Protein Quaternary Structure Research Articles

Identifying the region responsible for Brucella abortus MucR higher-order oligomer formation and examining its role in gene regulation

MucR is a member of the Ros/MucR family of prokaryotic zinc-finger proteins found in the α-proteobacteria which regulate the expression of genes required for the successful pathogenic and symbiotic interactions of these bacteria with the eukaryotic hosts. The structure and function of their distinctive zinc-finger domain has been well-studied, but only recently the quaternary structure of the full length proteins was investigated demonstrating their ability to form higher-order oligomers. The aim of this study was to identify the region of MucR involved in higher-order oligomer formation by analysing deletion and point mutants of this protein by Light Scattering, and to determine the role that MucR oligomerization plays in the regulatory function of this protein. Here we demonstrate that a conserved hydrophobic region at the N-terminus of MucR is responsible for higher-order oligomer formation and that MucR oligomerization is essential for its regulatory function in Brucella. All these features of MucR are shared by the histone-like nucleoid structuring protein, (H-NS), leading us to propose that the prokaryotic zinc-finger proteins in the MucR/Ros family control gene expression employing a mechanism similar to that used by the H-NS proteins, rather than working as classical transcriptional regulators.

Surface-Induced Dissociation: An Effective Method for Characterization of Protein Quaternary Structure.

Many mass spectrometry applications make use of tandem mass spectrometry, where two stages of m/z analysis are coupled. In between the two stages of m/z analysis, an activation or reaction step is carried out to cause either structurally-informative fragmentation or structurally-characteristic reaction of the precursor ion of interest. This review focuses on the use of collisions with a surface (surface-induced dissociation, SID) as the activation method in tandem mass spectrometry, with an emphasis on SID papers published over the past four years. SID is described and compared with other activation methods. The major application focused on in this review is the structural characterization of native protein complexes, complexes kinetically trapped that retain native-like solution structures upon transfer to the gas-phase and throughout the relatively short timeframe of the mass spectrometry experiment. Other SID applications currently under investigation are also briefly described. Pioneering work on SID ...

Modeling of Protein Tertiary and Quaternary Structures Based on Evolutionary Information.

Proteins are subject to evolutionary forces that shape their three-dimensional structure to meet specific functional demands. The knowledge of the structure of a protein is therefore instrumental to gain information about the molecular basis of its function. However, experimental structure determination is inherently time consuming and expensive, making it impossible to follow the explosion of sequence data deriving from genome-scale projects. As a consequence, computational structural modeling techniques have received much attention and established themselves as a valuable complement to experimental structural biology efforts. Among these, comparative modeling remains the method of choice to model the three-dimensional structure of a protein when homology to a protein of known structure can be detected.The general strategy consists of using experimentally determined structures of proteins as templates for the generation of three-dimensional models of related family members (targets) of which the structure is unknown. This chapter provides a description of the individual steps needed to obtain a comparative model using SWISS-MODEL, one of the most widely used automated servers for protein structure homology modeling.

Biophysical, docking, and cellular studies on the effects of cerium oxide nanoparticles on blood components: in vitro.

