionary enhanced Markov clustering (EE-MC) and it is a hybrid combination of an adaptive evolutionary algorithm and a state-of-the-art clustering algorithm named enhanced Markov clustering. EE-MC was compared with state-of-the-art methodologies when applied to datasets from the human and the yeast Saccharomyces cerevisiae organisms. Using public available datasets, EE-MC outperformed existing methodologies (in some datasets the separation metric was increased by 10-20%). Moreover, when applied to new human datasets its performance was encouraging in the prediction of protein complexes which consist of proteins with high functional similarity. In specific, 5737 protein complexes were predicted and 72.58% of them are enriched for at least one gene ontology (GO) function term. EE-MC is by design able to overcome intrinsic limitations of existing methodologies such as their inability to handle weighted PPI networks, their constraint to assign every protein in exactly one cluster and the difficulties they face concerning the parameter tuning. This fact was experimentally validated and moreover, new potentially true human protein complexes were suggested as candidates for further validation using experimental techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

CRISPR RNA and anti-CRISPR protein binding to the Xanthomonas albilineans Csy1-Csy2 heterodimer in the type I-F CRISPR-Cas system.

PubMed

Hong, Suji; Ka, Donghyun; Yoon, Seo Jeong; Suh, Nayoung; Jeong, Migyeong; Suh, Jeong-Yong; Bae, Euiyoung

2018-02-23

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide microbial adaptive immunity against bacteriophages. In type I-F CRISPR-Cas systems, multiple Cas proteins (Csy1-4) compose a surveillance complex (Csy complex) with CRISPR RNA (crRNA) for target recognition. Here, we report the biochemical characterization of the Csy1-Csy2 subcomplex from Xanthomonas albilineans , including the analysis of its interaction with crRNA and AcrF2, an anti-CRISPR (Acr) protein from a phage that infects Pseudomonas aeruginosa The X. albilineans Csy1 and Csy2 proteins (XaCsy1 and XaCsy2, respectively) formed a stable heterodimeric complex that specifically bound the 8-nucleotide (nt) 5'-handle of the crRNA. In contrast, the XaCsy1-XaCsy2 heterodimer exhibited reduced affinity for the 28-nt X. albilineans CRISPR repeat RNA containing the 5'-handle sequence. Chromatographic and calorimetric analyses revealed tight binding between the Acr protein from the P. aeruginosa phage and the heterodimeric subunit of the X. albilineans Csy complex, suggesting that AcrF2 recognizes conserved features of Csy1-Csy2 heterodimers. We found that neither XaCsy1 nor XaCsy2 alone forms a stable complex with AcrF2 and the 5'-handle RNA, indicating that XaCsy1-XaCsy2 heterodimerization is required for binding them. We also solved the crystal structure of AcrF2 to a resolution of 1.34 Å, enabling a more detailed structural analysis of the residues involved in the interactions with the Csy1-Csy2 heterodimer. Our results provide information about the order of events during the formation of the multisubunit crRNA-guided surveillance complex and suggest that the Acr protein inactivating type I-F CRISPR-Cas systems has broad specificity. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The BioPlex Network: A Systematic Exploration of the Human Interactome.

PubMed

Huttlin, Edward L; Ting, Lily; Bruckner, Raphael J; Gebreab, Fana; Gygi, Melanie P; Szpyt, John; Tam, Stanley; Zarraga, Gabriela; Colby, Greg; Baltier, Kurt; Dong, Rui; Guarani, Virginia; Vaites, Laura Pontano; Ordureau, Alban; Rad, Ramin; Erickson, Brian K; Wühr, Martin; Chick, Joel; Zhai, Bo; Kolippakkam, Deepak; Mintseris, Julian; Obar, Robert A; Harris, Tim; Artavanis-Tsakonas, Spyros; Sowa, Mathew E; De Camilli, Pietro; Paulo, Joao A; Harper, J Wade; Gygi, Steven P

2015-07-16

Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80%-100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally related proteins. Finally, BioPlex, in combination with other approaches, can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial amyotrophic lateral sclerosis perturb a defined community of interactors. Copyright © 2015 Elsevier Inc. All rights reserved.

The BioPlex Network: A Systematic Exploration of the Human Interactome

PubMed Central

Huttlin, Edward L.; Ting, Lily; Bruckner, Raphael J.; Gebreab, Fana; Gygi, Melanie P.; Szpyt, John; Tam, Stanley; Zarraga, Gabriela; Colby, Greg; Baltier, Kurt; Dong, Rui; Guarani, Virginia; Vaites, Laura Pontano; Ordureau, Alban; Rad, Ramin; Erickson, Brian K.; Wühr, Martin; Chick, Joel; Zhai, Bo; Kolippakkam, Deepak; Mintseris, Julian; Obar, Robert A.; Harris, Tim; Artavanis-Tsakonas, Spyros; Sowa, Mathew E.; DeCamilli, Pietro; Paulo, Joao A.; Harper, J. Wade; Gygi, Steven P.

2015-01-01

SUMMARY Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80-100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally-related proteins. Finally, BioPlex, in combination with other approaches can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial Amyotrophic Lateral Sclerosis perturb a defined community of interactors. PMID:26186194

A model of the complex between human {beta}-microseminoprotein and CRISP-3 based on NMR data

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

Ghasriani, Houman; Fernlund, Per; Udby, Lene

2009-01-09

{beta}-Microseminoprotein (MSP), a 10 kDa seminal plasma protein, forms a tight complex with cysteine-rich secretory protein 3 (CRISP-3) from granulocytes. The 3D structure of human MSP has been determined but there is as yet no 3D structure for CRISP-3. We have now studied the complex between human MSP and CRISP-3 with multidimensional NMR. {sup 15}N-HSQC spectra show substantial differences between free and complexed hMSP. Using several 3D-NMR spectra of triply labeled hMSP in complex with a recombinant N-terminal domain of CRISP-3, most of the backbone of hMSP could be assigned. The data show that only one side of hMSP, comprisingmore » {beta}-strands 1, 4, 5, and 8 are affected by the complex formation, indicating that {beta}-strands 1 and 8 form the main binding surface. Based on this we present a tentative structure for the hMSP-CRISP-3 complex using the known crystal structure of triflin as a model of CRISP-3.« less

The Hexamer Structure of the Rift Valley Fever Virus Nucleoprotein Suggests a Mechanism for its Assembly into Ribonucleoprotein Complexes

PubMed Central

Ferron, François; Li, Zongli; Danek, Eric I.; Luo, Dahai; Wong, Yeehwa; Coutard, Bruno; Lantez, Violaine; Charrel, Rémi; Canard, Bruno; Walz, Thomas; Lescar, Julien

2011-01-01

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs. PMID:21589902

Myocilin, a Component of a Membrane-Associated Protein Complex Driven by a Homologous Q-SNARE Domain

PubMed Central

Dismuke, W. Michael; McKay, Brian S.; Stamer, W. Daniel

2012-01-01

Myocilin is a widely expressed protein with no known function, however, mutations in myocilin appear to manifest uniquely as ocular hypertension and the blinding disease glaucoma. Using the protein homology/analogy recognition engine (PHYRE) we find that the olfactomedin domain of myocilin is similar in sequence motif and structure to a six-bladed, kelch repeat motif based on the known crystal structures of such proteins. Additionally, using sequence analysis we identify a coiled-coil segment of myocilin with homology to human Q-SNARE proteins. Using COS-7 cells expressing full length human myocilin and a version lacking the C-terminal olfactomedin domain, we identified a membrane-associated protein complex containing myocilin by hydrodynamic analysis. The myocilin construct that included the coiled-coil but lacked the olfactomedin domain formed complexes similar to the full-length protein, indicating that the coiled-coil domain of myocilin is sufficient for myocilin to bind to the large detergent resistant complex. In human retina and retinal pigment epithelium, which express myocilin, we detected the protein in a large, SDS-resistant, membrane-associated complex. We characterized the hydrodynamic properties of myocilin in human tissues as either a 15s complex with an Mr=405,000–440,000 yielding a slightly elongated globular shape similar to known SNARE complexes or a dimer of 6.4s and Mr=108,000. By identifying the Q-SNARE homology within the second coil of myocilin and documenting its participation in a SNARE-like complex, we provide evidence of a SNARE domain containing protein associated with a human disease. PMID:22463803

Construction and Structural Analysis of Tethered Lipid Bilayer Containing Photosynthetic Antenna Proteins for Functional Analysis

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

Sumino, Ayumi; Dewa, Takehisa; Takeuchi, Toshikazu

2011-07-11

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAY), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RCmore » within the tethered membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.« less

Purification of ribonucleoproteins by a novel approach: isolation of the SSB1 ribonucleoprotein from yeast and demonstration that it has no role in mRNA splicing.

PubMed

Cusick, M E

1992-12-29

A novel approach is described to purify potential ribonucleoproteins (RNP) of yeast. The method assays a yeast RNP complex, assembled in vitro on actin pre-mRNA, by low-ionic strength acrylamide gel electrophoresis. The minimal protein components of this RNP complex were three proteins, one of 30 kDa and two at 42-44 kDa, defined by formation of the complex on biotinylated-RNA, binding of this complex to avidin-agarose, and salt elution of the protein in the biotinylated-RNP complex. Using the assay for RNP complex formation, an RNP protein was purified to homogeneity on the basis of its affinity towards single-stranded DNA and RNA. This RNP protein turned out to be identical to a known RNP protein, the single-stranded binding protein 1 (ssb1) of yeast, on the basis of identical gel electrophoretic migration, antibody cross-reactivity, and identical properties on the gel complex formation assay. In vitro mRNA splicing was normal in extracts made from a yeast strain missing ssb1 (ssb1- strain). Addition of anti-ssb1 antibody to splicing extracts made from a wild type strain did not inhibit or diminish splicing. Instead, mRNA splicing was reproducibly stimulated several fold, indicating competition between ssb1 and splicing factors for binding to single-stranded RNA in the extracts. RNP complexes still formed in the ssb1- strain, demonstrating that it would be possible to purify other RNP proteins from this strain using the gel complex formation assay.

Proteins evolve on the edge of supramolecular self-assembly.

PubMed

Garcia-Seisdedos, Hector; Empereur-Mot, Charly; Elad, Nadav; Levy, Emmanuel D

2017-08-10

The self-association of proteins into symmetric complexes is ubiquitous in all kingdoms of life. Symmetric complexes possess unique geometric and functional properties, but their internal symmetry can pose a risk. In sickle-cell disease, the symmetry of haemoglobin exacerbates the effect of a mutation, triggering assembly into harmful fibrils. Here we examine the universality of this mechanism and its relation to protein structure geometry. We introduced point mutations solely designed to increase surface hydrophobicity among 12 distinct symmetric complexes from Escherichia coli. Notably, all responded by forming supramolecular assemblies in vitro, as well as in vivo upon heterologous expression in Saccharomyces cerevisiae. Remarkably, in four cases, micrometre-long fibrils formed in vivo in response to a single point mutation. Biophysical measurements and electron microscopy revealed that mutants self-assembled in their folded states and so were not amyloid-like. Structural examination of 73 mutants identified supramolecular assembly hot spots predictable by geometry. A subsequent structural analysis of 7,471 symmetric complexes showed that geometric hot spots were buffered chemically by hydrophilic residues, suggesting a mechanism preventing mis-assembly of these regions. Thus, point mutations can frequently trigger folded proteins to self-assemble into higher-order structures. This potential is counterbalanced by negative selection and can be exploited to design nanomaterials in living cells.

Proteins evolve on the edge of supramolecular self-assembly

NASA Astrophysics Data System (ADS)

Garcia-Seisdedos, Hector; Empereur-Mot, Charly; Elad, Nadav; Levy, Emmanuel D.

2017-08-01

The self-association of proteins into symmetric complexes is ubiquitous in all kingdoms of life. Symmetric complexes possess unique geometric and functional properties, but their internal symmetry can pose a risk. In sickle-cell disease, the symmetry of haemoglobin exacerbates the effect of a mutation, triggering assembly into harmful fibrils. Here we examine the universality of this mechanism and its relation to protein structure geometry. We introduced point mutations solely designed to increase surface hydrophobicity among 12 distinct symmetric complexes from Escherichia coli. Notably, all responded by forming supramolecular assemblies in vitro, as well as in vivo upon heterologous expression in Saccharomyces cerevisiae. Remarkably, in four cases, micrometre-long fibrils formed in vivo in response to a single point mutation. Biophysical measurements and electron microscopy revealed that mutants self-assembled in their folded states and so were not amyloid-like. Structural examination of 73 mutants identified supramolecular assembly hot spots predictable by geometry. A subsequent structural analysis of 7,471 symmetric complexes showed that geometric hot spots were buffered chemically by hydrophilic residues, suggesting a mechanism preventing mis-assembly of these regions. Thus, point mutations can frequently trigger folded proteins to self-assemble into higher-order structures. This potential is counterbalanced by negative selection and can be exploited to design nanomaterials in living cells.

Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Cytoplasm-dislocalized Leukemic Nucleophosmin Protein (NPMc)*

PubMed Central

Du, Wei; Li, Jie; Sipple, Jared; Chen, Jianjun; Pang, Qishen

2010-01-01

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis. PMID:20864535

Effect of CMC Molecular Weight on Acid-Induced Gelation of Heated WPI-CMC Soluble Complex.

PubMed

Huan, Yan; Zhang, Sha; Vardhanabhuti, Bongkosh

2016-02-01

Acid-induced gelation properties of heated whey protein isolate (WPI) and carboxymethylcellulose (CMC) soluble complex were investigated as a function of CMC molecular weight (270, 680, and 750 kDa) and concentrations (0% to 0.125%). Heated WPI-CMC soluble complex with 6% protein was made by heating biopolymers together at pH 7.0 and 85 °C for 30 min and diluted to 5% protein before acid-induced gelation. Acid-induced gel formed from heated WPI-CMC complexes exhibited increased hardness and decreased water holding capacity with increasing CMC concentrations but gel strength decreased at higher CMC content. The highest gel strength was observed with CMC 750 k at 0.05%. Gels with low CMC concentration showed homogenous microstructure which was independent of CMC molecular weight, while increasing CMC concentration led to microphase separation with higher CMC molecular weight showing more extensive phase separation. When heated WPI-CMC complexes were prepared at 9% protein the acid gels showed improved gel hardness and water holding capacity, which was supported by the more interconnected protein network with less porosity when compared to complexes heated at 6% protein. It is concluded that protein concentration and biopolymer ratio during complex formation are the major factors affecting gel properties while the effect of CMC molecular weight was less significant. © 2016 Institute of Food Technologists®

Multiple domains of fission yeast Cdc19p (MCM2) are required for its association with the core MCM complex.

PubMed

Sherman, D A; Pasion, S G; Forsburg, S L

1998-07-01

The members of the MCM protein family are essential eukaryotic DNA replication factors that form a six-member protein complex. In this study, we use antibodies to four MCM proteins to investigate the structure of and requirements for the formation of fission yeast MCM complexes in vivo, with particular regard to Cdc19p (MCM2). Gel filtration analysis shows that the MCM protein complexes are unstable and can be broken down to subcomplexes. Using coimmunoprecipitation, we find that Mis5p (MCM6) and Cdc21p (MCM4) are tightly associated with one another in a core complex with which Cdc19p loosely associates. Assembly of Cdc19p with the core depends upon Cdc21p. Interestingly, there is no obvious change in Cdc19p-containing MCM complexes through the cell cycle. Using a panel of Cdc19p mutants, we find that multiple domains of Cdc19p are required for MCM binding. These studies indicate that MCM complexes in fission yeast have distinct substructures, which may be relevant for function.

Multiple Domains of Fission Yeast Cdc19p (MCM2) Are Required for Its Association with the Core MCM Complex

PubMed Central

Sherman, Daniel A.; Pasion, Sally G.; Forsburg, Susan L.

1998-01-01

The members of the MCM protein family are essential eukaryotic DNA replication factors that form a six-member protein complex. In this study, we use antibodies to four MCM proteins to investigate the structure of and requirements for the formation of fission yeast MCM complexes in vivo, with particular regard to Cdc19p (MCM2). Gel filtration analysis shows that the MCM protein complexes are unstable and can be broken down to subcomplexes. Using coimmunoprecipitation, we find that Mis5p (MCM6) and Cdc21p (MCM4) are tightly associated with one another in a core complex with which Cdc19p loosely associates. Assembly of Cdc19p with the core depends upon Cdc21p. Interestingly, there is no obvious change in Cdc19p-containing MCM complexes through the cell cycle. Using a panel of Cdc19p mutants, we find that multiple domains of Cdc19p are required for MCM binding. These studies indicate that MCM complexes in fission yeast have distinct substructures, which may be relevant for function. PMID:9658174

α-SNAP interferes with the zippering of the SNARE protein membrane fusion machinery.

PubMed

Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

2014-06-06

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

DJ-1 is a redox sensitive adapter protein for high molecular weight complexes involved in regulation of catecholamine homeostasis

PubMed Central

Piston, Dominik; Alvarez-Erviti, Lydia; Bansal, Vikas; Gargano, Daniela; Yao, Zhi; Szabadkai, Gyorgy; Odell, Mark; Puno, M Rhyan; Björkblom, Benny; Maple-Grødem, Jodi; Breuer, Peter; Kaut, Oliver; Larsen, Jan Petter; Bonn, Stefan; Møller, Simon Geir; Wüllner, Ullrich; Schapira, Anthony H V

2017-01-01

Abstract DJ-1 is an oxidation sensitive protein encoded by the PARK7 gene. Mutations in PARK7 are a rare cause of familial recessive Parkinson’s disease (PD), but growing evidence suggests involvement of DJ-1 in idiopathic PD. The key clinical features of PD, rigidity and bradykinesia, result from neurotransmitter imbalance, particularly the catecholamines dopamine (DA) and noradrenaline. We report in human brain and human SH-SY5Y neuroblastoma cell lines that DJ-1 predominantly forms high molecular weight (HMW) complexes that included RNA metabolism proteins hnRNPA1 and PABP1 and the glycolysis enzyme GAPDH. In cell culture models the oxidation status of DJ-1 determined the specific complex composition. RNA sequencing indicated that oxidative changes to DJ-1 were concomitant with changes in mRNA transcripts mainly involved in catecholamine metabolism. Importantly, loss of DJ-1 function upon knock down (KD) or expression of the PD associated form L166P resulted in the absence of HMW DJ-1 complexes. In the KD model, the absence of DJ-1 complexes was accompanied by impairment in catecholamine homeostasis, with significant increases in intracellular DA and noraderenaline levels. These changes in catecholamines could be rescued by re-expression of DJ-1. This catecholamine imbalance may contribute to the particular vulnerability of dopaminergic and noradrenergic neurons to neurodegeneration in PARK7-related PD. Notably, oxidised DJ-1 was significantly decreased in idiopathic PD brain, suggesting altered complex function may also play a role in the more common sporadic form of the disease. PMID:29016861

Systematic analysis of protein–detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials

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

Meyer, Arne; Dierks, Karsten; XtalConcepts, Marlowring 19, 22525 Hamburg

Application of in situ dynamic light scattering to solutions of protein–detergent complexes permits characterization of these complexes in samples as small as 2 µl in volume. Detergents are widely used for the isolation and solubilization of membrane proteins to support crystallization and structure determination. Detergents are amphiphilic molecules that form micelles once the characteristic critical micelle concentration (CMC) is achieved and can solubilize membrane proteins by the formation of micelles around them. The results are presented of a study of micelle formation observed by in situ dynamic light-scattering (DLS) analyses performed on selected detergent solutions using a newly designed advancedmore » hardware device. DLS was initially applied in situ to detergent samples with a total volume of approximately 2 µl. When measured with DLS, pure detergents show a monodisperse radial distribution in water at concentrations exceeding the CMC. A series of all-transn-alkyl-β-d-maltopyranosides, from n-hexyl to n-tetradecyl, were used in the investigations. The results obtained verify that the application of DLS in situ is capable of distinguishing differences in the hydrodynamic radii of micelles formed by detergents differing in length by only a single CH{sub 2} group in their aliphatic tails. Subsequently, DLS was applied to investigate the distribution of hydrodynamic radii of membrane proteins and selected water-insoluble proteins in presence of detergent micelles. The results confirm that stable protein–detergent complexes were prepared for (i) bacteriorhodopsin and (ii) FetA in complex with a ligand as examples of transmembrane proteins. A fusion of maltose-binding protein and the Duck hepatitis B virus X protein was added to this investigation as an example of a non-membrane-associated protein with low water solubility. The increased solubility of this protein in the presence of detergent could be monitored, as well as the progress of proteolytic cleavage to separate the fusion partners. This study demonstrates the potential of in situ DLS to optimize solutions of protein–detergent complexes for crystallization applications.« less

Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells.

PubMed Central

Harlow, E; Pim, D C; Crawford, L V

1981-01-01

Mouse cells transformed by simian virus 40 (SV40) have been shown to contain a complex of the virus-coded large-T antigen with a host 53,000-molecular-weight (53K) protein. Initial attempts to detect a similar complex in lytically infected cells were unsuccessful, and it therefore seemed that the complex might be peculiar to transformed or abortively transformed nonpermissive cells. Immunoprecipitation of [32P]phosphate-labeled extracts of SV40-infected CV-1 African green monkey kidney cells with antibodies specific for large-T or the 53K protein revealed that the large-T-53K protein complex was formed during lytic infections. Only a minor fraction of the large-T present was associated with 53K protein, and large-T and the 53K host protein cosedimented during centrifugation through sucrose gradients. We used monospecific sera and monoclonal antibodies to study the rate of synthesis and phosphorylation of the 53K protein during lytic infections. Infection of CV-1 cells with SV40 increased the rate of synthesis of the 53K protein fivefold over that in mock-infected cells. At the same time, the rate of phosphorylation of the 53K protein increased more than 30-fold compared with control cultures. Monkey cells transformed by UV-irradiated SV40 (Gluzman et al., J. Virol. 22:256-266, 1977) also contained the large-T-53K protein complex. The formation of the complex is therefore not a peculiarity of SV40-transformed rodent cells but is a common feature of SV40 infections. Images PMID:6163871

Fluorescence correlation spectroscopy to study antibody binding and stoichiometry of complexes

NASA Astrophysics Data System (ADS)

Swift, Kerry M.; Matayoshi, Edmund D.

2008-02-01

FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor necrosis factor alpha (TNF-α) and two of its protein antagonists Humira (TM) (adalimumab), a fully humanized monoclonal antibody, and Enbrel (TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the protein drugs and their biological target, the trimeric soluble form of TNF-α. The bivalency of adalimumab and the trimeric nature of TNF-α potentially allow several forms of associative complexes that may differ in stoichiometry. Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our FCS data showed that a single trimeric TNF-α can bind up to three adalimumab molecules. Under some conditions even larger complexes are formed, apparently the result of cross-linking of TNF-α trimers by adalimumab. In addition, distinct differences between Humira and Enbrel were observed in their association with TNF-α.

Structural insights into NHEJ: building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time

PubMed Central

Williams, Gareth J.; Hammel, Michal; Radhakrishnan, Sarvan Kumar; Ramsden, Dale; Lees-Miller, Susan P.; Tainer, John A.

2014-01-01

Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: 1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), 2) lesion processing, and 3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation. PMID:24656613

Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex

PubMed Central

Kubota, Toru; Yokosawa, Noriko; Yokota, Shin-ichi; Fujii, Nobuhiro

2002-01-01

It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the β subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione S-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor. PMID:12438593

Bio-Organic Nanotechnology: Using Proteins and Synthetic Polymers for Nanoscale Devices

NASA Technical Reports Server (NTRS)

Molnar, Linda K.; Xu, Ting; Trent, Jonathan D.; Russell, Thomas P.

2003-01-01

While the ability of proteins to self-assemble makes them powerful tools in nanotechnology, in biological systems protein-based structures ultimately depend on the context in which they form. We combine the self-assembling properties of synthetic diblock copolymers and proteins to construct intricately ordered, three-dimensional polymer protein structures with the ultimate goal of forming nano-scale devices. This hybrid approach takes advantage of the capabilities of organic polymer chemistry to build ordered structures and the capabilities of genetic engineering to create proteins that are selective for inorganic or organic substrates. Here, microphase-separated block copolymers coupled with genetically engineered heat shock proteins are used to produce nano-scale patterning that maximizes the potential for both increased structural complexity and integrity.

The Role of Hexon Protein as a Molecular Mold in Patterning the Protein IX Organization in Human Adenoviruses.

PubMed

Reddy, Vijay S

2017-09-01

Adenoviruses are respiratory, ocular and enteric pathogens that form complex capsids, which are assembled from seven different structural proteins and composed of several core proteins that closely interact with the packaged dsDNA genome. The recent near-atomic resolution structures revealed that the interlacing continuous hexagonal network formed by the protein IX molecules is conserved among different human adenoviruses (HAdVs), but not in non-HAdVs. In this report, we propose a distinct role for the hexon protein as a "molecular mold" in enabling the formation of such hexagonal protein IX network that has been shown to preserve the stability and infectivity of HAdVs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Strategies for the structural analysis of multi-protein complexes: lessons from the 3D-Repertoire project.

PubMed

Collinet, B; Friberg, A; Brooks, M A; van den Elzen, T; Henriot, V; Dziembowski, A; Graille, M; Durand, D; Leulliot, N; Saint André, C; Lazar, N; Sattler, M; Séraphin, B; van Tilbeurgh, H

2011-08-01

Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce 'keystone' subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify 'well behaved' complexes. Herein, we recount our experiences gained at protein production and complex assembly during the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes. Copyright © 2011 Elsevier Inc. All rights reserved.

Chemical cross-linking of the urease complex from Helicobacter pylori and analysis by Fourier transform ion cyclotron resonance mass spectrometry and molecular modeling

NASA Astrophysics Data System (ADS)

Carlsohn, Elisabet; Ångström, Jonas; Emmett, Mark R.; Marshall, Alan G.; Nilsson, Carol L.

2004-05-01

Chemical cross-linking of proteins is a well-established method for structural mapping of small protein complexes. When combined with mass spectrometry, cross-linking can reveal protein topology and identify contact sites between the peptide surfaces. When applied to surface-exposed proteins from pathogenic organisms, the method can reveal structural details that are useful in vaccine design. In order to investigate the possibilities of applying cross-linking on larger protein complexes, we selected the urease enzyme from Helicobacter pylori as a model. This membrane-associated protein complex consists of two subunits: [alpha] (26.5 kDa) and [beta] (61.7 kDa). Three ([alpha][beta]) heterodimers form a trimeric ([alpha][beta])3 assembly which further associates into a unique dodecameric 1.1 MDa complex composed of four ([alpha][beta])3 units. Cross-linked peptides from trypsin-digested urease complex were analyzed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and molecular modeling. Two potential cross-linked peptides (present in the cross-linked sample but undetectable in [alpha], [beta], and native complex) were assigned. Molecular modeling of urease [alpha][beta] complex and trimeric urease units ([alpha][beta])3 revealed a linkage site between the [alpha]-subunit and the [beta]-subunit, and an internal cross-linkage in the [beta]-subunit.

The WD40 Domain Protein MSI1 Functions in a Histone Deacetylase Complex to Fine-Tune Abscisic Acid Signaling.

PubMed

Mehdi, Saher; Derkacheva, Maria; Ramström, Margareta; Kralemann, Lejon; Bergquist, Jonas; Hennig, Lars

2016-01-01

MSI1 belongs to a family of histone binding WD40-repeat proteins. Arabidopsis thaliana contains five genes encoding MSI1-like proteins, but their functions in diverse chromatin-associated complexes are poorly understood. Here, we show that MSI1 is part of a histone deacetylase complex. We copurified HISTONE DEACETYLASE19 (HDA19) with MSI1 and transcriptional regulatory SIN3-like proteins and provide evidence that MSI1 and HDA19 associate into the same complex in vivo. These data suggest that MSI1, HDA19, and HISTONE DEACETYLATION COMPLEX1 protein form a core complex that can integrate various SIN3-like proteins. We found that reduction of MSI1 or HDA19 causes upregulation of abscisic acid (ABA) receptor genes and hypersensitivity of ABA-responsive genes. The MSI1-HDA19 complex fine-tunes ABA signaling by binding to the chromatin of ABA receptor genes and by maintaining low levels of acetylation of histone H3 at lysine 9, thereby affecting the expression levels of ABA receptor genes. Reduced MSI1 or HDA19 levels led to increased tolerance to salt stress corresponding to the increased ABA sensitivity of gene expression. Together, our results reveal the presence of an MSI1-HDA19 complex that fine-tunes ABA signaling in Arabidopsis. © 2016 American Society of Plant Biologists. All rights reserved.

The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum.

PubMed

Wojtkowska, Małgorzata; Buczek, Dorota; Stobienia, Olgierd; Karachitos, Andonis; Antoniewicz, Monika; Slocinska, Małgorzata; Makałowski, Wojciech; Kmita, Hanna

2015-07-01

Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

PubMed

Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

2012-05-01

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

Glycolipid-anchored proteins in neuroblastoma cells form detergent- resistant complexes without caveolin

PubMed Central

1995-01-01

It has been known for a number of years that glycosyl- phosphatidylinositol (GPI)-anchored proteins, in contrast to many transmembrane proteins, are insoluble at 4 degrees C in nonionic detergents such as Triton X-100. Recently, it has been proposed that this behavior reflects the incorporation of GPI-linked proteins into large aggregates that are rich in sphingolipids and cholesterol, as well as in cytoplasmic signaling molecules such as heterotrimeric G proteins and src-family tyrosine kinases. It has been suggested that these lipid-protein complexes are derived from caveolae, non-clathrin- coated invaginations of the plasmalemma that are abundant in endothelial cells, smooth muscle, and lung. Caveolin, a proposed coat protein of caveolae, has been hypothesized to be essential for formation of the complexes. To further investigate the relationship between the detergent-resistant complexes and caveolae, we have characterized the behavior of GPI-anchored proteins in lysates of N2a neuroblastoma cells, which lack morphologically identifiable caveolae, and which do not express caveolin (Shyng, S.-L., J. E. Heuser, and D. A. Harris. 1994. J. Cell Biol. 125:1239-1250). We report here that the complexes prepared from N2a cells display the large size and low buoyant density characteristic of complexes isolated from sources that are rich in caveolae, and contain the same major constituents, including multiple GPI-anchored proteins, alpha and beta subunits of heterotrimeric G proteins, and the tyrosine kinases fyn and yes. Our results argue strongly that detergent-resistant complexes are not equivalent to caveolae in all cell types, and that in neuronal cells caveolin is not essential for the integrity of these complexes. PMID:7537273

Purification and Characterization of a Polygalacturonase-Inhibiting Protein from Phaseolus vulgaris L. 1

PubMed Central

Cervone, Felice; De Lorenzo, Giulia; Degrà, Luisa; Salvi, Giovanni; Bergami, Mario

1987-01-01

Homogeneous endo-polygalacturonase (PG) was covalently bound to cyanogen-bromide-activated Sepharose, and the resulting PG-Sepharose conjugate was utilized to purify, by affinity chromatography, a protein from Phaseolus vulgaris hypocotyls that binds to and inhibits PG. Isoelectric focusing of the purified PG-inhibiting protein (PGIP) showed a major protein band that coincided with PG-inhibiting activity. PGIP formed a complex with PG at pH 5.0 and at low salt concentrations. The complex dissociated in 0.5 m Na-acetate and pH values lower than 4.5 or higher than 6.0. Formation of the PG-PGIP complex resulted in complete inhibition of PG activity. PG activity was restored upon dissociation of the complex. The protein exhibited inhibitory activity toward PGs from Colletotrichum lindemuthianum, Fusarium moniliforme and Aspergillus niger. The possible role of PGIP in regulating the activity of fungal PG's and their ability to elicit plant defense reactions are discussed. Images Fig. 3 PMID:16665751

RNA stores tau reversibly in complex coacervates

PubMed Central

Lin, Yanxian; Eschmann, Neil A.; Zhou, Hongjun; Rauch, Jennifer N.; Hernandez, Israel; Guzman, Elmer; Kosik, Kenneth S.; Han, Songi

2017-01-01

Nonmembrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here, we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios, depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable β-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and predispose the protein toward the formation of insoluble fibrils. PMID:28683104

Fundamental Characteristics of AAA+ Protein Family Structure and Function.

PubMed

Miller, Justin M; Enemark, Eric J

2016-01-01

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins.

The 60 kDa heat shock proteins in the hyperthermophilic archaeon Sulfolobus shibatae.

PubMed

Kagawa, H K; Osipiuk, J; Maltsev, N; Overbeek, R; Quaite-Randall, E; Joachimiak, A; Trent, J D

1995-11-10

One of the most abundant proteins in the hyperthermophilic archaeon Sulfolobus shibatae is the 59 kDa heat shock protein (TF55) that is believed to form a homo-oligomeric double ring complex structurally similar to the bacterial chaperonins. We discovered a second protein subunit in the S. shibatae ring complex (referred to as alpha) that is stoichiometric with TF55 (renamed beta). The gene and flanking regions of alpha were cloned and sequenced and its inferred amino acid sequence has 54.4% identity and 74.4% similarity to beta. Transcription start sites for both alpha and beta were mapped and three potential transcription regulatory regions were identified. Northern analyses of cultures shifted from normal growth temperatures (70 to 75 degrees C) to heat shock temperatures (85 to 90 degrees C) indicated that the levels of alpha and beta mRNAs increased during heat shock, but at all temperatures their relative proportions remained constant. Monitoring protein synthesis by autoradiography of total proteins from cultures pulse labeled with L(-)[35S]methionine at normal and heat shock temperatures indicated significant increases in alpha and beta synthesis during heat shock. Under extreme heat shock conditions (> or = 90 degrees C) alpha and beta appeared to be the only two proteins synthesized. The purified alpha and beta subunits combined to form high molecular mass complexes with similar mobilities on native polyacrylamide gels to the complexes isolated directly from cells. Equal proportions of the two subunits gave the greatest yield of the complex, which we refer to as a "rosettasome". It is argued that the rosettasome consists of two homo-oligomeric rings; one of alpha and the other of beta. Polyclonal antibodies against alpha and beta from S. shibatae cross-reacted with proteins of similar molecular mass in 10 out of the 17 archaeal species tested, suggesting that the two rosettasome proteins are highly conserved among the archaea. The archaeal sequences were aligned with bacterial and eukaryotic chaperonins to generate a phylogenetic tree. The tree reveals the close relationship between the archaeal rosettasomes and the eukaryotic TCP1 protein family and the distant relationship to the bacterial GroEL/HSP60 proteins.

Structural Characterization of a Thrombin-Aptamer Complex by High Resolution Native Top-Down Mass Spectrometry

NASA Astrophysics Data System (ADS)

Zhang, Jiang; Loo, Rachel R. Ogorzalek; Loo, Joseph A.

2017-09-01

Native mass spectrometry (MS) with electrospray ionization (ESI) has evolved as an invaluable tool for the characterization of intact native proteins and non-covalently bound protein complexes. Here we report the structural characterization by high resolution native top-down MS of human thrombin and its complex with the Bock thrombin binding aptamer (TBA), a 15-nucleotide DNA with high specificity and affinity for thrombin. Accurate mass measurements revealed that the predominant form of native human α-thrombin contains a glycosylation mass of 2205 Da, corresponding to a sialylated symmetric biantennary oligosaccharide structure without fucosylation. Native MS showed that thrombin and TBA predominantly form a 1:1 complex under near physiological conditions (pH 6.8, 200 mM NH4OAc), but the binding stoichiometry is influenced by the solution ionic strength. In 20 mM ammonium acetate solution, up to two TBAs were bound to thrombin, whereas increasing the solution ionic strength destabilized the thrombin-TBA complex and 1 M NH4OAc nearly completely dissociated the complex. This observation is consistent with the mediation of thrombin-aptamer binding through electrostatic interactions and it is further consistent with the human thrombin structure that contains two anion binding sites on the surface. Electron capture dissociation (ECD) top-down MS of the thrombin-TBA complex performed with a high resolution 15 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer showed the primary binding site to be at exosite I located near the N-terminal sequence of the heavy chain, consistent with crystallographic data. High resolution native top-down MS is complementary to traditional structural biology methods for structurally characterizing native proteins and protein-DNA complexes. [Figure not available: see fulltext.

Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion

PubMed Central

Takáts, Szabolcs; Glatz, Gábor; Szenci, Győző; Boda, Attila; Horváth, Gábor V.; Hegedűs, Krisztina; Kovács, Attila L.

2018-01-01

The autophagosomal SNARE Syntaxin17 (Syx17) forms a complex with Snap29 and Vamp7/8 to promote autophagosome-lysosome fusion via multiple interactions with the tethering complex HOPS. Here we demonstrate that, unexpectedly, one more SNARE (Ykt6) is also required for autophagosome clearance in Drosophila. We find that loss of Ykt6 leads to large-scale accumulation of autophagosomes that are unable to fuse with lysosomes to form autolysosomes. Of note, loss of Syx5, the partner of Ykt6 in ER-Golgi trafficking does not prevent autolysosome formation, pointing to a more direct role of Ykt6 in fusion. Indeed, Ykt6 localizes to lysosomes and autolysosomes, and forms a SNARE complex with Syx17 and Snap29. Interestingly, Ykt6 can be outcompeted from this SNARE complex by Vamp7, and we demonstrate that overexpression of Vamp7 rescues the fusion defect of ykt6 loss of function cells. Finally, a point mutant form with an RQ amino acid change in the zero ionic layer of Ykt6 protein that is thought to be important for fusion-competent SNARE complex assembly retains normal autophagic activity and restores full viability in mutant animals, unlike palmitoylation or farnesylation site mutant Ykt6 forms. As Ykt6 and Vamp7 are both required for autophagosome-lysosome fusion and are mutually exclusive subunits in a Syx17-Snap29 complex, these data suggest that Vamp7 is directly involved in membrane fusion and Ykt6 acts as a non-conventional, regulatory SNARE in this process. PMID:29694367

Emerging players in the initiation of eukaryotic DNA replication

PubMed Central

2012-01-01

Faithful duplication of the genome in eukaryotes requires ordered assembly of a multi-protein complex called the pre-replicative complex (pre-RC) prior to S phase; transition to the pre-initiation complex (pre-IC) at the beginning of DNA replication; coordinated progression of the replisome during S phase; and well-controlled regulation of replication licensing to prevent re-replication. These events are achieved by the formation of distinct protein complexes that form in a cell cycle-dependent manner. Several components of the pre-RC and pre-IC are highly conserved across all examined eukaryotic species. Many of these proteins, in addition to their bona fide roles in DNA replication are also required for other cell cycle events including heterochromatin organization, chromosome segregation and centrosome biology. As the complexity of the genome increases dramatically from yeast to human, additional proteins have been identified in higher eukaryotes that dictate replication initiation, progression and licensing. In this review, we discuss the newly discovered components and their roles in cell cycle progression. PMID:23075259

Building a pseudo-atomic model of the anaphase-promoting complex.

PubMed

Kulkarni, Kiran; Zhang, Ziguo; Chang, Leifu; Yang, Jing; da Fonseca, Paula C A; Barford, David

2013-11-01

The anaphase-promoting complex (APC/C) is a large E3 ubiquitin ligase that regulates progression through specific stages of the cell cycle by coordinating the ubiquitin-dependent degradation of cell-cycle regulatory proteins. Depending on the species, the active form of the APC/C consists of 14-15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex.

Antioxidative capacity and binding affinity of the complex of green tea catechin and beta-lactoglobulin glycated by the Maillard reaction.

PubMed

Perusko, Marija; Al-Hanish, Ayah; Mihailovic, Jelena; Minic, Simeon; Trifunovic, Sara; Prodic, Ivana; Cirkovic Velickovic, Tanja

2017-10-01

Major green tea catechin, epigallocatechin-3-gallate (EGCG), binds non-covalently to numerous dietary proteins, including beta-lactoglobulin of cow's milk. The effects of glycation of proteins via Maillard reaction on the binding capacity for polyphenols and the antiradical properties of the formed complexes have not been studied previously. Binding constant of BLG glycated by milk sugar lactose to EGCG was measured by the method of fluorophore quenching. Binding of EGCG was confirmed by CD and FTIR. The antioxidative properties of the complexes were examined by measuring ABTS radical scavenging capacity, superoxide anion scavenging capacity and total reducing power assay. Glycation of BLG does not significantly influence the binding constant of EGCG for the protein. Conformational changes were observed for both native and glycated BLG upon complexation with EGCG. Masking effect of polyphenol complexation on the antioxidative potential of the protein was of the similar degree for both glycated BLG and native BLG. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark

A helix swap involving the fifth helix between two adjacently bound Tah1 molecules restores the normal binding environment of the conserved MEEVD peptide of Hsp90. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes with Hsp90 and Tah1. Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR)more » domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90){sub 2}–Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.« less

Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase

PubMed Central

Sareen, Archana; Chaudhury, Indrajit; Adams, Nicole; Sobeck, Alexandra

2012-01-01

Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2–FANCI complex versus the monomeric proteins are. We show that the FANCD2–FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2–FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to—and independently of—FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase. PMID:22753026

Artificial Loading of ASC Specks with Cytosolic Antigens

PubMed Central

Sahillioğlu, Ali Can; Özören, Nesrin

2015-01-01

Inflammasome complexes form upon interaction of Nod Like Receptor (NLR) proteins with pathogen associated molecular patterns (PAPMS) inside the cytosol. Stimulation of a subset of inflammasome receptors including NLRP3, NLRC4 and AIM2 triggers formation of the micrometer-sized spherical supramolecular complex called the ASC speck. The ASC speck is thought to be the platform of inflammasome activity, but the reason why a supramolecular complex is preferred against oligomeric platforms remains elusive. We observed that a set of cytosolic proteins, including the model antigen ovalbumin, tend to co-aggregate on the ASC speck. We suggest that co-aggregation of antigenic proteins on the ASC speck during intracellular infection might be instrumental in antigen presentation. PMID:26258904

Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1, membrane glycoprotein.

PubMed

Li, J; Quilty, J; Popov, M; Reithmeier, R A

2000-07-01

The human erythrocyte anion exchanger (AE)1 (Band 3) contains a single complex N-linked oligosaccharide that is attached to Asn(642) in the fourth extracellular loop of this polytopic membrane protein, while other isoforms (AE2, AE3 and trout AE1) are N-glycosylated on the preceding extracellular loop. Human AE1 expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells contained a high-mannose oligosaccharide. The lack of oligosaccharide processing was not due to retention of AE1 in the endoplasmic reticulum since biotinylation assays showed that approx. 30% of the protein was expressed at the cell surface. Moving the N-glycosylation site to the preceding extracellular loop in an AE1 glycosylation mutant (N555) resulted in processing of the oligosaccharide and production of a complex form of AE1. A double N-glycosylation mutant (N555/N642) contained both a high-mannose and a complex oligosaccharide chain. The complex form of the N555 mutant could be biotinylated showing that this form of the glycoprotein was at the cell surface. Pulse-chase experiments showed that the N555 mutant was efficiently converted from a high-mannose to a complex oligosaccharide with a half-time of approx. 4 h, which reflected the time course of trafficking of AE1 from the endoplasmic reticulum to the plasma membrane. The turnover of the complex form of the N555 mutant occurred with a half-life of approx. 15 h. The results show that the oligosaccharide attached to the endogenous site in extracellular loop 4 in human AE1 is not processed in HEK-293 or COS-7 cells, while the oligosaccharide attached to the preceding loop is converted into the complex form.

Unfolding and refolding details of lysozyme in the presence of β-casein micelles.