IntroductionThe application of nanoparticles (NPs) in medicine and biology has received great interest due to their novel features. However, their adverse effects on the biological system are not well understood.Materials and methodsThis study aims to evaluate the effect of cerium oxide nanoparticles (CNPs) on conformational changes of human hemoglobin (HHb) and lymphocytes by different spectroscopic (intrinsic and synchronous fluorescence spectroscopy and far and near circular dichroism [CD] spectroscopy), docking and cellular (MTT and flow cytometry) investigations.Results and discussionTransmission electron microscopy (TEM) showed that CNP diameter is ~30 nm. The infrared spectrum demonstrated a strong band around 783 cm−1 corresponding to the CNP stretching bond. Fluorescence data revealed that the CNP is able to quench the intrinsic fluorescence of HHb through both dynamic and static quenching mechanisms. The binding constant (Kb), number of binding sites (n), and thermodynamic parameters over three different temperatures indicated that hydrophobic interactions might play a considerable role in the interaction of CNPs with HHb. Synchronous fluorescence spectroscopy indicated that microenvironmental changes around Trp and Tyr residues remain almost unchanged. CD studies displayed that the regular secondary structure of HHb had no significant changes; however, the quaternary structure of protein is subjected to marginal structural changes. Docking studies showed the larger CNP cluster is more oriented toward experimental data, compared with smaller counterparts. Cellular assays revealed that CNP, at high concentrations (>50 µg/mL), initiated an antiproliferative response through apoptosis induction on lymphocytes.ConclusionThe findings may exhibit that, although CNPs did not significantly perturb the native conformation of HHb, they can stimulate some cellular adverse effects at high concentrations that may limit the medicinal and biological application of CNPs. In other words, CNP application in biological systems should be done at low concentrations.

Correction: Investigation of protein quaternary structure via stoichiometry and symmetry ınformation.

[This corrects the article DOI: 10.1371/journal.pone.0197176.].

A hydrophobic low-complexity region regulates aggregation of the yeast pyruvate kinase Cdc19 into amyloid-like aggregates in vitro

Cells form stress granules (SGs) upon stress stimuli to protect sensitive proteins and RNA from degradation. In the yeast Saccharomyces cerevisiae, specific stresses such as nutrient starvation and heat-shock trigger recruitment of the yeast pyruvate kinase Cdc19 into SGs. This RNA-binding protein was shown to form amyloid-like aggregates that are physiologically reversible and essential for cell cycle restart after stress. Cellular Cdc19 exists in an equilibrium between a homotetramer and monomer state. Here, we show that Cdc19 aggregation in vitro is governed by protein quaternary structure, and we investigate the physical-chemical basis of Cdc19's assembly properties. Equilibrium shift toward the monomer state exposes a hydrophobic low-complexity region (LCR), which is prone to induce intermolecular interactions with surrounding proteins. We further demonstrate that hydrophobic/hydrophilic interfaces can trigger Cdc19 aggregation in vitro Moreover, we performed in vitro biophysical analyses to compare Cdc19 aggregates with fibrils produced by two known dysfunctional amyloidogenic peptides. We show that the Cdc19 aggregates share several structural features with pathological amyloids formed by human insulin and the Alzheimer's disease-associated Aβ42 peptide, particularly secondary β-sheet structure, thermodynamic stability, and staining by the thioflavin T dye. However, Cdc19 aggregates could not seed aggregation. These results indicate that Cdc19 adopts an amyloid-like structure in vitro that is regulated by the exposure of a hydrophobic LCR in its monomeric form. Together, our results highlight striking structural similarities between functional and dysfunctional amyloids and reveal the crucial role of hydrophobic/hydrophilic interfaces in regulating Cdc19 aggregation.

Comparing protein structures with RINspector automation in Cytoscape

Residue interaction networks (RINs) have been shown to be relevant representations of the tertiary or quaternary structures of proteins, in particular thanks to network centrality analyses. We recently developed the RINspector 1.0.0 Cytoscape app, which couples centrality analyses with backbone flexibility predictions. This combined approach permits the identification of crucial residues for the folding or function of the protein that can constitute good targets for mutagenesis experiments. Here we present an application programming interface (API) for RINspector 1.1.0 that enables interplay between Cytoscape, RINspector and external languages, such as R or Python. This API provides easy access to batch centrality calculations and flexibility predictions, and allows for the easy comparison of results between different structures. These comparisons can lead to the identification of specific and conserved central residues, and show the impact of mutations to these and other residues on the flexibility of the proteins. We give two use cases to demonstrate the interest of these functionalities and provide the corresponding scripts: the first concerns NMR conformers, the second focuses on mutations in a structure.

Comparing protein structures with RINspector automation in Cytoscape

Residue interaction networks (RINs) have been shown to be relevant representations of the tertiary or quaternary structures of proteins, in particular thanks to network centrality analyses. We recently developed the RINspector 1.0.0 Cytoscape app, which couples centrality analyses with backbone flexibility predictions. This combined approach permits the identification of crucial residues for the folding or function of the protein that can constitute good targets for mutagenesis experiments. Here we present an application programming interface (API) for RINspector 1.1.0 that enables interplay between Cytoscape, RINspector and external languages, such as R or Python. This API provides easy access to batch centrality calculations and flexibility predictions, and allows for the easy comparison of results between different structures. These comparisons can lead to the identification of specific and conserved central residues, and show the impact of mutations to these and other residues on the flexibility of the proteins. We give two use cases to demonstrate the interest of these functionalities and provide the corresponding scripts: the first concerns NMR conformers, the second focuses on mutations in a structure.

Investigation of protein quaternary structure via stoichiometry and symmetry information.

The Protein Data Bank (PDB) is the single worldwide archive of experimentally-determined three-dimensional (3D) structures of proteins and nucleic acids. As of January 2017, the PDB housed more than 125,000 structures and was growing by more than 11,000 structures annually. Since the 3D structure of a protein is vital to understand the mechanisms of biological processes, diseases, and drug design, correct oligomeric assembly information is of critical importance. Unfortunately, the biologically relevant oligomeric form of a 3D structure is not directly obtainable by X-ray crystallography, whilst in solution methods (NMR or single particle EM) it is known from the experiment. Instead, this information may be provided by the PDB Depositor as metadata coming from additional experiments, be inferred by sequence-sequence comparisons with similar proteins of known oligomeric state, or predicted using software, such as PISA (Proteins, Interfaces, Structures and Assemblies) or EPPIC (Evolutionary Protein Protein Interface Classifier). Despite significant efforts by professional PDB Biocurators during data deposition, there remain a number of structures in the archive with incorrect quaternary structure descriptions (or annotations). Further investigation is, therefore, needed to evaluate the correctness of quaternary structure annotations. In this study, we aim to identify the most probable oligomeric states for proteins represented in the PDB. Our approach evaluated the performance of four independent prediction methods, including text mining of primary publications, inference from homologous protein structures, and two computational methods (PISA and EPPIC). Aggregating predictions to give consensus results outperformed all four of the independent prediction methods, yielding 83% correct, 9% wrong, and 8% inconclusive predictions, when tested with a well-curated benchmark dataset. We have developed a freely-available web-based tool to make this approach accessible to researchers and PDB Biocurators (http://quatstruct.rcsb.org/).

Structure of a cleavage-independent HIV Env recapitulates the glycoprotein architecture of the native cleaved trimer

Furin cleavage of the HIV envelope glycoprotein is an essential step for cell entry that enables formation of well-folded, native-like glycosylated trimers, releases constraints on the fusion peptide, and limits enzymatic processing of the N-glycan shield. Here, we show that a cleavage-independent, stabilized, soluble Env trimer mimic (BG505 NFL.664) exhibits a “closed-form”, native-like, prefusion conformation akin to furin-cleaved Env trimers. The crystal structure of BG505 NFL.664 at 3.39 Å resolution with two potent bNAbs also identifies the full epitopes of PGV19 and PGT122 that target the receptor binding site and N332 supersite, respectively. Quantitative site-specific analysis of the glycan shield reveals that native-like glycan processing is maintained despite furin-independent maturation in the secretory pathway. Thus, cleavage-independent NFL Env trimers exhibit quaternary protein and carbohydrate structures similar to the native viral spike that further validate their potential as vaccine immunogen candidates.