PubMed

Wu, Fu-Gen; Luo, Jun-Jie; Yu, Zhi-Wu

2011-02-28

In this work, we selected a small globular protein, lysozyme, to study how it unfolds and refolds in the presence of micelles composed of the unstructured β-casein proteins by using microcalorimetry and circular dichroism spectroscopy. It was found that a partially unfolded structure of lysozyme starts to form when the β-casein/lysozyme molar ratio is above 0.7, and the structure forms exclusively when the β-casein/lysozyme molar ratio is above 1.6. This partially unfolded state of lysozyme loses most of its tertiary structure and after heating, the denatured lysozyme molecules are trapped in the charged coatings of β-casein micelles and cannot refold upon cooling. The thus obtained protein complex can be viewed as a kind of special polyelectrolyte complex micelle. The net charge ratios of the two proteins and the ionic strength of the dispersions can significantly modulate the electrostatic and hydrophobic interactions between the two proteins. Our present work may have implications for the nanoparticle protein engineering therapy in the biomedicine field and may provide a better understanding of the principles governing the protein-protein interactions. Besides, the heating-cooling-reheating procedure employed in this work can also be used to study the unfolding and refolding details of the target protein in other protein-protein, protein-polymer and protein-small solute systems.

Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

DOEpatents

Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

2016-08-09

A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

DOEpatents

Thompson, Vicki S; Lacey, Jeffrey A; Gentillon, Cynthia A; Apel, William A

2015-03-03

A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

Construction of Matryoshka-type structures from supercharged protein nanocages.

PubMed

Beck, Tobias; Tetter, Stephan; Künzle, Matthias; Hilvert, Donald

2015-01-12

Designing nanoscaled hierarchical structures with increasing levels of complexity is challenging. Here we show that electrostatic interactions between two complementarily supercharged protein nanocages can be effectively utilized to create nested Matryoshka-type structures. Cage-within-cage complexes containing spatially ordered iron oxide nanoparticles spontaneously self-assemble upon mixing positively supercharged ferritin compartments with AaLS-13, a larger shell-forming protein with a negatively supercharged lumen. Exploiting engineered Coulombic interactions and protein dynamics in this way opens up new avenues for creating hierarchically organized supramolecular assemblies for application as delivery vehicles, reaction chambers, and artificial organelles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PCoM-DB Update: A Protein Co-Migration Database for Photosynthetic Organisms.

PubMed

Takabayashi, Atsushi; Takabayashi, Saeka; Takahashi, Kaori; Watanabe, Mai; Uchida, Hiroko; Murakami, Akio; Fujita, Tomomichi; Ikeuchi, Masahiko; Tanaka, Ayumi

2017-01-01

The identification of protein complexes is important for the understanding of protein structure and function and the regulation of cellular processes. We used blue-native PAGE and tandem mass spectrometry to identify protein complexes systematically, and built a web database, the protein co-migration database (PCoM-DB, http://pcomdb.lowtem.hokudai.ac.jp/proteins/top), to provide prediction tools for protein complexes. PCoM-DB provides migration profiles for any given protein of interest, and allows users to compare them with migration profiles of other proteins, showing the oligomeric states of proteins and thus identifying potential interaction partners. The initial version of PCoM-DB (launched in January 2013) included protein complex data for Synechocystis whole cells and Arabidopsis thaliana thylakoid membranes. Here we report PCoM-DB version 2.0, which includes new data sets and analytical tools. Additional data are included from whole cells of the pelagic marine picocyanobacterium Prochlorococcus marinus, the thermophilic cyanobacterium Thermosynechococcus elongatus, the unicellular green alga Chlamydomonas reinhardtii and the bryophyte Physcomitrella patens. The Arabidopsis protein data now include data for intact mitochondria, intact chloroplasts, chloroplast stroma and chloroplast envelopes. The new tools comprise a multiple-protein search form and a heat map viewer for protein migration profiles. Users can compare migration profiles of a protein of interest among different organelles or compare migration profiles among different proteins within the same sample. For Arabidopsis proteins, users can compare migration profiles of a protein of interest with putative homologous proteins from non-Arabidopsis organisms. The updated PCoM-DB will help researchers find novel protein complexes and estimate their evolutionary changes in the green lineage. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Dissociation of branched-chain alpha-keto acid dehydrogenase kinase (BDK) from branched-chain alpha-keto acid dehydrogenase complex (BCKDC) by BDK inhibitors.

PubMed

Murakami, Taro; Matsuo, Masayuki; Shimizu, Ayako; Shimomura, Yoshiharu

2005-02-01

Branched-chain alpha-keto acid dehydrogenase kinase (BDK) phosphorylates and inactivates the branched-chain alpha-keto acid dehydrogenase complex (BCKDC), which is the rate-limiting enzyme in the branched-chain amino acid catabolism. BDK has been believed to be bound to the BCKDC. However, recent our studies demonstrated that protein-protein interaction between BDK and BCKDC is one of the factors to regulate BDK activity. Furthermore, only the bound form of BDK appears to have its activity. In the present study, we examined effects of BDK inhibitors on the amount of BDK bound to the BCKDC using rat liver extracts. The bound form of BDK in the extracts of liver from low protein diet-fed rats was measured by an immunoprecipitation pull down assay with or without BDK inhibitors. Among the BDK inhibitors. alpha-ketoisocaproate, alpha-chloroisocaproate, and a-ketoisovalerate released the BDK from the complex. Furthermore, the releasing effect of these inhibitors on the BDK appeared to depend on their inhibition constants. On the other hand, clofibric acid and thiamine pyrophosphate had no effect on the protein-protein interaction between two enzymes. These results suggest that the dissociation of the BDK from the BCKDC is one of the mechanisms responsible for the action of some inhibitors to BDK.

Formation of HDL-like complexes from apolipoprotein A-I(M) and DMPC.

PubMed

Suurkuusk, M; Singh, S K

2000-01-20

Conditions for the preparation of reconstituted high density lipoproteins (HDLs) by incubation of the synthetic lipid dimyristoylphosphatidylcholine (DMPC) and recombinant apolipoprotein A-I(M) have been investigated as a function of ratio of incubation lipid to protein, incubation temperature and the lipid form (multilamellar (MLV) or small unilamellar (SUV) vesicles). The size distributions of the resultant lipid-protein complex particles from various incubations have been evaluated by native gel electrophoresis. Structural changes of the protein after incorporation into these complex particles have been estimated by CD. Thermal characteristics of the particles has been examined by DSC and correlated with CD results. Titration calorimetry has been used to obtain interaction parameters based on a simplified binding model. It is hypothesized that the major enthalpic step in the production of rHDLs is the primary association step between protein and lipid vesicles. It has been shown that by raising the temperature and incubation ratio, the formation of rHDL particles can be directed towards smaller size and a narrower size distribution. The results have been described on the basis of a model where formation of discoidal particles requires prior saturation of vesicle surface area by adsorbed protein, thus explaining differences between particles formed from MLVs and SUVs.

Deciphering the Binding between Nupr1 and MSL1 and Their DNA-Repairing Activity

PubMed Central

Doménech, Rosa; Pantoja-Uceda, David; Gironella, Meritxell; Santoro, Jorge; Velázquez-Campoy, Adrián; Neira, José L.; Iovanna, Juan L.

2013-01-01

The stress protein Nupr1 is a highly basic, multifunctional, intrinsically disordered protein (IDP). MSL1 is a histone acetyl transferase-associated protein, known to intervene in the dosage compensation complex (DCC). In this work, we show that both Nupr1 and MSL1 proteins were recruited and formed a complex into the nucleus in response to DNA-damage, which was essential for cell survival in reply to cisplatin damage. We studied the interaction of Nupr1 and MSL1, and their binding affinities to DNA by spectroscopic and biophysical methods. The MSL1 bound to Nupr1, with a moderate affinity (2.8 µM) in an entropically-driven process. MSL1 did not bind to non-damaged DNA, but it bound to chemically-damaged-DNA with a moderate affinity (1.2 µM) also in an entropically-driven process. The Nupr1 protein bound to chemically-damaged-DNA with a slightly larger affinity (0.4 µM), but in an enthalpically-driven process. Nupr1 showed different interacting regions in the formed complexes with Nupr1 or DNA; however, they were always disordered (“fuzzy”), as shown by NMR. These results underline a stochastic description of the functionality of the Nupr1 and its other interacting partners. PMID:24205110

Triamines and their derivatives as bifunctional chelating agents

DOEpatents

Troutner, David E.; John, Christy S.; Pillai, Maroor R. A.

1992-03-31

A group of functionalized triamine chelants and their derivatives that form complexes with radioactive metal ions are disclosed. The complexes can be covalently attached to a protein or an antibody or antibody fragment and used for therapeutic and/or diagnostic purposes.

The structure of the yeast plasma membrane SNARE complex reveals destabilizing water-filled cavities.

PubMed

Strop, Pavel; Kaiser, Stephen E; Vrljic, Marija; Brunger, Axel T

2008-01-11

SNARE proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7A resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.

Models for the Binary Complex of Bacteriophage T4 Gp59 Helicase Loading Protein. GP32 Single-Stranded DNA-Binding Protein and Ternary Complex with Pseudo-Y Junction DNA

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

Hinerman, Jennifer M.; Dignam, J. David; Mueser, Timothy C.

2012-04-05

The bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable withmore » that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Moreover, fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596–18607).« less

Independent evolution of functionally exchangeable mitochondrial outer membrane import complexes

PubMed Central

Dimmer, Kai S

2018-01-01

Assembly and/or insertion of a subset of mitochondrial outer membrane (MOM) proteins, including subunits of the main MOM translocase, require the fungi-specific Mim1/Mim2 complex. So far it was unclear which proteins accomplish this task in other eukaryotes. Here, we show by reciprocal complementation that the MOM protein pATOM36 of trypanosomes is a functional analogue of yeast Mim1/Mim2 complex, even though these proteins show neither sequence nor topological similarity. Expression of pATOM36 rescues almost all growth, mitochondrial biogenesis, and morphology defects in yeast cells lacking Mim1 and/or Mim2. Conversely, co-expression of Mim1 and Mim2 restores the assembly and/or insertion defects of MOM proteins in trypanosomes ablated for pATOM36. Mim1/Mim2 and pATOM36 form native-like complexes when heterologously expressed, indicating that additional proteins are not part of these structures. Our findings indicate that Mim1/Mim2 and pATOM36 are the products of convergent evolution and arose only after the ancestors of fungi and trypanosomatids diverged. PMID:29923829

Novel protein-protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani.

PubMed

Mishra, Arjun K; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J Venkatesh

2015-01-09

Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein-protein interaction between SpdSyn and AdoMetDc. The protein-protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes. Copyright © 2014 Elsevier Inc. All rights reserved.

Exposure of DNA bases induced by the interaction of DNA and calf thymus DNA helix-destabilizing protein.

PubMed Central

Kohwi-Shigematsu, T; Enomoto, T; Yamada, M A; Nakanishi, M; Tsuboi, M

1978-01-01

The reaction of chloroacetaldehyde with adenine bases in DNA to give a fluorescent product was used to study the availability to intermolecular reaction of positions 1 and 6 of adenine in DNA complexes with calf thymus DNA helix-destabilizing protein. No inhibition of this reaction was observed when heat-denatured DNA was complexed with the protein at a protein/DNA weight ratio of 10:1, compared to free DNA. On the contrary, the same reaction was inhibited markedly for denatured DNA in the presence of calf thymus histone HI at protein/DNA weight ratio of 2:1. Furthermore, the exchange rate for hydrogens of amino and imide groups of DNA bases in DNA strands with deuterium in the solvent was totally unaffected upon complexing of DNA with the DNA helix-destabilizing protein as examined by stopped-flow ultraviolet spectroscopy. These results indicate that the DNA helix-destabilizing protein forms a complex with single-stranded DNA, leaving DNA bases uncovered by the protein. The fluorescence intensity of DNA pretreated with chloroacetaldehyde was amplified by nearly 3-fold upon addition of the DNA helix-destabilizing protein. The possibility of "unstacking" of DNA bases induced by the protein is discussed. PMID:216994

Production, crystallization and preliminary X-ray diffraction of the Gαs α-helical domain in complex with a nanobody.

PubMed

Triest, Sarah; Wohlkönig, Alexandre; Pardon, Els; Steyaert, Jan

2014-11-01

GPCR-G-protein complexes are one of the most important components of cell-signalling cascades. Extracellular signals are sensed by membrane-associated G-protein-coupled receptors (GPCRs) and transduced via G proteins towards intracellular effector molecules. Structural studies of these transient complexes are crucial to understand the molecular details of these interactions. Although a nucleotide-free GPCR-G-protein complex is stable, it is not an ideal sample for crystallization owing to the intrinsic mobility of the Gαs α-helical domain (AHD). To stabilize GPCR-G-protein complexes in a nucleotide-free form, nanobodies were selected that target the flexible GαsAHD. One of these nanobodies, CA9177, was co-crystallized with the GαsAHD. Initial crystals were obtained using the sitting-drop method in a sparse-matrix screen and further optimized. The crystals diffracted to 1.59 Å resolution and belonged to the monoclinic space group P2₁, with unit-cell parameters a=44.07, b=52.55, c=52.66 Å, α=90.00, β=107.89, γ=90.00°. The structure of this specific nanobody reveals its binding epitope on GαsAHD and will help to determine whether this nanobody could be used as crystallization chaperone for GPCR-G-protein complexes.

A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

PubMed Central

Cestari, Igor; Kalidas, Savitha; Monnerat, Severine; Anupama, Atashi; Phillips, Margaret A.

2013-01-01

The genes for all cytoplasmic and potentially all mitochondrial aminoacyl-tRNA synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple aminoacyl-tRNA synthetase (MARS) complex contained at least six aaRS enzymes and three additional non-aaRS proteins. Steady-state kinetic studies showed that association in the MARS complex enhances tRNA-aminoacylation efficiency, which is in part dependent on a MARS complex-associated protein (MCP), named MCP2, that binds tRNAs and increases their aminoacylation by the complex. Conditional repression of MCP2 in T. brucei bloodstream forms resulted in reduced parasite growth and infectivity in mice. Thus, association in a MARS complex enhances tRNA-aminoacylation and contributes to parasite fitness. The MARS complex may be part of a cellular regulatory system and a target for drug development. PMID:24126051

A novel Pfs38 protein complex on the surface of Plasmodium falciparum blood-stage merozoites.

PubMed

Paul, Gourab; Deshmukh, Arunaditya; Kaur, Inderjeet; Rathore, Sumit; Dabral, Surbhi; Panda, Ashutosh; Singh, Susheel Kumar; Mohmmed, Asif; Theisen, Michael; Malhotra, Pawan

2017-02-16

The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface. Recombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-1 65 were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte binding assay with 6-cys proteins were undertaken to find out their possible role in host-parasite infection and seropositivity was assessed using Indian and Liberian sera. Immunoprecipitation of parasite-derived polypeptides, followed by LC-MS/MS analysis, identified a large Pfs38 complex comprising of 6-cys proteins: Pfs41, Pfs38, Pfs12 and other merozoite surface proteins: GLURP, SERA5 and MSP-1. The existence of such a complex was further corroborated by several protein-protein interaction tools, co-localization and co-sedimentation analysis. Pfs38 protein of Pfs38 complex binds to host red blood cells (RBCs) directly via glycophorin A as a receptor. Seroprevalence analysis showed that of the six antigens, prevalence varied from 40 to 99%, being generally highest for MSP-1 65 and GLURP proteins. Together the data show the presence of a large Pfs38 protein-associated complex on the parasite surface which is involved in RBC binding. These results highlight the complex molecular interactions among the P. falciparum merozoite surface proteins and advocate the development of a multi-sub-unit malaria vaccine based on some of these protein complexes on merozoite surface.

Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

PubMed Central

Qiu, Ji; Swartz, James R.; Georgiou, George

1998-01-01

The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding. PMID:9835579

Characterization, stoichiometry, and stability of salivary protein-tannin complexes by ESI-MS and ESI-MS/MS.

PubMed

Canon, Francis; Paté, Franck; Meudec, Emmanuelle; Marlin, Thérèse; Cheynier, Véronique; Giuliani, Alexandre; Sarni-Manchado, Pascale

2009-12-01

Numerous protein-polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP-tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP-tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5.EgCG complexes are maintained intact in the gas phase.

Stacking interactions in PUF-RNA complexes

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

Yiling Koh, Yvonne; Wang, Yeming; Qiu, Chen

2012-07-02

Stacking interactions between amino acids and bases are common in RNA-protein interactions. Many proteins that regulate mRNAs interact with single-stranded RNA elements in the 3' UTR (3'-untranslated region) of their targets. PUF proteins are exemplary. Here we focus on complexes formed between a Caenorhabditis elegans PUF protein, FBF, and its cognate RNAs. Stacking interactions are particularly prominent and involve every RNA base in the recognition element. To assess the contribution of stacking interactions to formation of the RNA-protein complex, we combine in vivo selection experiments with site-directed mutagenesis, biochemistry, and structural analysis. Our results reveal that the identities of stackingmore » amino acids in FBF affect both the affinity and specificity of the RNA-protein interaction. Substitutions in amino acid side chains can restrict or broaden RNA specificity. We conclude that the identities of stacking residues are important in achieving the natural specificities of PUF proteins. Similarly, in PUF proteins engineered to bind new RNA sequences, the identity of stacking residues may contribute to 'target' versus 'off-target' interactions, and thus be an important consideration in the design of proteins with new specificities.« less

Defining the RNA-Protein Interactions in the Trypanosome Preribosomal Complex

PubMed Central

Wang, Lei; Ciganda, Martin

2013-01-01

In eukaryotes, 5S rRNA is transcribed in the nucleoplasm and requires the ribosomal protein L5 to deliver it to the nucleolus for ribosomal assembly. The trypanosome-specific proteins P34 and P37 form a novel preribosomal complex with the eukaryotic conserved L5-5S rRNA complex in the nucleoplasm. Previous results suggested that P34 acts together with L5 to bridge the interaction with 5S rRNA and thus to stabilize 5S rRNA, an important role in the early steps of ribosomal biogenesis. Here, we have delineated the domains of the two protein components, L5 and P34, and regions of the RNA partner, 5S rRNA, that are critical for protein-RNA interactions within the complex. We found that the L18 domain of L5 and the N terminus and RNA recognition motif of P34 bind 5S rRNA. We showed that Trypanosoma brucei L5 binds the β arm of 5S rRNA, while P34 binds loop A/stem V of 5S rRNA. We demonstrated that 5S rRNA is able to enhance the association between the protein components of the complex, L5 and P34. Both loop A/stem V and the β arm of 5S rRNA can separately enhance the protein-protein association, but their effects are neither additive nor synergistic. Domains in the two proteins for protein-protein and protein-RNA interactions overlap or are close to each other. This suggests that 5S rRNA binding might cause conformational changes in L5 and P34 and might also bridge the interactions, thus enhancing binding between the protein partners of this novel complex. PMID:23397568

Interactions among tobacco sieve element occlusion (SEO) proteins.

PubMed

Jekat, Stephan B; Ernst, Antonia M; Zielonka, Sascia; Noll, Gundula A; Prüfer, Dirk

2012-12-01

Angiosperms transport their photoassimilates through sieve tubes, which comprise longitudinally-connected sieve elements. In dicots and also some monocots, the sieve elements contain parietal structural proteins known as phloem proteins or P-proteins. Following injury, P proteins disperse and accumulate as viscous plugs at the sieve plates to prevent the loss of valuable transport sugars. Tobacco (Nicotiana tabacum) P-proteins are multimeric complexes comprising subunits encoded by members of the SEO (sieve element occlusion) gene family. The existence of multiple subunits suggests that P-protein assembly involves interactions between SEO proteins, but this process is largely uncharacterized and it is unclear whether the different subunits perform unique roles or are redundant. We therefore extended our analysis of the tobacco P-proteins NtSEO1 and NtSEO2 to investigate potential interactions between them, and found that both proteins can form homomeric and heteromeric complexes in planta.

LINCing complex functions at the nuclear envelope

PubMed Central

Rothballer, Andrea; Schwartz, Thomas U.; Kutay, Ulrike

2013-01-01

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. PMID:23324460

Regulation of the protein-conducting channel by a bound ribosome

PubMed Central

Gumbart, James; Trabuco, Leonardo G.; Schreiner, Eduard; Villa, Elizabeth; Schulten, Klaus

2009-01-01

Summary During protein synthesis, it is often necessary for the ribosome to form a complex with a membrane-bound channel, the SecY/Sec61 complex, in order to translocate nascent proteins across a cellular membrane. Structural data on the ribosome-channel complex are currently limited to low-resolution cryo-electron microscopy maps, including one showing a bacterial ribosome bound to a monomeric SecY complex. Using that map along with available atomic-level models of the ribosome and SecY, we have determined, through molecular dynamics flexible fitting (MDFF), an atomic-resolution model of the ribosome-channel complex. We characterized computationally the sites of ribosome-SecY interaction within the complex and determined the effect of ribosome binding on the SecY channel. We also constructed a model of a ribosome in complex with a SecY dimer by adding a second copy of SecY to the MDFF-derived model. The study involved 2.7-million-atom simulations over altogether nearly 50 ns. PMID:19913480

Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-TolC.

PubMed

Xu, Yongbin; Song, Saemee; Moeller, Arne; Kim, Nahee; Piao, Shunfu; Sim, Se-Hoon; Kang, Mooseok; Yu, Wookyung; Cho, Hyun-Soo; Chang, Iksoo; Lee, Kangseok; Ha, Nam-Chul

2011-04-15

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.