Comparing protein structures with RINspector automation in Cytoscape

Residue interaction networks (RINs) have been shown to be relevant representations of the tertiary or quaternary structures of proteins, in particular thanks to network centrality analyses. We recently developed the RINspector Cytoscape app, which couples centrality analyses with backbone flexibility predictions. This combined approach permits the identification of crucial residues for the folding or function of the protein that can constitute good targets for mutagenesis experiments. Here we present an application programming interface (API) for RINspector that enables interplay between Cytoscape, RINspector and external languages, such as R or Python. This API provides easy access to batch centrality calculations and flexibility predictions, and allows for the easy comparison of results between different structures. These comparisons can lead to the identification of specific and conserved central residues, and show the impact of mutations to these and other residues on the flexibility of the proteins. We give two use cases to demonstrate the interest of these functionalities and provide the corresponding scripts: the first concerns NMR conformers, the second focuses on mutations in a structure.

Expanding the Scope of Cross-Link Identifications by Incorporating Collisional Activated Dissociation and Ultraviolet Photodissociation Methods.

With the advent of new cross-linking chemistries, analytical technologies, and search algorithms, cross-linking has become an increasingly popular strategy for evaluating tertiary and quaternary structures of proteins. Collisional activated dissociation remains the primary MS/MS method for identifications of peptide cross-links in high throughput workflows. Ultraviolet photodissociation (UVPD) at 193 nm has emerged as an alternative ion activation method well-suited for characterization of peptides and has been found in some cases to identify different peptides or provide distinctive sequence information than obtained by collisional activation methods. Complementary high energy collision dissociation (HCD) and UVPD were used in the present study to characterize protein cross-linking for bovine serum albumin, hemoglobin, and E. coli ribosome. Cross-links identified by HCD and UVPD using bis(sulfosuccinimidyl)suberate (BS3), a homobifunctional amine-to-amine cross-linker, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), a heterofunctional amine-to-carboxylic acid cross-linker, were evaluated in the present study. While more unique BS3 cross-links were identified by HCD, UVPD, and HCD provided a complementary panel of DMTMM cross-links which extended the degree of structural insight obtained for the proteins.

Automated evaluation of quaternary structures from protein crystals.

A correct assessment of the quaternary structure of proteins is a fundamental prerequisite to understanding their function, physico-chemical properties and mode of interaction with other proteins. Currently about 90% of structures in the Protein Data Bank are crystal structures, in which the correct quaternary structure is embedded in the crystal lattice among a number of crystal contacts. Computational methods are required to 1) classify all protein-protein contacts in crystal lattices as biologically relevant or crystal contacts and 2) provide an assessment of how the biologically relevant interfaces combine into a biological assembly. In our previous work we addressed the first problem with our EPPIC (Evolutionary Protein Protein Interface Classifier) method. Here, we present our solution to the second problem with a new method that combines the interface classification results with symmetry and topology considerations. The new algorithm enumerates all possible valid assemblies within the crystal using a graph representation of the lattice and predicts the most probable biological unit based on the pairwise interface scoring. Our method achieves 85% precision (ranging from 76% to 90% for different oligomeric types) on a new dataset of 1,481 biological assemblies with consensus of PDB annotations. Although almost the same precision is achieved by PISA, currently the most popular quaternary structure assignment method, we show that, due to the fundamentally different approach to the problem, the two methods are complementary and could be combined to improve biological assembly assignments. The software for the automatic assessment of protein assemblies (EPPIC version 3) has been made available through a web server at http://www.eppic-web.org.

In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy.

Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA-binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53-MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of p53-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3α and MI773 efficiently inhibited the p53-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein-protein interactions and evaluation of inhibitors in living cells.