Functional Implications of an Intermeshing Cogwheel-like Interaction between TolC and MacA in the Action of Macrolide-specific Efflux Pump MacAB-TolC*

PubMed Central

Xu, Yongbin; Song, Saemee; Moeller, Arne; Kim, Nahee; Piao, Shunfu; Sim, Se-Hoon; Kang, Mooseok; Yu, Wookyung; Cho, Hyun-Soo; Chang, Iksoo; Lee, Kangseok; Ha, Nam-Chul

2011-01-01

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse Gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel. PMID:21325274

Analysis of Photosynthetic Antenna Function in a Mutant of Arabidopsis thaliana (L.) Lacking trans-Hexadecenoic Acid 1

PubMed Central

McCourt, Peter; Browse, John; Watson, Jan; Arntzen, Charles J.; Somerville, Chris R.

1985-01-01

Several lines of evidence support the proposal that the unusual chloroplast-specific lipid acyl group Δ3,trans-hexadecenoic acid (trans-C16:1) stimulates the formation or maintenance of the oligomeric form of the light-harvesting chlorophyll a/b complex (LHCP). To assess the functional significance of this apparent association we have analyzed LHCP structure and function in a mutant of Arabidopsis thaliana (L.) which lacks trans-C16:1 by electrophoretic analysis of the protein-chlorophyll complexes and by measurements of chlorophyll fluorescence under a variety of conditions. By these criteria the putative oligomeric form of LHCP appears to be slightly more labile to detergent-mediated dissociation in the mutant. The oligomeric PSI chlorophyll-protein complex, associated with PSI, was also more labile to detergent-mediated dissociation in the mutant, suggesting a previously unsuspected association of trans-C16:1 with the PSI complex. However, no significant effect of the mutation on the efficiency of energy transfer from LHCP to the photochemical reaction centers was observed under any of the various conditions imposed. Also, the stability of the chlorophyll-protein complexes to temperature-induced dissociation was unaffected in the mutant. The role of trans-C16:1 is very subtle or is only conditionally expressed. Images Fig. 1 PMID:16664340

In Vitro Identification of Histatin 5 Salivary Complexes

PubMed Central

Moffa, Eduardo B.; Machado, Maria A. A. M.; Mussi, Maria C. M.; Xiao, Yizhi; Garrido, Saulo S.; Giampaolo, Eunice T.; Siqueira, Walter L.

2015-01-01

With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein’s function is the identification of its interaction partners. Although in saliva some proteins may act primarily as single monomeric units, a significant percentage of all salivary proteins, if not the majority, appear to act in complexes with partners to execute their diverse functions. Coimmunoprecipitation (Co-IP) and pull-down assays were used to identify the heterotypic complexes between histatin 5, a potent natural antifungal protein, and other salivary proteins in saliva. Classical protein–protein interaction methods in combination with high-throughput mass spectrometric techniques were carried out. Co-IP using protein G magnetic Sepharose TM beads suspension was able to capture salivary complexes formed between histatin 5 and its salivary protein partners. Pull-down assay was used to confirm histatin 5 protein partners. A total of 52 different proteins were identified to interact with histatin 5. The present study used proteomic approaches in conjunction with classical biochemical methods to investigate protein–protein interaction in human saliva. Our study demonstrated that when histatin 5 is complexed with salivary amylase, one of the 52 proteins identified as a histatin 5 partner, the antifungal activity of histatin 5 is reduced. We expected that our proteomic approach could serve as a basis for future studies on the mechanism and structural-characterization of those salivary protein interactions to understand their clinical significance. PMID:26544073

The herpes simplex virus 1 UL51 protein interacts with the UL7 protein and plays a role in its recruitment into the virion.

PubMed

Roller, Richard J; Fetters, Rachel

2015-03-01

The alphaherpesvirus UL51 protein is a tegument component that interacts with the viral glycoprotein E and functions at multiple steps in virus assembly and spread in epithelial cells. We show here that pUL51 forms a complex in infected cells with another conserved tegument protein, pUL7. This complex can form in the absence of other viral proteins and is largely responsible for recruitment of pUL7 to cytoplasmic membranes and into the virion tegument. Incomplete colocalization of pUL51 and pUL7 in infected cells, however, suggests that a significant fraction of the population of each protein is not complexed with the other and that they may accomplish independent functions. The ability of herpesviruses to spread from cell to cell in the face of an immune response is critical for disease and shedding following reactivation from latency. Cell-to-cell spread is a conserved ability of herpesviruses, and the identification of conserved viral genes that mediate this process will aid in the design of attenuated vaccines and of novel therapeutics. The conserved UL51 gene of herpes simplex virus 1 plays important roles in cell-to-cell spread and in virus assembly in the cytoplasm, both of which likely depend on specific interactions with other viral and cellular proteins. Here we identify one of those interactions with the product of another conserved herpesvirus gene, UL7, and show that formation of this complex mediates recruitment of UL7 to membranes and to the virion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris

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

Gizatullina, Albina K.; Moscow Institute of Physics and Technology; Finkina, Ekaterina I.

2013-10-04

Highlights: •Lipid transfer protein from lentil seeds (Lc-LTP2) was overexpressed in E. coli. •Antimicrobial activity and spatial structure of the recombinant Lc-LTP2 were examined. •Internal tunnel-like lipid-binding cavity occupies ∼7% of the total Lc-LTP2 volume. •Binding of DMPG lipid induces moderate rearrangements in the Lc-LTP2 structure. •Lc-LTP2/DMPG complex has limited lifetime and dissociates within tens of hours. -- Abstract: Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7 Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeledmore » analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600 Å{sup 3}). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours.« less

Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis.

PubMed

Maruta, Natsumi; Trusov, Yuri; Brenya, Eric; Parekh, Urvi; Botella, José Ramón

2015-03-01

In animals, heterotrimeric G proteins, comprising Ga, Gb, and Gg subunits, are molecular switches whose function tightly depends on Ga and Gbg interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gbg, but not Ga. We report here that the Gbg dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gb, and Gg are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gbg functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gb-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gbg dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Ga subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gbg dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Ga subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.

A trypanosomal orthologue of an intermembrane space chaperone has a non-canonical function in biogenesis of the single mitochondrial inner membrane protein translocase.

PubMed

Wenger, Christoph; Oeljeklaus, Silke; Warscheid, Bettina; Schneider, André; Harsman, Anke

2017-08-01

Mitochondrial protein import is essential for Trypanosoma brucei across its life cycle and mediated by membrane-embedded heterooligomeric protein complexes, which mainly consist of trypanosomatid-specific subunits. However, trypanosomes contain orthologues of small Tim chaperones that escort hydrophobic proteins across the intermembrane space. Here we have experimentally analyzed three novel trypanosomal small Tim proteins, one of which contains only an incomplete Cx3C motif. RNAi-mediated ablation of TbERV1 shows that their import, as in other organisms, depends on the MIA pathway. Submitochondrial fractionation combined with immunoprecipitation and BN-PAGE reveals two pools of small Tim proteins: a soluble fraction forming 70 kDa complexes, consistent with hexamers and a second fraction that is tightly associated with the single trypanosomal TIM complex. RNAi-mediated ablation of the three proteins leads to a growth arrest and inhibits the formation of the TIM complex. In line with these findings, the changes in the mitochondrial proteome induced by ablation of one small Tim phenocopy the effects observed after ablation of TbTim17. Thus, the trypanosomal small Tims play an unexpected and essential role in the biogenesis of the single TIM complex, which for one of them is not linked to import of TbTim17.

Islet-to-LMO stoichiometries control the function of transcription complexes that specify motor neuron and V2a interneuron identity

PubMed Central

Song, Mi-Ryoung; Sun, Yunfu; Bryson, Ami; Gill, Gordon N.; Evans, Sylvia M.; Pfaff, Samuel L.

2009-01-01

Summary LIM transcription factors bind to nuclear LIM interactor (Ldb/NLI/Clim) in specific ratios to form higher-order complexes that regulate gene expression. Here we examined how the dosage of LIM homeodomain proteins Isl1 and Isl2 and LIM-only protein Lmo4 influences the assembly and function of complexes involved in the generation of spinal motor neurons (MNs) and V2a interneurons (INs). Reducing the levels of Islet proteins using a graded series of mutations favored V2a IN differentiation at the expense of MN formation. Although LIM-only proteins (LMOs) are predicted to antagonize the function of Islet proteins, we found that the presence or absence of Lmo4 had little influence on MN or V2a IN specification. We did find, however, that the loss of MNs resulting from reduced Islet levels was rescued by eliminating Lmo4, unmasking a functional interaction between these proteins. Our findings demonstrate that MN and V2a IN fates are specified by distinct complexes that are sensitive to the relative stoichiometries of the constituent factors and we present a model to explain how LIM domain proteins modulate these complexes and, thereby, this binary-cell-fate decision. PMID:19666821

Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins.

PubMed

Bano, Fouzia; Tammi, Markku I; Kang, David W; Harris, Edward N; Richter, Ralf P

2018-06-19

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin⋅biotin bond. Implications for the molecular mechanism of unbinding of HA⋅hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA⋅hyaladherin complexes and HA-rich extracellular matrices. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum.

PubMed

Yeoman, Jeffrey A; Hanssen, Eric; Maier, Alexander G; Klonis, Nectarios; Maco, Bohumil; Baum, Jake; Turnbull, Lynne; Whitchurch, Cynthia B; Dixon, Matthew W A; Tilley, Leann

2011-04-01

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

PubMed

Loris, R; De Greve, H; Dao-Thi, M H; Messens, J; Imberty, A; Wyns, L

2000-08-25

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework. Copyright 2000 Academic Press.

Structure/Function of the Novel Proteins LCIB and LCIC in the Chlamydomonas CCM

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

Martin, Spalding H.

2017-05-09

The goal of this project was to investigate the function of two novel proteins, LCIB and LCIC, which together form an essential protein complex that is required for function of a carbon-dioxide-concentrating mechanism (CCM) required by microalgae to grow in environments where carbon dioxide levels are at or below air equilibration levels.

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure.

PubMed

Wan, William; Stöhr, Jan; Kendall, Amy; Stubbs, Gerald

2015-01-01

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure

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

Wan, William; Stöhr, Jan; Kendall, Amy

2015-09-01

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled themore » complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.« less

Single gene deletions of mrpA to mrpG and mrpE point mutations affect activity of the Mrp Na+/H+ antiporter of alkaliphilic Bacillus and formation of hetero-oligomeric Mrp complexes.

PubMed

Morino, Masato; Natsui, Shinsuke; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

2008-06-01

Mrp antiporters catalyze secondary Na(+)(Li(+))/H(+) antiport and/or K(+)/H(+) antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex of MrpA, MrpB, MrpC, and MrpD was formed in the absence of MrpE, MrpF, or MrpG. By contrast, MrpE, MrpF, and MrpG were not observed in membranes lacking MrpA, MrpB, MrpC, or MrpD. Although MrpA and MrpD have been hypothesized to be the antiporter proteins, the MrpA-to-D complex was inactive. Every Mrp protein was required for an activity level near that of the wild-type Na(+)/H(+) antiporter, but a very low activity level was observed in the absence of MrpE. The introduction of an MrpE(P114G) mutation into the full Mrp complex led to antiport activity with a greatly increased apparent K(m) value for Na(+). The results suggested that interactions among the proteins of heterooligomeric Mrp complexes strongly impact antiporter properties.

RNA polymerase II components and Rrn7 form a preinitiation complex on the HomolD box to promote ribosomal protein gene expression in Schizosaccharomyces pombe.

PubMed

Montes, Matías; Moreira-Ramos, Sandra; Rojas, Diego A; Urbina, Fabiola; Käufer, Norbert F; Maldonado, Edio

2017-02-01

In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription. © 2017 Federation of European Biochemical Societies.

Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein.

PubMed

Osipiuk, J; Georgopoulos, C; Zylicz, M

1993-03-05

It is known that the initiation of bacteriophage lambda replication requires the orderly assembly of the lambda O.lambda P.DnaB helicase protein preprimosomal complex at the ori lambda DNA site. The DnaK, DnaJ, and GrpE heat shock proteins act together to destabilize the lambda P.DnaB complex, thus freeing DnaB and allowing it to unwind lambda DNA near the ori lambda site. The first step of this disassembly reaction is the binding of DnaK to the lambda P protein. In this report, we examined the influence of the DnaJ and GrpE proteins on the stability of the lambda P.DnaK complex. We present evidence for the existence of the following protein-protein complexes: lambda P.DnaK, lambda P.DnaJ, DnaJ.DnaK, DnaK.GrpE, and lambda P.DnaK.GrpE. Our results suggest that the presence of GrpE alone destabilizes the lambda P.DnaK complex, whereas the presence of DnaJ alone stabilizes the lambda P.DnaK complex. Using immunoprecipitation, we show that in the presence of GrpE, DnaK exhibits a higher affinity for the lambda P.DnaJ complex than it does alone. Using cross-linking with glutaraldehyde, we show that oligomeric forms of DnaK exhibit a higher affinity for lambda P than monomeric DnaK. However, in the presence of GrpE, monomeric DnaK can efficiently bind lambda P protein. These findings help explain our previous results, namely that in the GrpE-dependent lambda DNA replication system, the DnaK protein requirement can be reduced up to 10-fold.

Triamines and their derivatives as bifunctional chelating agents

DOEpatents

Troutner, D.E.; John, C.S.; Pillai, M.R.A.

1992-03-31

A group of functionalized triamine chelants and their derivatives that form complexes with radioactive metal ions are disclosed. The complexes can be covalently attached to a protein or an antibody or antibody fragment and used for therapeutic and/or diagnostic purposes. No Drawings

Study of base pair mutations in proline-rich homeodomain (PRH)-DNA complexes using molecular dynamics.

PubMed

Jalili, Seifollah; Karami, Leila; Schofield, Jeremy

2013-06-01

Proline-rich homeodomain (PRH) is a regulatory protein controlling transcription and gene expression processes by binding to the specific sequence of DNA, especially to the sequence 5'-TAATNN-3'. The impact of base pair mutations on the binding between the PRH protein and DNA is investigated using molecular dynamics and free energy simulations to identify DNA sequences that form stable complexes with PRH. Three 20-ns molecular dynamics simulations (PRH-TAATTG, PRH-TAATTA and PRH-TAATGG complexes) in explicit solvent water were performed to investigate three complexes structurally. Structural analysis shows that the native TAATTG sequence forms a complex that is more stable than complexes with base pair mutations. It is also observed that upon mutation, the number and occupancy of the direct and water-mediated hydrogen bonds decrease. Free energy calculations performed with the thermodynamic integration method predict relative binding free energies of 0.64 and 2 kcal/mol for GC to AT and TA to GC mutations, respectively, suggesting that among the three DNA sequences, the PRH-TAATTG complex is more stable than the two mutated complexes. In addition, it is demonstrated that the stability of the PRH-TAATTA complex is greater than that of the PRH-TAATGG complex.

Selective association of a fragment of the knob protein with spectrin, actin and the red cell membrane.

PubMed

Kilejian, A; Rashid, M A; Aikawa, M; Aji, T; Yang, Y F

1991-02-01

The knob protein of Plasmodium falciparum is essential for the formation of knob-like protrusions on the host erythrocyte membrane. A functional domain of the knob protein was identified. This peptide formed stable complexes with the two major red cell skeletal proteins, spectrin and actin. When introduced into resealed normal erythrocytes, the peptide associated selectively with the cytoplasmic surface of the membrane and formed knob-like electron dense deposits. Knobs are thought to play an important role in the immunopathology of P. falciparum infections. Our findings provide a first step towards understanding the molecular basis for selective membrane changes at knobs.

Effect of polymer molecular weight on chitosan-protein interaction.

PubMed

Bekale, L; Agudelo, D; Tajmir-Riahi, H A

2015-01-01

We present a comprehensive study of the interactions between chitosan nanoparticles (15, 100 and 200 kDa with the same degree of deacetylation 90%) and two model proteins, i.e., bovine (BSA) and human serum albumins (HSA), with the aim of correlating chitosan molecular weight (Mw) and the binding affinity of these naturally occurring polymers to protein. The effect of chitosan on the protein secondary structure and the influence of protein complexation on the shape of chitosan nanoparticles are discussed. A combination of multiple spectroscopic methods, transmission electron microscopy (TEM) and thermodynamic analysis were used to assess the polymer-protein complex formation. Results revealed that the three chitosan nanoparticles interact with BSA to form chitosan-BSA complexes, mainly through hydrophobic contacts with the affinity order: 200>100>15 kDa. However, HSA-chitosan complexation is mainly via electrostatic interactions with the stability order: 100>200>15 kDa. Furthermore, the association between polymer and protein causes a partial protein conformational change by a major reduction of α-helix from 63% (free BSA) to 57% (chitosan-BSA) and 57% (free HSA) to 51% (chitosan-HSA). Finally, TEM micrographs clearly revealed that the binding of serum albumins with chitosan nanoparticles induces a significant change in protein morphology and the shape of the polymer. Copyright © 2014 Elsevier B.V. All rights reserved.

Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase

PubMed Central

Burchett, Gina G.; Folsom, Charles G.; Lane, Kimberly T.

2015-01-01

β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins. PMID:26121040

Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase.

PubMed

Burchett, Gina G; Folsom, Charles G; Lane, Kimberly T

2015-01-01

β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins.

Crystal Structure of the ERp44-Peroxiredoxin 4 Complex Reveals the Molecular Mechanisms of Thiol-Mediated Protein Retention.

PubMed

Yang, Kai; Li, De-Feng; Wang, Xi'e; Liang, Jinzhao; Sitia, Roberto; Wang, Chih-Chen; Wang, Xi

2016-10-04

ERp44 controls the localization and transport of diverse proteins in the early secretory pathway. The mechanisms that allow client recognition and the source of the oxidative power for forming intermolecular disulfides are as yet unknown. Here we present the structure of ERp44 bound to a client, peroxiredoxin 4. Our data reveal that ERp44 binds the oxidized form of peroxiredoxin 4 via thiol-disulfide interchange reactions. The structure explains the redox-dependent recognition and characterizes the essential non-covalent interactions at the interface. The ERp44-Prx4 covalent complexes can be reduced by glutathione and protein disulfide isomerase family members in the ER, allowing the two components to recycle. This work provides insights into the mechanisms of thiol-mediated protein retention and indicates the key roles of ERp44 in this biochemical cycle to optimize oxidative folding and redox homeostasis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular architecture of the human GINS complex

PubMed Central

Boskovic, Jasminka; Coloma, Javier; Aparicio, Tomás; Zhou, Min; Robinson, Carol V; Méndez, Juan; Montoya, Guillermo

2007-01-01

Chromosomal DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of these is the GINS complex, which is required for initiation and elongation phases in eukaryotic DNA replication. The GINS complex consists of four paralogous subunits. At the G1/S transition, GINS is recruited to the origins of replication where it assembles with cell-division cycle protein (Cdc)45 and the minichromosome maintenance mutant (MCM)2–7 to form the Cdc45/Mcm2–7/GINS (CMG) complex, the presumed replicative helicase. We isolated the human GINS complex and have shown that it can bind to DNA. By using single-particle electron microscopy and three-dimensional reconstruction, we obtained a medium-resolution volume of the human GINS complex, which shows a horseshoe shape. Analysis of the protein interactions using mass spectrometry and monoclonal antibody mapping shows the subunit organization within the GINS complex. The structure and DNA-binding data suggest how GINS could interact with DNA and also its possible role in the CMG helicase complex. PMID:17557111

A pore-forming protein implements VLR-activated complement cytotoxicity in lamprey.

PubMed

Wu, Fenfang; Feng, Bo; Ren, Yong; Wu, Di; Chen, Yue; Huang, Shengfeng; Chen, Shangwu; Xu, Anlong

2017-01-01

Lamprey is a basal vertebrate with a unique adaptive immune system, which uses variable lymphocyte receptors (VLRs) for antigen recognition. Our previous study has shown that lamprey possessed a distinctive complement pathway activated by VLR. In this study, we identified a natterin family member-lamprey pore-forming protein (LPFP) with a jacalin-like lectin domain and an aerolysin-like pore-forming domain. LPFP had a high affinity with mannan and could form oligomer in the presence of mannan. LPFP could deposit on the surface of target cells, form pore-like complex resembling a wheel with hub and spokes, and mediate powerful cytotoxicity on target cells. These pore-forming proteins along with VLRs and complement molecules were essential for the specific cytotoxicity against exogenous pathogens and tumor cells. This unique cytotoxicity implemented by LPFP might emerge before or in parallel with the IgG-based classical complement lytic pathway completed by polyC9.

Crystallization of Proteins from Crude Bovine Rod Outer Segments☆

PubMed Central

Baker, Bo Y.; Gulati, Sahil; Shi, Wuxian; Wang, Benlian; Stewart, Phoebe L.; Palczewski, Krzysztof

2015-01-01

Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques. PMID:25950977

[Identification of C(2)M interacting proteins by yeast two-hybrid screening].

PubMed

Yue, Shan-shan; Xia, Lai-xin

2015-11-01

The synaptonemal complex (SC) is a huge structure which assembles between the homologous chromosomes during meiotic prophase I. Drosophila germ cell-specific nucleoprotein C(2)M clustering at chromosomes can induce SC formation. To further study the molecular function and mechanism of C(2)M in meiosis, we constructed a bait vector for C(2)M and used the yeast two-hybrid system to identify C(2)M interacting proteins. Forty interacting proteins were obtained, including many DNA and histone binding proteins, ATP synthases and transcription factors. Gene silencing assays in Drosophila showed that two genes, wech and Psf1, may delay the disappearance of SC. These results indicate that Wech and Psf1 may form a complex with C(2)M to participate in the formation or stabilization of the SC complex.

Rescuing the Rescuer: On the Protein Complex between the Human Mitochondrial Acyl Carrier Protein and ISD11.

PubMed

Herrera, María Georgina; Pignataro, María Florencia; Noguera, Martín Ezequiel; Cruz, Karen Magalí; Santos, Javier

2018-05-16

Iron-sulfur clusters are essential cofactors in many biochemical processes. ISD11, one of the subunits of the protein complex that carries out the cluster assembly in mitochondria, is necessary for cysteine desulfurase NFS1 stability and function. Several authors have recently provided evidence showing that ISD11 interacts with the acyl carrier protein (ACP). We carried out the coexpression of human mitochondrial ACP and ISD11 in E. coli. This work shows that ACP and ISD11 form a soluble, structured, and stable complex able to bind to the human NFS1 subunit modulating its activity. Results suggest that ACP plays a key-role in ISD11 folding and stability in vitro. These findings offer the opportunity to study the mechanism of interaction between ISD11 and NFS1.

Influence of endurance training on skeletal muscle mitophagy regulatory proteins in type 2 diabetic men.

PubMed

Brinkmann, Christian; Przyklenk, Axel; Metten, Alexander; Schiffer, Thorsten; Bloch, Wilhelm; Brixius, Klara; Gehlert, Sebastian

2017-11-01

Mitophagy is a form of autophagy for the elimination of mitochondria. Mitochondrial content and function are reduced in the skeletal muscle of patients with type 2 diabetes mellitus (T2DM). Physical training has been shown to restore mitochondrial capacity in T2DM patients, but the role of mitophagy has not been examined in this context. This study analyzes the impact of a 3-month endurance training on important skeletal muscle mitophagy regulatory proteins and oxidative phosphorylation (OXPHOS) complexes in T2DM patients. Muscle biopsies were obtained from eight overweight/obese T2DM men (61±10 years) at T1 (6 weeks pre-training), T2 (1 week pre-training), and T3 (3 to 4 days post-training). Protein contents were determined by Western blotting. The training increased mitochondrial complex II significantly (T2-T3: +29%, p = 0.037). The protein contents of mitophagy regulatory proteins (phosphorylated form of forkhead box O3A (pFOXO3A), mitochondrial E3 ubiquitin protein ligase-1 (MUL1), Bcl-2/adenovirus E1B 19-kD interacting protein-3 (BNIP3), microtubule-associated protein 1 light chain-3B (the ratio LC3B-II/LC3B-I was determined)) did not differ significantly between T1, T2, and T3. The results imply that training-induced changes in OXPHOS subunits (significant increase in complex II) are not accompanied by changes in mitophagy regulatory proteins in T2DM men. Future studies should elucidate whether acute exercise might affect mitophagic processes in T2DM patients (and whether a transient regulation of mitophagy regulatory proteins is evident) to fully clarify the role of physical activity and mitophagy for mitochondrial health in this particular patient group.

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts.

PubMed

Chen, Kuan-Yu; Li, Hsou-min

2007-01-01

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts

PubMed Central

Chen, Kuan-Yu; Li, Hsou-min

2007-01-01

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex. PMID:17144891

The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation

PubMed Central

Yamashita, Takayuki; Kupfer, Gary M.; Naf, Dieter; Suliman, Ahmed; Joenje, Hans; Asano, Shigetaka; D’Andrea, Alan D.

1998-01-01

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A–H). Two FA genes, corresponding to complementation groups A and C, have been cloned, but the function of the FAA and FAC proteins remains unknown. We have recently shown that the FAA and FAC proteins bind and form a nuclear complex. In the current study, we analyzed the FAA and FAC proteins in normal lymphoblasts and lymphoblasts from multiple FA complementation groups. In contrast to normal controls, FA cells derived from groups A, B, C, E, F, G, and H were defective in the formation of the FAA/FAC protein complex, the phosphorylation of the FAA protein, and the accumulation of the FAA/FAC protein complex in the nucleus. These biochemical events seem to define a signaling pathway required for the maintenance of genomic stability and normal hematopoiesis. Our results support the idea that multiple gene products cooperate in the FA Pathway. PMID:9789045

STAM Adaptor Proteins Interact with COPII Complexes and Function in ER-to-Golgi Trafficking

PubMed Central

Rismanchi, Neggy; Puertollano, Rosa; Blackstone, Craig

2009-01-01

Signal transducing adaptor molecules (STAMs) are involved in growth factor and cytokine signaling as well as receptor degradation, and they form complexes with a number of endocytic proteins, including Hrs and Eps15. Here we demonstrate that STAM proteins also localize prominently to early exocytic compartments and profoundly regulate Golgi morphology. Upon STAM overexpression in cells the Golgi apparatus becomes extensively fragmented and dispersed, but when STAMs are depleted the Golgi becomes highly condensed. Under both scenarios, vesicular stomatitis virus G protein (VSVG)-GFP trafficking to the plasma membrane is markedly inhibited, and recovery of Golgi morphology after brefeldin A treatment is substantially impaired in STAM-depleted cells. Furthermore, STAM proteins interact with COPII proteins, probably at endoplasmic reticulum (ER) exit sites, and Sar1 activity is required to maintain the localization of STAMs at discrete sites. Thus, in addition to their roles in signaling and endocytosis, STAMs function prominently in ER-to-Golgi trafficking, most likely through direct interactions with the COPII complex. PMID:19054391

Fundamental Characteristics of AAA+ Protein Family Structure and Function

PubMed Central

2016-01-01

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins. PMID:27703410

VIP21-caveolin, a membrane protein constituent of the caveolar coat, oligomerizes in vivo and in vitro.

PubMed Central

Monier, S; Parton, R G; Vogel, F; Behlke, J; Henske, A; Kurzchalia, T V

1995-01-01

VIP21-caveolin is a membrane protein, proposed to be a component of the striated coat covering the cytoplasmic surface of caveolae. To investigate the biochemical composition of the caveolar coat, we used our previous observation that VIP21-caveolin is present in large complexes and insoluble in the detergents CHAPS or Triton X-114. The mild treatment of these insoluble structures with sodium dodecyl sulfate leads to the detection of high molecular mass complexes of approximately 200, 400, and 600 kDa. The 400-kDa complex purified to homogeneity from dog lung is shown to consist exclusive of the two isoforms of VIP21-caveolin. Pulse-chase experiments indicate that the oligomers form early after the protein is synthesized in the endoplasmic reticulum (ER). VIP21-caveolin does indeed insert into the ER membrane through the classical translocation machinery. Its hydrophobic domain adopts an unusual loop configuration exposing the N- and C-flanking regions to the cytoplasm. Similar high molecular mass complexes can be produced from the in vitro-synthesized VIP21-caveolin. The complex formation occurs only if VIP21-caveolin isoforms are properly inserted into the membrane; formation is cytosol-dependent and does not involve a vesicle fusion step. We propose that high molecular mass oligomers of VIP21-caveolin represent the basic units forming the caveolar coat. They are formed in the ER and later, between the ER and the plasma membrane, these oligomers could associate into larger detergent-insoluble structures. Images PMID:7579702

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

Kolstoe, Simon E.; Jenvey, Michelle C.; Purvis, Alan

Serum amyloid P component is a pentameric plasma glycoprotein that recognizes and binds to amyloid fibres in a calcium-dependent fashion and is likely to contribute to their deposition and persistence in vivo. Five molecules of the drug CPHPC avidly cross-link pairs of protein pentamers and the decameric complex is rapidly cleared in vivo. Crystal structures of the protein in complex with a bivalent drug and cadmium ions, which improve crystal quality, allow the definition of the preferred bound drug isomers. Under physiological conditions, the pentameric human plasma protein serum amyloid P component (SAP) binds hexanoyl bis(d-proline) (R-1-(6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl) pyrrolidine-2-carboxylic acid; CPHPC)more » through its d-proline head groups in a calcium-dependent interaction. Cooperative effects in binding lead to a substantial enhancement of affinity. Five molecules of the bivalent ligand cross-link and stabilize pairs of SAP molecules, forming a decameric complex that is rapidly cleared from the circulation by the liver. Here, it is reported that X-ray analysis of the SAP complex with CPHPC and cadmium ions provides higher resolution detail of the interaction than is observed with calcium ions. Conformational isomers of CPHPC observed in solution by HPLC and by X-ray analysis are compared with the protein-bound form. These are discussed in relation to the development of CPHPC to provide SAP depletion for the treatment of amyloidosis and other indications.« less

Water-Soluble Chlorophyll Protein (WSCP) Stably Binds Two or Four Chlorophylls.

PubMed

Palm, Daniel M; Agostini, Alessandro; Tenzer, Stefan; Gloeckle, Barbara M; Werwie, Mara; Carbonera, Donatella; Paulsen, Harald

2017-03-28

Water-soluble chlorophyll proteins (WSCPs) of class IIa from Brassicaceae form tetrameric complexes containing one chlorophyll (Chl) per apoprotein but no carotenoids. The complexes are remarkably stable toward dissociation and protein denaturation even at 100 °C and extreme pH values, and the Chls are partially protected against photooxidation. There are several hypotheses that explain the biological role of WSCPs, one of them proposing that they function as a scavenger of Chls set free upon plant senescence or pathogen attack. The biochemical properties of WSCP described in this paper are consistent with the protein acting as an efficient and flexible Chl scavenger. At limiting Chl concentrations, the recombinant WSCP apoprotein binds substoichiometric amounts of Chl (two Chls per tetramer) to form complexes that are as stable toward thermal dissociation, denaturation, and photodamage as the fully pigmented ones. If more Chl is added, these two-Chl complexes can bind another two Chls to reach the fully pigmented state. The protection of WSCP Chls against photodamage has been attributed to the apoprotein serving as a diffusion barrier for oxygen, preventing its access to triplet excited Chls and, thus, the formation of singlet oxygen. By contrast, the sequential binding of Chls by WSCP suggests a partially open or at least flexible structure, raising the question of how WSCP photoprotects its Chls without the help of carotenoids.

The structure of the core NuRD repression complex provides insights into its interaction with chromatin

PubMed Central

Millard, Christopher J; Varma, Niranjan; Saleh, Almutasem; Morris, Kyle; Watson, Peter J; Bottrill, Andrew R; Fairall, Louise; Smith, Corinne J; Schwabe, John WR

2016-01-01

The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin. DOI: http://dx.doi.org/10.7554/eLife.13941.001 PMID:27098840

Complexing of Green Tea Catechins with Food Constituents and Degradation of the Complexes by Lactobacillus plantarum

PubMed Central

HAYASHI, Taeko; UEDA, Shuhei; TSURUTA, Hiroki; KUWAHARA, Hiroshige; OSAWA, Ro

2012-01-01

Complexing of green tea catechins with food constituents and their hydrolysis by tannase-producing Lactobacillus plantarum strains, were investigated. Our observations indicated that 1) epigallocatechin gallate (EGCg) and other catechin galloyl esters bound with food ingredients (i.e., proteins) to form a complex that is likely to be unabsorbable through the intestinal wall, whereas most catechins not esterified with gallic acid (GA) remain in free form, not complexing with food ingredients; 2) tannase activity of L. plantarum is strain dependent, possibly grouped into those with high tannase activity hydrolyzing EGCg to epigallocatechin and GA and those with the low activity; and 3) L. plantarum strains with high tannase activity are capable of hydrolyzing not only intact EGCg but also EGCg and other catechin galloyl esters complexed with dietary proteins to free non-galloyl ester catechins and GA. The evidence suggests that L. plantarum with high tannase activity, if it colonizes the human intestine, would release free non-galloyl-ester catechins and GA that are readily absorbed through the human intestinal epithelia from the complexes, thereby ensuring maximum delivery of the bioactive polyphenols of green tea to the host. PMID:24936346

Predicting overlapping protein complexes from weighted protein interaction graphs by gradually expanding dense neighborhoods.

PubMed

Dimitrakopoulos, Christos; Theofilatos, Konstantinos; Pegkas, Andreas; Likothanassis, Spiros; Mavroudi, Seferina

2016-07-01

Proteins are vital biological molecules driving many fundamental cellular processes. They rarely act alone, but form interacting groups called protein complexes. The study of protein complexes is a key goal in systems biology. Recently, large protein-protein interaction (PPI) datasets have been published and a plethora of computational methods that provide new ideas for the prediction of protein complexes have been implemented. However, most of the methods suffer from two major limitations: First, they do not account for proteins participating in multiple functions and second, they are unable to handle weighted PPI graphs. Moreover, the problem remains open as existing algorithms and tools are insufficient in terms of predictive metrics. In the present paper, we propose gradually expanding neighborhoods with adjustment (GENA), a new algorithm that gradually expands neighborhoods in a graph starting from highly informative "seed" nodes. GENA considers proteins as multifunctional molecules allowing them to participate in more than one protein complex. In addition, GENA accepts weighted PPI graphs by using a weighted evaluation function for each cluster. In experiments with datasets from Saccharomyces cerevisiae and human, GENA outperformed Markov clustering, restricted neighborhood search and clustering with overlapping neighborhood expansion, three state-of-the-art methods for computationally predicting protein complexes. Seven PPI networks and seven evaluation datasets were used in total. GENA outperformed existing methods in 16 out of 18 experiments achieving an average improvement of 5.5% when the maximum matching ratio metric was used. Our method was able to discover functionally homogeneous protein clusters and uncover important network modules in a Parkinson expression dataset. When used on the human networks, around 47% of the detected clusters were enriched in gene ontology (GO) terms with depth higher than five in the GO hierarchy. In the present manuscript, we introduce a new method for the computational prediction of protein complexes by making the realistic assumption that proteins participate in multiple protein complexes and cellular functions. Our method can detect accurate and functionally homogeneous clusters. Copyright © 2016 Elsevier B.V. All rights reserved.

Highly branched penta-saccharide-bearing amphiphiles for membrane protein studies

PubMed Central

Ehsan, Muhammad; Du, Yang; Scull, Nicola J.; Tikhonova, Elena; Tarrasch, Jeffrey; Mortensen, Jonas S.; Loland, Claus J.; Skiniotis, Georgios; Guan, Lan; Byrne, Bernadette; Kobilka, Brian K.; Chae, Pil Seok

2016-01-01

Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most of them have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA’Es with a novel highly branched penta-saccharide hydrophilic group. The PSA’Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA’Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins. PMID:26966956

Proteomic analysis in non-denaturing condition of the secretome reveals the presence of multienzyme complexes in Penicillium purpurogenum.

PubMed

Gonzalez-Vogel, Alvaro; Eyzaguirre, Jaime; Oleas, Gabriela; Callegari, Eduardo; Navarrete, Mario

2011-01-01

Proteins secreted by filamentous fungi play key roles in different aspects of their biology. The fungus Penicillium purpurogenum, used as a model organism, is able to degrade hemicelluloses and pectins by secreting a variety of enzymes to the culture medium. This work shows that these enzymes interact with each other to form high molecular weight, catalytically active complexes. By using a proteomics approach, we were able to identify several protein complexes in the secretome of this fungus. The expression and assembly of these complexes depend on the carbon source used and display molecular masses ranging from 300 to 700 kDa. These complexes are composed of a variety of enzymes, including arabinofuranosidases, acetyl xylan esterases, feruloyl esterases, β-glucosidases and xylanases. The protein-protein interactions in these multienzyme complexes were confirmed by coimmunoprecipitation assays. One of the complexes was purified from sugar beet pulp cultures and the subunits identified by tandem mass spectrometry. A better understanding of the biological significance of these kinds of interactions will help in the comprehension of the degradation mechanisms used by fungi and may be of special interest to the biotechnology industry.

A Sulfhydryl-Reactive Ruthenium (II) Complex and Its Conjugation to Protein G as a Universal Reagent for Fluorescent Immunoassays

PubMed Central

Goud, Thirumani Venkatshwar; Huang, Bor-Rong; Lin, Tzu-Chau; Biellmann, Jean-François; Chen, Chien-Sheng

2012-01-01

To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2′-dipyridine Ruthenium bis (hexafluorophosphate). The synthesized Ru(II) complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II)-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The emission peak wavelength of the Ru(II)-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II) complex, indicating that Ru(II)-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG) binding assay was conducted. The result showed that Ru(II)-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II)-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II)-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays. PMID:22563441

Identification of Nucleic Acid Binding Sites on Translin-Associated Factor X (TRAX) Protein

PubMed Central

Gupta, Gagan Deep; Kumar, Vinay

2012-01-01

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity. PMID:22427937

c-Abl interacts with the WAVE2 signaling complex to induce membrane ruffling and cell spreading.

PubMed

Stuart, Jeremy R; Gonzalez, Francis H; Kawai, Hidehiko; Yuan, Zhi-Min

2006-10-20

The Wiskott-Aldrich syndrome-related protein WAVE2 promotes Arp2/3-dependent actin polymerization downstream of Rho-GTPase activation. The Abelson-interacting protein-1 (Abi-1) forms the core of the WAVE2 complex and is necessary for proper stimulation of WAVE2 activity. Here we have shown that the Abl-tyrosine kinase interacts with the WAVE2 complex and that Abl kinase activity facilitates interaction between Abl and WAVE2 complex members. We have characterized various interactions between Abl and members of the WAVE2 complex and revealed that Abi-1 promotes interaction between Abl and WAVE2 members. We have demonstrated that Abl-dependent phosphorylation of WAVE2 is necessary for its activation in vivo, which is highlighted by the findings that RNA interference of WAVE2 expression in Abl/Arg-/- cells has no additive effect on the amount of membrane ruffling. Furthermore, Abl phosphorylates WAVE2 on tyrosine 150, and WAVE2-deficient cells rescued with a Y150F mutant fail to regain their ability to ruffle and form microspikes, unlike cells rescued with wild-type WAVE2. Together, these data show that c-Abl activates WAVE2 via tyrosine phosphorylation to promote actin remodeling in vivo and that Abi-1 forms the crucial link between these two factors.

Signaling pathways that regulate life and cell death: evolution of apoptosis in the context of self-defense.

PubMed

Muñoz-Pinedo, Cristina

2012-01-01

Programmed Cell Death is essential for the life cycle of many organisms. Cell death in multicellular organisms can occur as a consequence of massive damage (necrosis) or in a controlled form, through engagement of diverse biochemical programs. The best well known form of programmed cell death is apoptosis. Apoptosis occurs in animals as a consequence of a variety of stimuli including stress and social signals and it plays essential roles in morphogenesis and immune defense. The machinery of apoptosis is well conserved among animals and it is composed of caspases (the proteases which execute cell death), adapter proteins (caspase activators), Bcl-2 family proteins and Inhibitor of Apoptosis Proteins (IAPs). We will describe in this chapter the main apoptotic pathways in animals: the extrinsic (death receptor-mediated), the intrinsic/mitochondrial and the Granzyme B pathway. Other forms of non-apoptotic Programmed Cell Death which occur in animals will also be discussed. We will summarize the current knowledge about apoptotic-like and other forms of cell death in other organisms such as plants and protists.Additionally, we will discuss the hypothesis that apoptosis originated as part of a host defense mechanism. We will explore the similarities between the protein complexes which mediate apoptosis (apoptosomes) and complexes involved in immunity: inflammasomes. Additional functions of apoptotic proteins related to immune function will be summarized, in an effort to explore the evolutionary origins of cell death.

Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1.

PubMed

Schneider, G J; Geiduschek, E P

1990-06-25

The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers.

JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes.

PubMed

Yin, T; Tsang, M L; Yang, Y C

1994-10-28

Interleukin (IL)-4 and IL-9 regulate the proliferation of T lymphocytes through interactions with their receptors. Previous studies have shown that unknown tyrosine kinases are involved in the proliferative signaling triggered by IL-4 and IL-9. Here we show that IL-4 and IL-9 induce overlapping (170, 130, and 125 kilodalton (kDa)) and distinct (45 and 88/90 kDa, respectively) protein tyrosine phosphorylation in T lymphocytes. We further identify the 170-kDa tyrosine-phosphorylated protein as 4PS/insulin receptor substrate-1-like (IRS-1L) protein and 130-kDa protein as JAK1 kinase. Furthermore, we demonstrate for the first time that JAK1 forms complexes with the IL-4 receptor and 4PS/IRS-1L protein following ligand-receptor interaction. In addition, we demonstrate that IL-9, but not IL-4, induced tyrosine phosphorylation of Stat 91 transcriptional factor. The overlapping and distinct protein tyrosine phosphorylation and activation of the same JAK1 kinase in T lymphocytes strongly suggests that IL-4 and IL-9 share the common signal transduction pathways and that the specificity for each cytokine could be achieved through the unique tyrosine-phosphorylated proteins triggered by individual cytokines.

Modulating surface rheology by electrostatic protein/polysaccharide interactions.

PubMed

Ganzevles, Renate A; Zinoviadou, Kyriaki; van Vliet, Ton; Cohen, Martien A; de Jongh, Harmen H

2006-11-21

There is a large interest in mixed protein/polysaccharide layers at air-water and oil-water interfaces because of their ability to stabilize foams and emulsions. Mixed protein/polysaccharide adsorbed layers at air-water interfaces can be prepared either by adsorption of soluble protein/polysaccharide complexes or by sequential adsorption of complexes or polysaccharides to a previously formed protein layer. Even though the final protein and polysaccharide bulk concentrations are the same, the behavior of the adsorbed layers can be very different, depending on the method of preparation. The surface shear modulus of a sequentially formed beta-lactoglobulin/pectin layer can be up to a factor of 6 higher than that of a layer made by simultaneous adsorption. Furthermore, the surface dilatational modulus and surface shear modulus strongly (up to factors of 2 and 7, respectively) depend on the bulk -lactoglobulin/pectin mixing ratio. On the basis of the surface rheological behavior, a mechanistic understanding of how the structure of the adsorbed layers depends on the protein/polysaccharide interaction in bulk solution, mixing ratio, ionic strength, and order of adsorption to the interface (simultaneous or sequential) is derived. Insight into the effect of protein/polysaccharide interactions on the properties of adsorbed layers provides a solid basis to modulate surface rheological behavior.

Protein-protein interaction predictions using text mining methods.