Role of viral RNA in virion stability and disassembly

Viruses are metastable macromolecular entities that are assembled into compact particles. Entry into a target host cell initiates quaternary conformational changes in response to distinct host‐specific environmental changes including temperature, pH, osmolality, divalent cation concentrations, which promote disassembly and release of the virion's genome into the host cell. This host stimulus‐triggered disassembly is critical for initiating the infectious phase of the particle. How viruses use their quaternary coat protein structures as thermodynamic sensors for detecting entry into a conducive host cell environment is largely unknown. The process of viral disassembly is largely impervious to static structural methods as these only capture end‐state conformations. Amide Hydrogen Deuterium Exchange Mass Spectrometry (HDXMS) can be used to monitor whole viral particle dynamics in solution. The disassembling virion represents the virus at its most vulnerable state and is consequently a transient dynamic intermediate. A map of the quaternary structural changes in this state offers critical insights into cryptic epitopes as novel vaccine targets. Our research has pioneered structural mass spectrometry characterization of temperature and divalent cation‐dependent conformational changes on Dengue virus and Turnip Crinkle Virus (TCV) and mapped the specific disassembly processes in these two model virus systems. We have extended these studies to deconstruct the whole viral particle‐antibody (DENV2‐2D22) complex. Our results reveal the high intrinsic dynamics of the RNA packing capsid C‐protein which underpins viral transition from compact to expanded states in the process of disassembly. We have also uncovered new disassembly intermediates triggered by varying monovalent and divalent cation concentrations. Viral disassembly processes in TCV and Dengue reveal conformations of disassembly intermediates which represent powerful new targets for therapeutic antibody design as well as offer fundamental insights into strategically timed viral disassembly processes which can be extended to all viruses.Support or Funding InformationSingapore Ministry of Education Tier 3 grantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PClass: Protein Quaternary Structure Classification by Using Bootstrapping Strategy as Model Selection.

Protein quaternary structure complex is also known as a multimer, which plays an important role in a cell. The dimer structure of transcription factors is involved in gene regulation, but the trimer structure of virus-infection-associated glycoproteins is related to the human immunodeficiency virus. The classification of the protein quaternary structure complex for the post-genome era of proteomics research will be of great help. Classification systems among protein quaternary structures have not been widely developed. Therefore, we designed the architecture of a two-layer machine learning technique in this study, and developed the classification system PClass. The protein quaternary structure of the complex is divided into five categories, namely, monomer, dimer, trimer, tetramer, and other subunit classes. In the framework of the bootstrap method with a support vector machine, we propose a new model selection method. Each type of complex is classified based on sequences, entropy, and accessible surface area, thereby generating a plurality of feature modules. Subsequently, the optimal model of effectiveness is selected as each kind of complex feature module. In this stage, the optimal performance can reach as high as 70% of Matthews correlation coefficient (MCC). The second layer of construction combines the first-layer module to integrate mechanisms and the use of six machine learning methods to improve the prediction performance. This system can be improved over 10% in MCC. Finally, we analyzed the performance of our classification system using transcription factors in dimer structure and virus-infection-associated glycoprotein in trimer structure. PClass is available via a web interface at http://predictor.nchu.edu.tw/PClass/.

Determination of protein oligomeric structure from small-angle X-ray scattering.

Small-angle X-ray scattering (SAXS) is useful for determining the oligomeric states and quaternary structures of proteins in solution. The average molecular mass in solution can be calculated directly from a single SAXS curve collected on an arbitrary scale from a sample of unknown protein concentration without the need for beamline calibration or protein standards. The quaternary structure in solution can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from proposed models of the oligomer. This approach is especially robust when the crystal structure of the target protein is known, and the candidate oligomer models are derived from the crystal lattice. When SAXS data are obtained at multiple protein concentrations, this analysis can provide insight into dynamic self-association equilibria. Herein, we summarize the computational methods that are used to determine protein molecular mass and quaternary structure from SAXS data. These methods are organized into a workflow and demonstrated with four case studies using experimental SAXS data from the published literature.

Virion Capsid Dynamics and Quaternary Conformational Changes Upon Host Entry

Viruses are metastable macromolecular entities that are assembled into compact particles. Entry into a target host cell initiates quaternary conformational changes in response to distinct host-specific environmental changes including temperature, pH, osmolality, divalent cation concentrations, which promote disassembly and release of the virion's genome into the host cell. This host stimulus-triggered disassembly is critical for initiating the infectious phase of the particle. How viruses use their quaternary coat protein structures as thermodynamic sensors for detecting entry into a conducive host cell environment is largely unknown. The process of viral disassembly is largely impervious to static structural methods as these only capture end-state conformations. Amide Hydrogen Deuterium Exchange Mass Spectrometry (HDXMS) can be used to monitor whole viral particle dynamics in solution. The disassembling virion represents the virus at its most vulnerable state and is consequently a transient dynamic intermediate. A map of the quaternary structural changes in this state offers critical insights into cryptic epitopes as novel vaccine targets. Our research has pioneered structural mass spectrometry characterization of temperature and divalent cation-dependent conformational changes on Dengue virus and Turnip Crinkle Virus (TCV) and mapped the specific disassembly processes in these two model virus systems. We have extended these studies to deconstruct the whole viral particle-antibody (DENV2-2D22) complex. Our results reveal the high intrinsic dynamics of the RNA packing capsid C-protein which underpins viral transition from compact to expanded states in the process of disassembly. We have also uncovered new disassembly intermediates triggered by varying monovalent and divalent cation concentrations. Viral disassembly processes in TCV and Dengue reveal conformations of disassembly intermediates which represent powerful new targets for therapeutic antibody design as well as offer fundamental insights into strategically timed viral disassembly processes which can be extended to all viruses.

M60-like metalloprotease domain of the Escherichia coli YghJ protein forms amyloid fibrils.

Amyloids are protein fibrils with a characteristic spatial structure. Amyloids were long perceived as the pathogens involved in a set of lethal diseases in humans and animals. In recent decades, it has become clear that amyloids represent a quaternary protein structure that is not only pathological but also functionally important and is widely used by different organisms, ranging from archaea to animals, to implement diverse biological functions. The greatest biological variety of amyloids is found in prokaryotes, where they control the formation of biofilms and cell wall sheaths, facilitate the overcoming of surface tension, and regulate the metabolism of toxins. Several amyloid proteins were identified in the important model, biotechnological and pathogenic bacterium Escherichia coli. In previous studies, using a method for the proteomic screening and identification of amyloids, we identified 61 potentially amyloidogenic proteins in the proteome of E. coli. Among these proteins, YghJ was the most enriched with bioinformatically predicted amyloidogenic regions. YghJ is a lipoprotein with a zinc metalloprotease M60-like domain that is involved in mucin degradation in the intestine as well as in proinflammatory responses. In this study, we analyzed the amyloid properties of the YghJ M60-like domain and demonstrated that it forms amyloid-like fibrils in vitro and in vivo.

How Pairwise Coevolutionary Models Capture the Collective Residue Variability in Proteins?

Global coevolutionary models of homologous protein families, as constructed by direct coupling analysis (DCA), have recently gained popularity in particular due to their capacity to accurately predict residue-residue contacts from sequence information alone, and thereby to facilitate tertiary and quaternary protein structure prediction. More recently, they have also been used to predict fitness effects of amino-acid substitutions in proteins, and to predict evolutionary conserved protein-protein interactions. These models are based on two currently unjustified hypotheses: 1) correlations in the amino-acid usage of different positions are resulting collectively from networks of direct couplings; and 2) pairwise couplings are sufficient to capture the amino-acid variability. Here, we propose a highly precise inference scheme based on Boltzmann-machine learning, which allows us to systematically address these hypotheses. We show how correlations are built up in a highly collective way by a large number of coupling paths, which are based on the proteins three-dimensional structure. We further find that pairwise coevolutionary models capture the collective residue variability across homologous proteins even for quantities which are not imposed by the inference procedure, like three-residue correlations, the clustered structure of protein families in sequence space or the sequence distances between homologs. These findings strongly suggest that pairwise coevolutionary models are actually sufficient to accurately capture the residue variability in homologous protein families.