PubMed

Papanikolaou, Nikolas; Pavlopoulos, Georgios A; Theodosiou, Theodosios; Iliopoulos, Ioannis

2015-03-01

It is beyond any doubt that proteins and their interactions play an essential role in most complex biological processes. The understanding of their function individually, but also in the form of protein complexes is of a great importance. Nowadays, despite the plethora of various high-throughput experimental approaches for detecting protein-protein interactions, many computational methods aiming to predict new interactions have appeared and gained interest. In this review, we focus on text-mining based computational methodologies, aiming to extract information for proteins and their interactions from public repositories such as literature and various biological databases. We discuss their strengths, their weaknesses and how they complement existing experimental techniques by simultaneously commenting on the biological databases which hold such information and the benchmark datasets that can be used for evaluating new tools. Copyright © 2014 Elsevier Inc. All rights reserved.

Bacterial formate hydrogenlyase complex.

PubMed

McDowall, Jennifer S; Murphy, Bonnie J; Haumann, Michael; Palmer, Tracy; Armstrong, Fraser A; Sargent, Frank

2014-09-23

Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a [NiFe] hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor the core complex to the cytoplasmic side of the membrane. As isolated, the FHL complex retains formate hydrogenlyase activity in vitro. Protein film electrochemistry experiments on Hyd-3 demonstrate that it has a unique ability among [NiFe] hydrogenases to catalyze production of H2 even at high partial pressures of H2. Understanding and harnessing the activity of the FHL complex is critical to advancing future biohydrogen research efforts.

Residue-residue contacts: application to analysis of secondary structure interactions.

PubMed

Potapov, Vladimir; Edelman, Marvin; Sobolev, Vladimir

2013-01-01

Protein structures and their complexes are formed and stabilized by interactions, both inside and outside of the protein. Analysis of such interactions helps in understanding different levels of structures (secondary, super-secondary, and oligomeric states). It can also assist molecular biologists in understanding structural consequences of modifying proteins and/or ligands. In this chapter, our definition of atom-atom and residue-residue contacts is described and applied to analysis of protein-protein interactions in dimeric β-sandwich proteins.

Complex coacervation of supercharged proteins with polyelectrolytes.

PubMed

Obermeyer, Allie C; Mills, Carolyn E; Dong, Xue-Hui; Flores, Romeo J; Olsen, Bradley D

2016-04-21

Complexation of proteins with polyelectrolytes or block copolymers can lead to phase separation to generate a coacervate phase or self-assembly of coacervate core micelles. However, many proteins do not coacervate at conditions near neutral pH and physiological ionic strength. Here, protein supercharging is used to systematically explore the effect of protein charge on the complex coacervation with polycations. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase separated with strong polycations when the ratio of negatively charged residues to positively charged residues on the protein (α) was greater than 1.1-1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5-2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of positive charge in the coacervate phase. The composition of protein and polymer in the coacervate phase was determined using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromolecular charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3-1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temperatures.

Macrocyclic metal complexes for metalloenzyme mimicry and sensor development.

PubMed

Joshi, Tanmaya; Graham, Bim; Spiccia, Leone

2015-08-18

Examples of proteins that incorporate one or more metal ions within their structure are found within a broad range of classes, including oxidases, oxidoreductases, reductases, proteases, proton transport proteins, electron transfer/transport proteins, storage proteins, lyases, rusticyanins, metallochaperones, sporulation proteins, hydrolases, endopeptidases, luminescent proteins, iron transport proteins, oxygen storage/transport proteins, calcium binding proteins, and monooxygenases. The metal coordination environment therein is often generated from residues inherent to the protein, small exogenous molecules (e.g., aqua ligands) and/or macrocyclic porphyrin units found, for example, in hemoglobin, myoglobin, cytochrome C, cytochrome C oxidase, and vitamin B12. Thus, there continues to be considerable interest in employing macrocyclic metal complexes to construct low-molecular weight models for metallobiosites that mirror essential features of the coordination environment of a bound metal ion without inclusion of the surrounding protein framework. Herein, we review and appraise our research exploring the application of the metal complexes formed by two macrocyclic ligands, 1,4,7-triazacyclononane (tacn) and 1,4,7,10-tetraazacyclododecane (cyclen), and their derivatives in biological inorganic chemistry. Taking advantage of the kinetic inertness and thermodynamic stability of their metal complexes, these macrocyclic scaffolds have been employed in the development of models that aid the understanding of metal ion-binding natural systems, and complexes with potential applications in biomolecule sensing, diagnosis, and therapy. In particular, the focus has been on "coordinatively unsaturated" metal complexes that incorporate a kinetically inert and stable metal-ligand moiety, but which also contain one or more weakly bound ligands, allowing for the reversible binding of guest molecules via the formation and dissociation of coordinate bonds. With regards to mimicking metallobiosites, examples are presented from our work on tacn-based complexes developed as simplified structural models for multimetallic enzyme sites. In particular, structural comparisons are made between multinuclear copper(II) complexes formed by such ligands and multicopper enzymes featuring type-2 and type-3 copper centers, such as ascorbate oxidase (AO) and laccase (Lc). Likewise, with the aid of relevant examples, we highlight the importance of cooperativity between either multiple metal centers or a metal center and a proximal auxiliary unit appended to the macrocyclic ligand in achieving efficient phosphate ester cleavage. Finally, the critical importance of the Zn(II)-imido and Zn(II)-phosphate interactions in Zn-cyclen-based systems for delivering highly sensitive electrochemical and fluorescent chemosensors is also showcased. The Account additionally highlights some of the factors that limit the performance of these synthetic nucleases and the practical application of the biosensors, and then identifies some avenues for the development of more effective macrocyclic constructs in the future.

Hepatitis Delta Antigen Requires a Flexible Quasi-Double-Stranded RNA Structure To Bind and Condense Hepatitis Delta Virus RNA in a Ribonucleoprotein Complex

PubMed Central

Griffin, Brittany L.; Chasovskikh, Sergey; Dritschilo, Anatoly

2014-01-01

ABSTRACT The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2′OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the in vitro binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed in vitro revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg. IMPORTANCE RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication. Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed with internal loops and bulges. We analyzed the role of the HDV RNA sequence and secondary structure in the formation of a minimal RNP and visualized the structure of this RNP using atomic force microscopy. Our results indicate that HDAg does not recognize the primary sequence of the RNA; rather, the principle contribution of unpaired bases in HDV RNA to HDAg binding is to allow flexibility in the unbranched quasi-double-stranded RNA structure. Visualization of RNPs by atomic force microscopy indicated that the RNA is significantly bent or condensed in the complex. PMID:24741096

Complex formation between the protein components of methane monooxygenase from Methylosinus trichosporium OB3b. Identification of sites of component interaction.

PubMed

Fox, B G; Liu, Y; Dege, J E; Lipscomb, J D

1991-01-05

Kinetic, spectroscopic, and chemical evidence for the formation of specific catalytically essential complexes between the three protein components of the soluble form of methane monooxygenase from Methylosinus trichosporium OB3b is reported. The effects of the concentrations of the reductase and component B on the hydroxylation activity of the reconstituted enzyme system has been numerically simulated based on a kinetic model which assumes formation of multiple high affinity complexes with the hydroxylase component during catalysis. The formation of several of these complexes has been directly demonstrated. By using EPR spectroscopy, the binding of approximately 2 mol of component B/mol of hydroxylase (subunit structure (alpha beta gamma)2) is shown to significantly change the electronic environment of the mu-(H/R)-oxo-bridged binuclear iron cluster of the hydroxylase in both the mixed valent (Fe(II).Fe(III)) and fully reduced (Fe(II).Fe(II)) states. Protein-protein complexes between the reductase and component B as well as between the reductase and hydroxylase have been shown to form by monitoring quenching of the tryptophan fluorescence spectrum of either the component B (KD approximately 0.4 microM) or hydroxylase (two binding sites, KDa approximately 10 nM, KDb approximately 8 microM). The observed KD values are in agreement with the best fit values from the kinetic simulation. Through the use of the covalent zero length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), the binding sites of the component B and reductase were shown to be on the hydroxylase alpha and beta subunits, respectively. The alpha and beta subunits of the hydroxylase are cross-linked by EDC suggesting that they are juxtaposed. EDC also caused the rapid loss of the ability of the monomeric component B to stimulate the hydroxylation reaction suggesting that cross-linking of reactive groups on the protein surface had occurred. This effect was inhibited by the presence of hydroxylase and was accompanied by a loss of the ability of the component B to bind to the hydroxylase. Thus, formation of a component B-hydroxylase complex is apparently required for effective catalysis linked to NADH oxidation. When present in concentrations greater than required to saturate the initial hydroxylase complex, component B inhibited both the rate of the enzymic reaction and the cross-linking of the reductase to the hydroxylase. This suggests that a second complex involving component B can form that negatively regulates catalysis by preventing formation of the reductase-hydroxylase complex.

A photo-cross-linking approach to monitor folding and assembly of newly synthesized proteins in a living cell.

PubMed

Miyazaki, Ryoji; Myougo, Naomi; Mori, Hiroyuki; Akiyama, Yoshinori

2018-01-12

Many proteins form multimeric complexes that play crucial roles in various cellular processes. Studying how proteins are correctly folded and assembled into such complexes in a living cell is important for understanding the physiological roles and the qualitative and quantitative regulation of the complex. However, few methods are suitable for analyzing these rapidly occurring processes. Site-directed in vivo photo-cross-linking is an elegant technique that enables analysis of protein-protein interactions in living cells with high spatial resolution. However, the conventional site-directed in vivo photo-cross-linking method is unsuitable for analyzing dynamic processes. Here, by combining an improved site-directed in vivo photo-cross-linking technique with a pulse-chase approach, we developed a new method that can analyze the folding and assembly of a newly synthesized protein with high spatiotemporal resolution. We demonstrate that this method, named the pulse-chase and in vivo photo-cross-linking experiment (PiXie), enables the kinetic analysis of the formation of an Escherichia coli periplasmic (soluble) protein complex (PhoA). We also used our new technique to investigate assembly/folding processes of two membrane complexes (SecD-SecF in the inner membrane and LptD-LptE in the outer membrane), which provided new insights into the biogenesis of these complexes. Our PiXie method permits analysis of the dynamic behavior of various proteins and enables examination of protein-protein interactions at the level of individual amino acid residues. We anticipate that our new technique will have valuable utility for studies of protein dynamics in many organisms. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Challenges and opportunities of using liquid chromatography and mass spectrometry methods to develop complex vaccine antigens as pharmaceutical dosage forms.

PubMed

Hickey, John M; Sahni, Neha; Toth, Ronald T; Kumru, Ozan S; Joshi, Sangeeta B; Middaugh, C Russell; Volkin, David B

2016-10-01

Liquid chromatographic methods, combined with mass spectrometry, offer exciting and important opportunities to better characterize complex vaccine antigens including recombinant proteins, virus-like particles, inactivated viruses, polysaccharides, and protein-polysaccharide conjugates. The current abilities and limitations of these physicochemical methods to complement traditional in vitro and in vivo vaccine potency assays are explored in this review through the use of illustrative case studies. Various applications of these state-of-the art techniques are illustrated that include the analysis of influenza vaccines (inactivated whole virus and recombinant hemagglutinin), virus-like particle vaccines (human papillomavirus and hepatitis B), and polysaccharide linked to protein carrier vaccines (pneumococcal). Examples of utilizing these analytical methods to characterize vaccine antigens in the presence of adjuvants, which are often included to boost immune responses as part of the final vaccine dosage form, are also presented. Some of the challenges of using chromatographic and LC-MS as physicochemical assays to routinely test complex vaccine antigens are also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Phosphorylation of the IDP KID Modulates Affinity for KIX by Increasing the Lifetime of the Complex.

PubMed

Dahal, Liza; Shammas, Sarah L; Clarke, Jane

2017-12-19

Intrinsically disordered proteins (IDPs) are known to undergo a range of posttranslational modifications, but by what mechanism do such modifications affect the binding of an IDP to its partner protein? We investigate this question using one such IDP, the kinase inducible domain (KID) of the transcription factor CREB, which interacts with the KIX domain of CREB-binding protein upon phosphorylation. As with many other IDPs, KID undergoes coupled folding and binding to form α-helical structure upon interacting with KIX. This single site phosphorylation plays an important role in the control of transcriptional activation in vivo. Here we show that, contrary to expectation, phosphorylation has no effect on association rates-unphosphorylated KID binds just as rapidly as pKID, the phosphorylated form-but rather, acts by increasing the lifetime of the complex. We propose that by controlling the lifetime of the bound complex of pKID:KIX via altering the dissociation rate, phosphorylation can facilitate effective control of transcription regulation. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Hot spot computational identification: Application to the complex formed between the hen egg white lysozyme (HEL) and the antibody HyHEL-10

NASA Astrophysics Data System (ADS)

Moreira, I. S.; Fernandes, P. A.; Ramos, M. J.

The definition and comprehension of the hot spots in an interface is a subject of primary interest for a variety of fields, including structure-based drug design. Therefore, to achieve an alanine mutagenesis computational approach that is at the same time accurate and predictive, capable of reproducing the experimental mutagenesis values is a major challenge in the computational biochemistry field. Antibody/protein antigen complexes provide one of the greatest models to study protein-protein recognition process because they have three fundamentally features: specificity, high complementary association and a small epitope restricted to the diminutive complementary determining regions (CDR) region, while the remainder of the antibody is largely invariant. Thus, we apply a computational mutational methodological approach to the study of the antigen-antibody complex formed between the hen egg white lysozyme (HEL) and the antibody HyHEL-10. A critical evaluation that focuses essentially on the limitations and advantages between different computational methods for hot spot determination, as well as between experimental and computational methodological approaches, is presented.

Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A.

PubMed

Canela, Núria; Orzáez, Mar; Fucho, Raquel; Mateo, Francesca; Gutierrez, Ricardo; Pineda-Lucena, Antonio; Bachs, Oriol; Pérez-Payá, Enrique

2006-11-24

The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity

PubMed Central

Lasker, Keren; Ahrens, Daniel G.; Eckart, Michael R.

2017-01-01

ABSTRACT Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. In the asymmetrically dividing bacterium Caulobacter crescentus, cell polarity stems from the cell cycle-regulated localization and turnover of signaling protein complexes in these hubs, and yet the mechanisms that establish the identity of the two cell poles have not been established. Here, we recapitulate the tripartite assembly of a cell fate signaling complex that forms during the G1-S transition. Using in vivo and in vitro analyses of dynamic polar protein complex formation, we show that a polymeric cell polarity protein, SpmX, serves as a direct bridge between the PopZ polymeric network and the cell fate-directing DivJ histidine kinase. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. The relative copy numbers of these proteins are essential for complex formation, as overexpression of SpmX in Caulobacter reorganizes the polarity of the cell, generating ectopic cell poles containing PopZ and DivJ. Hierarchical formation of higher-order SpmX oligomers nucleates new PopZ microdomain assemblies at the incipient lateral cell poles, driving localized outgrowth. By comparison to self-assembling protein networks and polar cell growth mechanisms in other bacterial species, we suggest that the cooligomeric PopZ-SpmX protein complex in Caulobacter illustrates a paradigm for coupling cell cycle progression to the controlled geometry of cell pole establishment. PMID:28246363

Evidence for the Existence of One Antenna-Associated, Lipid-Dissolved and Two Protein-Bound Pools of Diadinoxanthin Cycle Pigments in Diatoms[C][W

PubMed Central

Lepetit, Bernard; Volke, Daniela; Gilbert, Matthias; Wilhelm, Christian; Goss, Reimund

2010-01-01

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls. PMID:20935178

MobB protein stimulates nicking at the R1162 origin of transfer by increasing the proportion of complexed plasmid DNA.

PubMed Central

Perwez, T; Meyer, R

1996-01-01

An essential early step in conjugal mobilization of R1162, nicking of the DNA strand that is subsequently transferred, is carried out in the relaxosome, a complex of two plasmid-encoded proteins and DNA at the origin of transfer (oriT). A third protein, MobB, is also required for efficient mobilization. We show that in the cell this protein increases the proportion of molecules specifically nicked at oriT, resulting in lower yields of covalently closed molecules after alkaline extraction. These nicked molecules largely remain supercoiled, with unwinding presumably constrained by the relaxosome. MobB enhances the sensitivity of the oriT DNA to oxidation by permanganate, indicating that the protein acts by increasing the fraction of complexed molecules. Mutations that significantly reduce the amount of complexed DNA in the cell were isolated. However, plasmids with these mutations were mobilized at nearly the normal frequency, were nicked at a commensurate level, and still required MobB. Our results indicate that the frequency of transfer is determined both by the amount of time each molecule is in the nicked form and by the proportion of complexed molecules in the total population. PMID:8824623

A script to highlight hydrophobicity and charge on protein surfaces

PubMed Central

Hagemans, Dominique; van Belzen, Ianthe A. E. M.; Morán Luengo, Tania; Rüdiger, Stefan G. D.

2015-01-01

The composition of protein surfaces determines both affinity and specificity of protein-protein interactions. Matching of hydrophobic contacts and charged groups on both sites of the interface are crucial to ensure specificity. Here, we propose a highlighting scheme, YRB, which highlights both hydrophobicity and charge in protein structures. YRB highlighting visualizes hydrophobicity by highlighting all carbon atoms that are not bound to nitrogen and oxygen atoms. The charged oxygens of glutamate and aspartate are highlighted red and the charged nitrogens of arginine and lysine are highlighted blue. For a set of representative examples, we demonstrate that YRB highlighting intuitively visualizes segments on protein surfaces that contribute to specificity in protein-protein interfaces, including Hsp90/co-chaperone complexes, the SNARE complex and a transmembrane domain. We provide YRB highlighting in form of a script that runs using the software PyMOL. PMID:26528483

The multi-replication protein A (RPA) system--a new perspective.

PubMed

Sakaguchi, Kengo; Ishibashi, Toyotaka; Uchiyama, Yukinobu; Iwabata, Kazuki

2009-02-01

Replication protein A (RPA) complex has been shown, using both in vivo and in vitro approaches, to be required for most aspects of eukaryotic DNA metabolism: replication, repair, telomere maintenance and homologous recombination. Here, we review recent data concerning the function and biological importance of the multi-RPA complex. There are distinct complexes of RPA found in the biological kingdoms, although for a long time only one type of RPA complex was believed to be present in eukaryotes. Each complex probably serves a different role. In higher plants, three distinct large and medium subunits are present, but only one species of the smallest subunit. Each of these protein subunits forms stable complexes with their respective partners. They are paralogs as complex. Humans possess two paralogs and one analog of RPA. The multi-RPA system can be regarded as universal in eukaryotes. Among eukaryotic kingdoms, paralogs, orthologs, analogs and heterologs of many DNA synthesis-related factors, including RPA, are ubiquitous. Convergent evolution seems to be ubiquitous in these processes. Using recent findings, we review the composition and biological functions of RPA complexes.

Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion.

PubMed

Zick, Michael; Stroupe, Christopher; Orr, Amy; Douville, Deborah; Wickner, William T

2014-01-01

Like other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans-SNARE pairing but cannot rearrange their lipids for fusion. DOI: http://dx.doi.org/10.7554/eLife.01879.001.

Reversibly constraining spliceosome-substrate complexes by engineering disulfide crosslinks.

PubMed

McCarthy, Patrick; Garside, Erin; Meschede-Krasa, Yonatan; MacMillan, Andrew; Pomeranz Krummel, Daniel

2017-08-01

The spliceosome is a highly dynamic mega-Dalton enzyme, formed in part by assembly of U snRNPs onto its pre-mRNA substrate transcripts. Early steps in spliceosome assembly are challenging to study biochemically and structurally due to compositional and conformational dynamics. We detail an approach to covalently and reversibly constrain or trap non-covalent pre-mRNA/protein spliceosome complexes. This approach involves engineering a single disulfide bond between a thiol-bearing cysteine sidechain and a proximal backbone phosphate of the pre-mRNA, site-specifically modified with an N-thioalkyl moiety. When distance and angle between reactants is optimal, the sidechain will react with the single N-thioalkyl to form a crosslink upon oxidation. We provide protocols detailing how this has been applied successfully to trap an 11-subunit RNA-protein assembly, the human U1 snRNP, in complex with a pre-mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

PubMed

Thomas, Jemima C; Matak-Vinkovic, Dijana; Van Molle, Inge; Ciulli, Alessio

2013-08-06

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

Multimeric Complexes among Ankyrin-Repeat and SOCS-box Protein 9 (ASB9), ElonginBC, and Cullin 5: Insights into the Structure and Assembly of ECS-type Cullin-RING E3 Ubiquitin Ligases

PubMed Central

2013-01-01

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC–Cullin–SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM–MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9–EloBC–Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM–MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems. PMID:23837592

Structural characterization of POM6 Fab and mouse prion protein complex identifies key regions for prions conformational conversion.

PubMed

Baral, Pravas Kumar; Swayampakula, Mridula; Aguzzi, Adriano; James, Michael N G

2018-05-01

Conversion of the cellular prion protein PrP C into its pathogenic isoform PrP S c is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti-prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis-folding events. The structural data of moPrP:POM6 Fab complex are available in the PDB under the accession number www.rcsb.org/pdb/search/structidSearch.do?structureId=6AQ7. © 2018 Federation of European Biochemical Societies.

Profiling the Serum Protein Corona of Fibrillar Human Islet Amyloid Polypeptide.

PubMed

Pilkington, Emily H; Gustafsson, Ove J R; Xing, Yanting; Hernandez-Fernaud, Juan; Zampronio, Cleidi; Kakinen, Aleksandr; Faridi, Ava; Ding, Feng; Wilson, Paul; Ke, Pu Chun; Davis, Thomas P

2018-05-16

Amyloids may be regarded as native nanomaterials that form in the presence of complex protein mixtures. By drawing an analogy with the physicochemical properties of nanoparticles in biological fluids, we hypothesized that amyloids should form a protein corona in vivo that would imbue the underlying amyloid with a modified biological identity. To explore this hypothesis, we characterized the protein corona of human islet amyloid polypeptide (IAPP) fibrils in fetal bovine serum using two complementary methodologies developed herein: quartz crystal microbalance and "centrifugal capture", coupled with nanoliquid chromatography tandem mass spectroscopy. Clear evidence for a significant protein corona was obtained. No trends were identified for amyloid corona proteins based on their physicochemical properties, whereas strong binding with IAPP fibrils occurred for linear proteins or multidomain proteins with structural plasticity. Proteomic analysis identified amyloid-enriched proteins that are known to play significant roles in mediating cellular machinery and processing, potentially leading to pathological outcomes and therapeutic targets.

Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay.

PubMed

Wang, Simon; Yoo, SooHyun; Kim, Hae-Yoon; Wang, Mannan; Zheng, Clare; Parkhouse, Wade; Krieger, Charles; Harden, Nicholas

2015-01-20

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development. Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex.

PubMed

Kruse, Thomas; Bork-Jensen, Jette; Gerdes, Kenn

2005-01-01

MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B. subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became spherical, enlarged and finally lysed. Depletion of each mre gene separately conferred similar gross changes in cell morphology and viability. Thus, the three proteins encoded by mreBCD are all essential and function in the same morphogenetic pathway. Interestingly, the presence of a multicopy plasmid carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and MreD. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology.

Identifying the molecular functions of electron transport proteins using radial basis function networks and biochemical properties.

PubMed

Le, Nguyen-Quoc-Khanh; Nguyen, Trinh-Trung-Duong; Ou, Yu-Yen

2017-05-01

The electron transport proteins have an important role in storing and transferring electrons in cellular respiration, which is the most proficient process through which cells gather energy from consumed food. According to the molecular functions, the electron transport chain components could be formed with five complexes with several different electron carriers and functions. Therefore, identifying the molecular functions in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. This work includes two phases for discriminating electron transport proteins from transport proteins and classifying categories of five complexes in electron transport proteins. In the first phase, the performances from PSSM with AAIndex feature set were successful in identifying electron transport proteins in transport proteins with achieved sensitivity of 73.2%, specificity of 94.1%, and accuracy of 91.3%, with MCC of 0.64 for independent data set. With the second phase, our method can approach a precise model for identifying of five complexes with different molecular functions in electron transport proteins. The PSSM with AAIndex properties in five complexes achieved MCC of 0.51, 0.47, 0.42, 0.74, and 1.00 for independent data set, respectively. We suggest that our study could be a power model for determining new proteins that belongs into which molecular function of electron transport proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro.

PubMed

Canetta, Elisabetta; Kim, Sang Hyon; Kalinina, Natalia O; Shaw, Jane; Adya, Ashok K; Gillespie, Trudi; Brown, John W S; Taliansky, Michael

2008-02-29

Fibrillarin, one of the major proteins of the nucleolus, has methyltransferase activity directing 2'-O-ribose methylation of rRNA and snRNAs and is required for rRNA processing. The ability of the plant umbravirus, groundnut rosette virus, to move long distances through the phloem, the specialized plant vascular system, has been shown to strictly depend on the interaction of one of its proteins, the ORF3 protein (protein encoded by open reading frame 3), with fibrillarin. This interaction is essential for several stages in the groundnut rosette virus life cycle such as nucleolar import of the ORF3 protein via Cajal bodies, relocalization of some fibrillarin from the nucleolus to cytoplasm, and assembly of cytoplasmic umbraviral ribonucleoprotein particles that are themselves required for the long-distance spread of the virus and systemic infection. Here, using atomic force microscopy, we determine the architecture of these complexes as single-layered ringlike structures with a diameter of 18-22 nm and a height of 2.0+/-0.4 nm, which consist of several (n=6-8) distinct protein granules. We also estimate the molar ratio of fibrillarin to ORF3 protein in the complexes as approximately 1:1. Based on these data, we propose a model of the structural organization of fibrillarin-ORF3 protein complexes and discuss potential mechanistic and functional implications that may also apply to other viruses.

Thermodynamics of complex structures formed between single-stranded DNA oligomers and the KH domains of the far upstream element binding protein

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

Chakraborty, Kaushik; Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in

The noncovalent interaction between protein and DNA is responsible for regulating the genetic activities in living organisms. The most critical issue in this problem is to understand the underlying driving force for the formation and stability of the complex. To address this issue, we have performed atomistic molecular dynamics simulations of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein (FBP) complexed with two single-stranded DNA (ss-DNA) oligomers in aqueous media. Attempts have been made to calculate the individual components of the net entropy change for the complexation process by adopting suitablemore » statistical mechanical approaches. Our calculations reveal that translational, rotational, and configurational entropy changes of the protein and the DNA components have unfavourable contributions for this protein-DNA association process and such entropy lost is compensated by the entropy gained due to the release of hydration layer water molecules. The free energy change corresponding to the association process has also been calculated using the Free Energy Perturbation (FEP) method. The free energy gain associated with the KH4–DNA complex formation has been found to be noticeably higher than that involving the formation of the KH3–DNA complex.« less

Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

DOE PAGES

Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.; ...

2015-03-16

Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A 1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A 1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less

Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

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

Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.

Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A 1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A 1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less

Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis

PubMed Central

Zheng, Xiangdong; Gooi, Li Ming; Wason, Arpit; Gabriel, Elke; Mehrjardi, Narges Zare; Yang, Qian; Zhang, Xingrun; Debec, Alain; Basiri, Marcus L.; Avidor-Reiss, Tomer; Pozniakovsky, Andrei; Poser, Ina; Šarić, Tomo; Hyman, Anthony A.; Li, Haitao; Gopalakrishnan, Jay

2014-01-01

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4’s tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an “extended surface-like” platform to tether the Sas-4–PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9–10th β-strands (β9–10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9–10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9–10 surface in mediating protein–protein interactions for efficient Sas-4–PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication. PMID:24385583

Replication protein A, the laxative that keeps DNA regular: The importance of RPA phosphorylation in maintaining genome stability.

PubMed

Byrne, Brendan M; Oakley, Gregory G

2018-04-20

The eukaryotic ssDNA-binding protein, Replication protein A (RPA), was first discovered almost three decades ago. Since then, much progress has been made to elucidate the critical roles for RPA in DNA metabolic pathways that help promote genomic stability. The canonical RPA heterotrimer (RPA1-3) is an essential coordinator of DNA metabolism that interacts with ssDNA and numerous protein partners to coordinate its roles in DNA replication, repair, recombination and telomere maintenance. An alternative form of RPA, termed aRPA, is formed by a complex of RPA4 with RPA1 and RPA3. aRPA is expressed differentially in cells compared to canonical RPA and has been shown to inhibit canonical RPA function while allowing for regular maintenance of cell viability. Interestingly, while aRPA is defective in DNA replication and cell cycle progression, it was shown to play a supporting role in nucleotide excision repair and recombination. The binding domains of canonical RPA interact with a growing number of partners involved in numerous genome maintenance processes. The protein interactions of the RPA-ssDNA complex are not only governed by competition between the binding proteins but also by post-translation modifications such as phosphorylation. Phosphorylation of RPA2 is an important post-translational modification of the RPA complex, and is essential for directing context-specific functions of the RPA complex in the DNA damage response. Due to the importance of RPA in cellular metabolism, it was identified as an appealing target for chemotherapeutic drug development that could be used in future cancer treatment regimens. Copyright © 2018 Elsevier Ltd. All rights reserved.

Distribution of the Pyruvate Dehydrogenase Complex in Developing Soybean Cotyledons

USDA-ARS?s Scientific Manuscript database

The somewhat surprising report that storage proteins and oil are non-uniformly distributed in the cotyledons of developing soybeans prompted us to determine the spatial distribution of the mitochondrial and plastidial forms of the pyruvate dehydrogenase complex (PDC). It has been proposed that pla...

Ferulic acid enhances IgE binding to peanut allergens in western blots.

USDA-ARS?s Scientific Manuscript database

Phenolic compounds at high concentrations are known to form insoluble complexes with proteins. We hypothesized that this complex formation could interfere with Western blot and ELISA assays for peanut allergens. To verify this, three simple phenolic compounds (ferulic, caffeic, and chlorogenic acids...

Hypotensive Effect and Accumulation of Dinitrosyl Iron Complexes in Blood and Tissues after Intravenous and Subcutaneous Injection.

PubMed

Timoshin, A A; Lakomkin, V L; Abramov, A A; Ruuge, E K; Vanin, A F

2016-12-01

Subcutaneous injection of Oxacom with glutathione-bound dinitrosyl iron complex as the active principle produced a slower drop of mean BP and longer accumulation of protein-bound dinitrosyl iron complexes in whole blood and tissues than intravenous injection of this drug, while durations of hypotensive effect in both cases were practically identical. In contrast to intravenous injection of the drug, its subcutaneous administration was not characterized by a high concentration of protein-bound dinitrosyl iron complexes in the blood at the onset of experiment; in addition, accumulation of these NO forms in the lungs was more pronounced after subcutaneous injection than after intravenous one.

Shape Complementarity of Protein-Protein Complexes at Multiple Resolutions

PubMed Central

Zhang, Qing; Sanner, Michel; Olson, Arthur J.

2010-01-01

Biological complexes typically exhibit intermolecular interfaces of high shape complementarity. Many computational docking approaches use this surface complementarity as a guide in the search for predicting the structures of protein-protein complexes. Proteins often undergo conformational changes in order to create a highly complementary interface when associating. These conformational changes are a major cause of failure for automated docking procedures when predicting binding modes between proteins using their unbound conformations. Low resolution surfaces in which high frequency geometric details are omitted have been used to address this problem. These smoothed, or blurred, surfaces are expected to minimize the differences between free and bound structures, especially those that are due to side chain conformations or small backbone deviations. In spite of the fact that this approach has been used in many docking protocols, there has yet to be a systematic study of the effects of such surface smoothing on the shape complementarity of the resulting interfaces. Here we investigate this question by computing shape complementarity of a set of 66 protein-protein complexes represented by multi-resolution blurred surfaces. Complexed and unbound structures are available for these protein-protein complexes. They are a subset of complexes from a non-redundant docking benchmark selected for rigidity (i.e. the proteins undergo limited conformational changes between their bound and unbound states). In this work we construct the surfaces by isocontouring a density map obtained by accumulating the densities of Gaussian functions placed at all atom centers of the molecule. The smoothness or resolution is specified by a Gaussian fall-off coefficient, termed “blobbyness”. Shape complementarity is quantified using a histogram of the shortest distances between two proteins' surface mesh vertices for both the crystallographic complexes and the complexes built using the protein structures in their unbound conformation. The histograms calculated for the bound complex structures demonstrate that medium resolution smoothing (blobbyness=−0.9) can reproduce about 88% of the shape complementarity of atomic resolution surfaces. Complexes formed from the free component structures show a partial loss of shape complementarity (more overlaps and gaps) with the atomic resolution surfaces. For surfaces smoothed to low resolution (blobbyness=−0.3), we find more consistency of shape complementarity between the complexed and free cases. To further reduce bad contacts without significantly impacting the good contacts we introduce another blurred surface, in which the Gaussian densities of flexible atoms are reduced. From these results we discuss the use of shape complementarity in protein-protein docking. PMID:18837463

The structure of Erb1-Ytm1 complex reveals the functional importance of a high-affinity binding between two β-propellers during the assembly of large ribosomal subunits in eukaryotes

PubMed Central

Wegrecki, Marcin; Rodríguez-Galán, Olga; de la Cruz, Jesús; Bravo, Jeronimo

2015-01-01

Ribosome biogenesis is one of the most essential pathways in eukaryotes although it is still not fully characterized. Given the importance of this process in proliferating cells, it is obvious that understanding the macromolecular details of the interactions that take place between the assembly factors, ribosomal proteins and nascent pre-rRNAs is essentially required for the development of new non-genotoxic treatments for cancer. Herein, we have studied the association between the WD40-repeat domains of Erb1 and Ytm1 proteins. These are essential factors for the biogenesis of 60S ribosomal subunits in eukaryotes that form a heterotrimeric complex together with the also essential Nop7 protein. We provide the crystal structure of a dimer formed by the C-terminal part of Erb1 and Ytm1 from Chaetomium thermophilum at 2.1 Å resolution. Using a multidisciplinary approach we show that the β-propeller domains of these proteins interact in a novel manner that leads to a high-affinity binding. We prove that a point mutation within the interface of the complex impairs the interaction between the two proteins and negatively affects growth and ribosome production in yeast. Our study suggests insights into the association of the Erb1-Ytm1 dimer with pre-ribosomal particles. PMID:26476442

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

NASA Technical Reports Server (NTRS)

Cho, G.; Keefe, A. D.; Liu, R.; Wilson, D. S.; Szostak, J. W.

2000-01-01

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. Copyright 2000 Academic Press.

Electrostatic Forces as Dominant Interactions Between Proteins and Polyanions: an ESI MS Study of Fibroblast Growth Factor Binding to Heparin Oligomers

NASA Astrophysics Data System (ADS)

Minsky, Burcu Baykal; Dubin, Paul L.; Kaltashov, Igor A.

2017-04-01

The interactions between fibroblast growth factors (FGFs) and their receptors (FGFRs) are facilitated by heparan sulfate (HS) and heparin (Hp), highly sulfated biological polyelectrolytes. The molecular basis of FGF interactions with these polyelectrolytes is highly complex due to the structural heterogeneity of HS/Hp, and many details still remain elusive, especially the significance of charge density and minimal chain length of HS/Hp in growth factor recognition and multimerization. In this work, we use electrospray ionization mass spectrometry (ESI MS) to investigate the association of relatively homogeneous oligoheparins (octamer, dp8, and decamer, dp10) with acidic fibroblast growth factor (FGF-1). This growth factor forms 1:1, 2:1, and 3:1 protein/heparinoid complexes with both dp8 and dp10, and the fraction of bound protein is highly dependent on protein/heparinoid molar ratio. Multimeric complexes are preferentially formed on the highly sulfated Hp oligomers. Although a variety of oligomers appear to be binding-competent, there is a strong correlation between the affinity and the overall level of sulfation (the highest charge density polyanions binding FGF most strongly via multivalent interactions). These results show that the interactions between FGF-1 and Hp oligomers are primarily directed by electrostatics, and also demonstrate the power of ESI MS as a tool to study multiple binding equilibria between proteins and structurally heterogeneous polyanions.

Dissociation of a Dynamic Protein Complex Studied by All-Atom Molecular Simulations.

PubMed

Zhang, Liqun; Borthakur, Susmita; Buck, Matthias

2016-02-23

The process of protein complex dissociation remains to be understood at the atomic level of detail. Computers now allow microsecond timescale molecular-dynamics simulations, which make the visualization of such processes possible. Here, we investigated the dissociation process of the EphA2-SHIP2 SAM-SAM domain heterodimer complex using unrestrained all-atom molecular-dynamics simulations. Previous studies on this system have shown that alternate configurations are sampled, that their interconversion can be fast, and that the complex is dynamic by nature. Starting from different NMR-derived structures, mutants were designed to stabilize a subset of configurations by swapping ion pairs across the protein-protein interface. We focused on two mutants, K956D/D1235K and R957D/D1223R, with attenuated binding affinity compared with the wild-type proteins. In contrast to calculations on the wild-type complexes, the majority of simulations of these mutants showed protein dissociation within 2.4 μs. During the separation process, we observed domain rotation and pivoting as well as a translation and simultaneous rolling, typically to alternate and weaker binding interfaces. Several unsuccessful recapturing attempts occurred once the domains were moderately separated. An analysis of protein solvation suggests that the dissociation process correlates with a progressive loss of protein-protein contacts. Furthermore, an evaluation of internal protein dynamics using quasi-harmonic and order parameter analyses indicates that changes in protein internal motions are expected to contribute significantly to the thermodynamics of protein dissociation. Considering protein association as the reverse of the separation process, the initial role of charged/polar interactions is emphasized, followed by changes in protein and solvent dynamics. The trajectories show that protein separation does not follow a single distinct pathway, but suggest that the mechanism of dissociation is common in that it initially involves transitions to surfaces with fewer, less favorable contacts compared with those seen in the fully formed complex. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Filamin 2 (Fln2)

PubMed Central

Thompson, Terri G.; Chan, Yiu-Mo; Hack, Andrew A.; Brosius, Melissa; Rajala, Michael; Lidov, Hart G.W.; McNally, Elizabeth M.; Watkins, Simon; Kunkel, Louis M.

2000-01-01

Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin–glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin–glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a γ- and δ-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy. PMID:10629222

Adiponectin Receptors Form Homomers and Heteromers Exhibiting Distinct Ligand Binding and Intracellular Signaling Properties*

PubMed Central

Almabouada, Farid; Diaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R.; Vazquez-Martinez, Rafael; Malagon, Maria M.

2013-01-01

Adiponectin binds to two widely expressed receptors (AdipoR1 and AdipoR2) that contain seven transmembrane domains but, unlike G-protein coupled receptors, present an extracellular C terminus and a cytosolic N terminus. Recently, AdipoR1 was found to associate in high order complexes. However, it is still unknown whether AdipoR2 may also form homomers or heteromers with AdipoR1 or if such interactions may be functionally relevant. Herein, we have analyzed the oligomerization pattern of AdipoRs by FRET and immunoprecipitation and evaluated both the internalization of AdipoRs in response to various adiponectin isoforms and the effect of adiponectin binding to different AdipoR combinations on AMP-activated protein kinase phosphorylation and peroxisome proliferator-activated receptor α activation. Transfection of HEK293AD cells with AdipoR1 and AdipoR2 showed that both receptors colocalize at both the plasma membrane and the endoplasmic reticulum. Co-transfection with the different AdipoR pairs yielded high FRET efficiencies in non-stimulated cells, which indicates that AdipoR1 and AdipoR2 form homo- and heteromeric complexes under resting conditions. Live FRET imaging suggested that both homo- and heteromeric AdipoR complexes dissociate in response to adiponectin, but heteromers separate faster than homomers. Finally, phosphorylation of AMP-activated protein kinase in response to adiponectin was delayed in cells wherein heteromer formation was favored. In sum, our findings indicate that AdipoR1 and AdipoR2 form homo- and heteromers that present unique interaction behaviors and signaling properties. This raises the possibility that the pleiotropic, tissue-dependent functions of adiponectin depend on the expression levels of AdipoR1 and AdipoR2 and, therefore, on the steady-state proportion of homo- and heteromeric complexes. PMID:23255609

Adiponectin receptors form homomers and heteromers exhibiting distinct ligand binding and intracellular signaling properties.

PubMed

Almabouada, Farid; Diaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R; Vazquez-Martinez, Rafael; Malagon, Maria M

2013-02-01

Adiponectin binds to two widely expressed receptors (AdipoR1 and AdipoR2) that contain seven transmembrane domains but, unlike G-protein coupled receptors, present an extracellular C terminus and a cytosolic N terminus. Recently, AdipoR1 was found to associate in high order complexes. However, it is still unknown whether AdipoR2 may also form homomers or heteromers with AdipoR1 or if such interactions may be functionally relevant. Herein, we have analyzed the oligomerization pattern of AdipoRs by FRET and immunoprecipitation and evaluated both the internalization of AdipoRs in response to various adiponectin isoforms and the effect of adiponectin binding to different AdipoR combinations on AMP-activated protein kinase phosphorylation and peroxisome proliferator-activated receptor α activation. Transfection of HEK293AD cells with AdipoR1 and AdipoR2 showed that both receptors colocalize at both the plasma membrane and the endoplasmic reticulum. Co-transfection with the different AdipoR pairs yielded high FRET efficiencies in non-stimulated cells, which indicates that AdipoR1 and AdipoR2 form homo- and heteromeric complexes under resting conditions. Live FRET imaging suggested that both homo- and heteromeric AdipoR complexes dissociate in response to adiponectin, but heteromers separate faster than homomers. Finally, phosphorylation of AMP-activated protein kinase in response to adiponectin was delayed in cells wherein heteromer formation was favored. In sum, our findings indicate that AdipoR1 and AdipoR2 form homo- and heteromers that present unique interaction behaviors and signaling properties. This raises the possibility that the pleiotropic, tissue-dependent functions of adiponectin depend on the expression levels of AdipoR1 and AdipoR2 and, therefore, on the steady-state proportion of homo- and heteromeric complexes.

The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent.

PubMed Central

Austin, S; Dixon, R

1992-01-01

The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent. We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites. Images PMID:1534752

Protein crystallization aboard the Space Shuttle and the Mir space station

NASA Technical Reports Server (NTRS)

Delbaere, Louis T. J.; Vandonselaar, Margaret; Prasad, Lata; Quail, J. W.; Birnbaum, George I.; Delucas, Lawrence J.; Moore, Karen; Bugg, Charles E.

1993-01-01

Two different protein crystallizations, namely ,the free Fab fragment of the Je142 monoclonal antibody and the complex of Fab fragment/HPr with antigen, were performed aboard the Discovery Space Shuttle flights and the Mir space station, respectively. Medium sized crystals of the Je142 Fab fragment were obtained. The Je142 Fab fragment/Hpr complex produced two medium-sized crystals after two months aboard the Mir space station. Microgravity was found to eliminate the tendency of these crystals to form clusters.

Fiber Diffraction of the Prion-Forming Domain HET-s(218-289) Shows Dehydration-Induced Deformation of a Complex Amyloid Structure

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

Wan, William; Stubbs, Gerald

2014-05-01

Amyloids are filamentous protein aggregates that can be formed by many different proteins and are associated with both disease and biological functions. The pathogenicities or biological functions of amyloids are determined by their particular molecular structures, making accurate structural models a requirement for understanding their biological effects. One potential factor that can affect amyloid structures is hydration. Previous studies of simple stacked β-sheet amyloids have suggested that dehydration does not impact structure, but other studies indicated dehydration-related structural changes of a putative water-filled nanotube. Our results show that dehydration significantly affects the molecular structure of the fungal prion-forming domain HET-s(218–289),more » which forms a β-solenoid with no internal solvent-accessible regions. The dehydration-related structural deformation of HET-s(218–289) indicates that water can play a significant role in complex amyloid structures, even when no obvious water-accessible cavities are present.« less

BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

PubMed Central

Di Pietro, Santiago M.; Falcón-Pérez, Juan M.; Tenza, Danièle; Setty, Subba R.G.; Marks, Michael S.; Raposo, Graça

2006-01-01

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery. PMID:16837549

A Novel c-Jun N-terminal Kinase (JNK) Signaling Complex Involved in Neuronal Migration during Brain Development.

PubMed

Zhang, Feng; Yu, Jingwen; Yang, Tao; Xu, Dan; Chi, Zhixia; Xia, Yanheng; Xu, Zhiheng

2016-05-27

Disturbance of neuronal migration may cause various neurological disorders. Both the transforming growth factor-β (TGF-β) signaling and microcephaly-associated protein WDR62 are important for neuronal migration during brain development; however, the underlying molecular mechanisms involved remain unclear. We show here that knock-out or knockdown of Tak1 (TGFβ-activated kinase 1) and Jnk2 (c-Jun N-terminal kinase 2) perturbs neuronal migration during cortical development and that the migration defects incurred by knock-out and/or knockdown of Tβr2 (type II TGF-β receptor) or Tak1 can be partially rescued by expression of TAK1 and JNK2, respectively. Furthermore, TAK1 forms a protein complex with RAC1 and two scaffold proteins of the JNK pathway, the microcephaly-associated protein WDR62 and the RAC1-interacting protein POSH (plenty of Src homology). Components of the complex coordinate with each other in the regulation of TAK1 as well as JNK activities. We suggest that unique JNK protein complexes are involved in the diversified biological and pathological functions during brain development and pathogenesis of diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification and characterization of multiple rubisco activases in chemoautotrophic bacteria

PubMed Central

Tsai, Yi-Chin Candace; Lapina, Maria Claribel; Bhushan, Shashi; Mueller-Cajar, Oliver

2015-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is responsible for almost all biological CO2 assimilation, but forms inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. The distantly related AAA+ proteins rubisco activase and CbbX remodel inhibited rubisco complexes to effect inhibitor release in plants and α-proteobacteria, respectively. Here we characterize a third class of rubisco activase in the chemolithoautotroph Acidithiobacillus ferrooxidans. Two sets of isoforms of CbbQ and CbbO form hetero-oligomers that function as specific activases for two structurally diverse rubisco forms. Mutational analysis supports a model wherein the AAA+ protein CbbQ functions as motor and CbbO is a substrate adaptor that binds rubisco via a von Willebrand factor A domain. Understanding the mechanisms employed by nature to overcome rubisco's shortcomings will increase our toolbox for engineering photosynthetic carbon dioxide fixation. PMID:26567524

Monitoring of the Enzymatic Degradation of Protein Corona and Evaluating the Accompanying Cytotoxicity of Nanoparticles.

PubMed

Ma, Zhifang; Bai, Jing; Jiang, Xiue

2015-08-19

Established nanobio interactions face the challenge that the formation of nanoparticle-protein corona complexes shields the inherent properties of the nanoparticles and alters the manner of the interactions between nanoparticles and biological systems. Therefore, many studies have focused on protein corona-mediated nanoparticle binding, internalization, and intracellular transportation. However, there are a few studies to pay attention to if the corona encounters degradation after internalization and how the degradation of the protein corona affects cytotoxicity. To fill this gap, we prepared three types of off/on complexes based on gold nanoparticles (Au NPs) and dye-labeled serum proteins and studied the extracellular and intracellular proteolytic processes of protein coronas as well as their accompanying effects on cytotoxicity through multiple evaluation mechanisms, including cell viability, adenosine triphosphate (ATP) content, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS). The proteolytic process was confirmed by recovery of the fluorescence of the dye-labeled protein molecules that was initially quenched by Au NPs. Our results indicate that the degradation rate of protein corona is dependent on the type of the protein based on systematical evaluation of the extracellular and intracellular degradation processes of the protein coronas formed by human serum albumin (HSA), γ-globulin (HGG), and serum fibrinogen (HSF). Degradation is the fastest for HSA corona and the slowest for HSF corona. Notably, we also find that the Au NP-HSA corona complex induces lower cell viability, slower ATP production, lower MMP, and higher ROS levels. The cytotoxicity of the nanoparticle-protein corona complex may be associated with the protein corona degradation process. All of these results will enrich the database of cytotoxicity induced by nanomaterial-protein corona complexes.

How curvature-generating proteins build scaffolds on membrane nanotubes

PubMed Central

Evergren, Emma; Golushko, Ivan; Prévost, Coline; Renard, Henri-François; Johannes, Ludger; McMahon, Harvey T.; Lorman, Vladimir; Voth, Gregory A.; Bassereau, Patricia

2016-01-01

Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein–membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30–40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes. PMID:27655892

Human neuronal uncoupling proteins 4 and 5 (UCP4 and UCP5): structural properties, regulation, and physiological role in protection against oxidative stress and mitochondrial dysfunction.

PubMed

Ramsden, David B; Ho, Philip W-L; Ho, Jessica W-M; Liu, Hui-Fang; So, Danny H-F; Tse, Ho-Man; Chan, Koon-Ho; Ho, Shu-Leong

2012-07-01

Uncoupling proteins (UCPs) belong to a large family of mitochondrial solute carriers 25 (SLC25s) localized at the inner mitochondrial membrane. UCPs transport protons directly from the intermembrane space to the matrix. Of five structural homologues (UCP1 to 5), UCP4 and 5 are principally expressed in the central nervous system (CNS). Neurons derived their energy in the form of ATP that is generated through oxidative phosphorylation carried out by five multiprotein complexes (Complexes I-V) embedded in the inner mitochondrial membrane. In oxidative phosphorylation, the flow of electrons generated by the oxidation of substrates through the electron transport chain to molecular oxygen at Complex IV leads to the transport of protons from the matrix to the intermembrane space by Complex I, III, and IV. This movement of protons to the intermembrane space generates a proton gradient (mitochondrial membrane potential; MMP) across the inner membrane. Complex V (ATP synthase) uses this MMP to drive the conversion of ADP to ATP. Some electrons escape to oxygen-forming harmful reactive oxygen species (ROS). Proton leakage back to the matrix which bypasses Complex V resulting in a major reduction in ROS formation while having a minimal effect on MMP and hence, ATP synthesis; a process termed "mild uncoupling." UCPs act to promote this proton leakage as means to prevent excessive build up of MMP and ROS formation. In this review, we discuss the structure and function of mitochondrial UCPs 4 and 5 and factors influencing their expression. Hypotheses concerning the evolution of the two proteins are examined. The protective mechanisms of the two proteins against neurotoxins and their possible role in regulating intracellular calcium movement, particularly with regard to the pathogenesis of Parkinson's disease are discussed.

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

PubMed

Paca-Uccaralertkun, S; Zhao, L J; Adya, N; Cross, J V; Cullen, B R; Boros, I M; Giam, C Z

1994-01-01

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.

Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue

PubMed Central

Köhler, Simone; Wojcik, Michal; Dernburg, Abby F.

2017-01-01

When cells enter meiosis, their chromosomes reorganize as linear arrays of chromatin loops anchored to a central axis. Meiotic chromosome axes form a platform for the assembly of the synaptonemal complex (SC) and play central roles in other meiotic processes, including homologous pairing, recombination, and chromosome segregation. However, little is known about the 3D organization of components within the axes, which include cohesin complexes and additional meiosis-specific proteins. Here, we investigate the molecular organization of meiotic chromosome axes in Caenorhabditis elegans through STORM (stochastic optical reconstruction microscopy) and PALM (photo-activated localization microscopy) superresolution imaging of intact germ-line tissue. By tagging one axis protein (HIM-3) with a photoconvertible fluorescent protein, we established a spatial reference for other components, which were localized using antibodies against epitope tags inserted by CRISPR/Cas9 genome editing. Using 3D averaging, we determined the position of all known components within synapsed chromosome axes to high spatial precision in three dimensions. We find that meiosis-specific HORMA domain proteins span a gap between cohesin complexes and the central region of the SC, consistent with their essential roles in SC assembly. Our data further suggest that the two different meiotic cohesin complexes are distinctly arranged within the axes: Although cohesin complexes containing the kleisin REC-8 protrude above and below the plane defined by the SC, complexes containing COH-3 or -4 kleisins form a central core, which may physically separate sister chromatids. This organization may help to explain the role of the chromosome axes in promoting interhomolog repair of meiotic double-strand breaks by inhibiting intersister repair. PMID:28559338

Nucleolin forms a specific complex with a fragment of the viral (minus) strand of minute virus of mice DNA.

PubMed Central

Barrijal, S; Perros, M; Gu, Z; Avalosse, B L; Belenguer, P; Amalric, F; Rommelaere, J

1992-01-01

Nucleolin, a major nucleolar protein, forms a specific complex with the genome (a single-stranded DNA molecule of minus polarity) of parvovirus MVMp in vitro. By means of South-western blotting experiments, we mapped the binding site to a 222-nucleotide motif within the non-structural transcription unit, referred to as NUBE (nucleolin-binding element). The specificity of the interaction was confirmed by competitive gel retardation assays. DNaseI and nuclease S1 probing showed that NUBE folds into a secondary structure, in agreement with a computer-assisted conformational prediction. The whole NUBE may be necessary for the interaction with nucleolin, as suggested by the failure of NUBE subfragments to bind the protein and by the nuclease footprinting experiments. The present work extends the previously reported ability of nucleolin to form a specific complex with ribosomal RNA, to a defined DNA substrate. Considering the tropism of MVMp DNA replication for host cell nucleoli, these data raise the possibility that nucleolin may contribute to the regulation of the parvoviral life-cycle. Images PMID:1408821

Protein complex formation and intranuclear dynamics of NAC1 in cancer cells.

PubMed

Nakayama, Naomi; Kato, Hiroaki; Sakashita, Gyosuke; Nariai, Yuko; Nakayama, Kentaro; Kyo, Satoru; Urano, Takeshi

2016-09-15

Nucleus accumbens-associated protein 1 (NAC1) is a cancer-related transcription regulator protein that is also involved in the pluripotency and differentiation of embryonic stem cells. NAC1 is overexpressed in various carcinomas including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation and the dynamics of intranuclear NAC1 in cancer cells remain poorly understood. In this study, analysis of HeLa cell lysates by fast protein liquid chromatography (FPLC) on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300-500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Furthermore, live cell photobleaching analyses with green fluorescent protein (GFP)-fused NAC1 proteins revealed the intranuclear dynamics of NAC1. Collectively our results demonstrate that NAC1 forms a protein complex to function as a transcriptional regulator in cancer cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Cellular Chaperonin CCTγ Contributes to Rabies Virus Replication during Infection

PubMed Central

Zhang, Jinyang; Wu, Xiaopeng; Zan, Jie; Wu, Yongping; Ye, Chengjin; Ruan, Xizhen

2013-01-01

Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit. PMID:23637400

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

PubMed Central

Zhang, Yi; Ng, Huck-Hui; Erdjument-Bromage, Hediye; Tempst, Paul; Bird, Adrian; Reinberg, Danny

1999-01-01

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. PMID:10444591

Can small hydrophobic gold nanoparticles inhibit β2-microglobulin fibrillation?

NASA Astrophysics Data System (ADS)

Brancolini, Giorgia; Toroz, Dimitrios; Corni, Stefano

2014-06-01

Inorganic nanoparticles stabilized by a shell of organic ligands can enhance or suppress the natural propensity of proteins to form fibrils. Functionalization facilitates targeted delivery of the nanoparticles to various cell types, bioimaging, drug delivery and other therapeutic and diagnostic applications. In this study, we provide a computational model of the effect of a prototypical thiol-protected gold nanoparticle, Au25L18- (L = S(CH2)2Ph) on the β2-microglobulin natural fibrillation propensity. To reveal the molecular basis of the protein-nanoparticle association process, we performed various simulations at multiple levels (Classical Molecular Dynamics and Brownian Dynamics) that cover multiple length- and timescales. The results provide a model of the ensemble of structures constituting the protein-gold nanoparticle complexes, and insights into the driving forces for the binding of β2-microglobulin to hydrophobic small size gold nanoparticles. We have found that the small nanoparticles can bind the protein to form persistent complexes. This binding of nanoparticles is able to block the active sites of domains from binding to another protein, thus leading to potential inhibition of the fibrillation activity. A comparison with the binding patches identified for the interaction of the protein with a known inhibitor of fibrillation, supports our conclusion.Inorganic nanoparticles stabilized by a shell of organic ligands can enhance or suppress the natural propensity of proteins to form fibrils. Functionalization facilitates targeted delivery of the nanoparticles to various cell types, bioimaging, drug delivery and other therapeutic and diagnostic applications. In this study, we provide a computational model of the effect of a prototypical thiol-protected gold nanoparticle, Au25L18- (L = S(CH2)2Ph) on the β2-microglobulin natural fibrillation propensity. To reveal the molecular basis of the protein-nanoparticle association process, we performed various simulations at multiple levels (Classical Molecular Dynamics and Brownian Dynamics) that cover multiple length- and timescales. The results provide a model of the ensemble of structures constituting the protein-gold nanoparticle complexes, and insights into the driving forces for the binding of β2-microglobulin to hydrophobic small size gold nanoparticles. We have found that the small nanoparticles can bind the protein to form persistent complexes. This binding of nanoparticles is able to block the active sites of domains from binding to another protein, thus leading to potential inhibition of the fibrillation activity. A comparison with the binding patches identified for the interaction of the protein with a known inhibitor of fibrillation, supports our conclusion. Electronic supplementary information (ESI) available: Details on the molecular dynamics simulation results. Table S1 reports results of the MD trajectories with a single NP at different initial velocities (d1, d2, d3, and d4) (three-dimensional structures and contact residues). Table S2 reports results of the MD trajectories with a couple of NPs at different initial velocities (initial orientations, three-dimensional structures, contact residues and root-mean-square deviations). Table S3 reports root-mean-square fluctuations and divergence of the protein structure with respect to the NMR model. Table S4 describes the average energy of the final complexes. See DOI: 10.1039/c4nr01514b

Rapid self-assembly of complex biomolecular architectures during mussel byssus biofabrication

PubMed Central

Priemel, Tobias; Degtyar, Elena; Dean, Mason N.; Harrington, Matthew J.

2017-01-01

Protein-based biogenic materials provide important inspiration for the development of high-performance polymers. The fibrous mussel byssus, for instance, exhibits exceptional wet adhesion, abrasion resistance, toughness and self-healing capacity–properties that arise from an intricate hierarchical organization formed in minutes from a fluid secretion of over 10 different protein precursors. However, a poor understanding of this dynamic biofabrication process has hindered effective translation of byssus design principles into synthetic materials. Here, we explore mussel byssus assembly in Mytilus edulis using a synergistic combination of histological staining and confocal Raman microspectroscopy, enabling in situ tracking of specific proteins during induced thread formation from soluble precursors to solid fibres. Our findings reveal critical insights into this complex biological manufacturing process, showing that protein precursors spontaneously self-assemble into complex architectures, while maturation proceeds in subsequent regulated steps. Beyond their biological importance, these findings may guide development of advanced materials with biomedical and industrial relevance. PMID:28262668

Physicochemical characterization of mineral (iron/zinc) bound caseinate and their mineral uptake in Caco-2 cells.

PubMed

Shilpashree, B G; Arora, Sumit; Kapila, Suman; Sharma, Vivek

2018-08-15

Milk proteins (especially caseins) are widely accepted as good vehicle for the delivery of various bioactive compounds including minerals. Succinylation is one of the most acceptable chemical modification techniques to enhance the mineral binding ability of caseins. Addition of minerals to succinylated proteins may alter their physicochemical and biochemical properties. Physicochemical characteristics of succinylated sodium caseinate (S.NaCN)-mineral (iron/zinc) complexes were elucidated. Chromatographic behaviour and fluorescence intensity confirmed the structural modification of S.NaCN upon binding with minerals. The bound mineral from protein complexes showed significantly higher (P < 0.05) in vitro bioavailability (mineral uptake) than mineral salts in Caco-2 cells. Also, iron bound S.NaCN showed higher cellular ferritin formation than iron in its free form. These mineral bound protein complexes with improved bioavailability could safely replace inorganic fortificants in various functional food formulations. Copyright © 2018 Elsevier Ltd. All rights reserved.

Interaction Network and Localization of Brucella abortus Membrane Proteins Involved in the Synthesis, Transport, and Succinylation of Cyclic β-1,2-Glucans

PubMed Central

Guidolin, Leticia S.; Morrone Seijo, Susana M.; Guaimas, Francisco F.

2015-01-01

ABSTRACT Cyclic β-1,2-glucans (CβG) are periplasmic homopolysaccharides that play an important role in the virulence and interaction of Brucella with the host. Once synthesized in the cytoplasm by the CβG synthase (Cgs), CβG are transported to the periplasm by the CβG transporter (Cgt) and succinylated by the CβG modifier enzyme (Cgm). Here, we used a bacterial two-hybrid system and coimmunoprecipitation techniques to study the interaction network between these three integral inner membrane proteins. Our results indicate that Cgs, Cgt, and Cgm can form both homotypic and heterotypic interactions. Analyses carried out with Cgs mutants revealed that the N-terminal region of the protein (Cgs region 1 to 418) is required to sustain the interactions with Cgt and Cgm as well as with itself. We demonstrated by single-cell fluorescence analysis that in Brucella, Cgs and Cgt are focally distributed in the membrane, particularly at the cell poles, whereas Cgm is mostly distributed throughout the membrane with a slight accumulation at the poles colocalizing with the other partners. In summary, our results demonstrate that Cgs, Cgt, and Cgm form a membrane-associated biosynthetic complex. We propose that the formation of a membrane complex could serve as a mechanism to ensure the fidelity of CβG biosynthesis by coordinating their synthesis with the transport and modification. IMPORTANCE In this study, we analyzed the interaction and localization of the proteins involved in the synthesis, transport, and modification of Brucella abortus cyclic β-1,2-glucans (CβG), which play an important role in the virulence and interaction of Brucella with the host. We demonstrate that these proteins interact, forming a complex located mainly at the cell poles; this is the first experimental evidence of the existence of a multienzymatic complex involved in the metabolism of osmoregulated periplasmic glucans in bacteria and argues for another example of pole differentiation in Brucella. We propose that the formation of this membrane complex could serve as a mechanism to ensure the fidelity of CβG biosynthesis by coordinating synthesis with the transport and modification. PMID:25733613

Comparing anterior and posterior Hox complex formation reveals guidelines for predicting cis-regulatory elements

PubMed Central

Uhl, Juli D.; Cook, Tiffany A.; Gebelein, Brian

2010-01-01

Hox transcription factors specify numerous cell fates along the anterior-posterior axis by regulating the expression of downstream target genes. While expression analysis has uncovered large numbers of de-regulated genes in cells with altered Hox activity, determining which are direct versus indirect targets has remained a significant challenge. Here, we characterize the DNA binding activity of Hox transcription factor complexes on eight experimentally verified cis-regulatory elements. Hox factors regulate the activity of each element by forming protein complexes with two cofactor proteins, Extradenticle (Exd) and Homothorax (Hth). Using comparative DNA binding assays, we found that a number of flexible arrangements of Hox, Exd, and Hth binding sites mediate cooperative transcription factor complexes. Moreover, analysis of a Distal-less regulatory element (DMXR) that is repressed by abdominal Hox factors revealed that suboptimal binding sites can be combined to form high affinity transcription complexes. Lastly, we determined that the anterior Hox factors are more dependent upon Exd and Hth for complex formation than posterior Hox factors. Based upon these findings, we suggest a general set of guidelines to serve as a basis for designing bioinformatics algorithms aimed at identifying Hox regulatory elements using the wealth of recently sequenced genomes. PMID:20398649

LAR-RPTP Clustering Is Modulated by Competitive Binding between Synaptic Adhesion Partners and Heparan Sulfate

PubMed Central

Won, Seoung Youn; Kim, Cha Yeon; Kim, Doyoun; Ko, Jaewon; Um, Ji Won; Lee, Sung Bae; Buck, Matthias; Kim, Eunjoon; Heo, Won Do; Lee, Jie-Oh; Kim, Ho Min

2017-01-01

The leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are cellular receptors of heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans that direct axonal growth and neuronal regeneration. LAR-RPTPs are also synaptic adhesion molecules that form trans-synaptic adhesion complexes by binding to various postsynaptic adhesion ligands, such as Slit- and Trk-like family of proteins (Slitrks), IL-1 receptor accessory protein-like 1 (IL1RAPL1), interleukin-1 receptor accessory protein (IL-1RAcP) and neurotrophin receptor tyrosine kinase C (TrkC), to regulate synaptogenesis. Here, we determined the crystal structure of the human LAR-RPTP/IL1RAPL1 complex and found that lateral interactions between neighboring LAR-RPTP/IL1RAPL1 complexes in crystal lattices are critical for the higher-order assembly and synaptogenic activity of these complexes. Moreover, we found that LAR-RPTP binding to the postsynaptic adhesion ligands, Slitrk3, IL1RAPL1 and IL-1RAcP, but not TrkC, induces reciprocal higher-order clustering of trans-synaptic adhesion complexes. Although LAR-RPTP clustering was induced by either HS or postsynaptic adhesion ligands, the dominant binding of HS to the LAR-RPTP was capable of dismantling pre-established LAR-RPTP-mediated trans-synaptic adhesion complexes. These findings collectively suggest that LAR-RPTP clustering for synaptogenesis is modulated by a complex synapse-organizing protein network. PMID:29081732

Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing

PubMed Central

Wongpalee, Somsakul Pop; Vashisht, Ajay; Sharma, Shalini; Chui, Darryl; Wohlschlegel, James A; Black, Douglas L

2016-01-01

Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest. DOI: http://dx.doi.org/10.7554/eLife.19743.001 PMID:27882870