RESUMO
It has been recently suggested that a significant fraction of homomer protein-protein interfaces evolve neutrally, without contributing to function, due to a hydrophobic bias in missense mutations. However, the fraction of such gratuitous complexes is currently unknown. Here, we quantified the fraction of homodimers where multimerization is unlikely to contribute to their biochemical function. We show that: 1) ligand binding-site structure predicts whether a homomer is functional or not; the vast majority of homodimers with multichain binding-sites (MBS) are likely to be functional, while in homodimers with single-chain binding-sites (SBS) and small to medium interfaces, quaternary structure is unlikely to be functional in a significant fraction-35%, even up to 42%-of complexes; 2) the hydrophobicity of interfaces changes little with the strength of selection, and the amino acid composition of interfaces is shaped by the "hydrophobic ratchet" in both types, but they are not in a strict equilibrium with mutations; particularly cysteines are much more abundant in mutations than in interfaces or surfaces; 3) in MBS homomers, the interfaces are conserved, while in a high fraction of SBS homomers, the interface is not more conserved than the solvent-accessible surface; and 4) MBS homomer interfaces coevolve more strongly with ligand binding sites than the interfaces of SBS homomers, and MBS complexes have higher capacity to transfer information from ligands across the interfaces than SBS homomers, explaining the enrichment of allostery in the former.
Assuntos
Aminoácidos , Proteínas , Ligantes , Proteínas/metabolismo , Sítios de Ligação/genética , Domínios Proteicos , Aminoácidos/química , Ligação Proteica/genética , Estrutura Quaternária de ProteínaAssuntos
Carpas/genética , Carpas/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Fator de Transcrição STAT5/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Carpas/imunologia , Chlorocebus aethiops , Clonagem Molecular , Proteínas de Peixes/química , Técnicas In Vitro , Fosforilação , Filogenia , Estrutura Quaternária de Proteína , Receptores de Interleucina-2/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT5/químicaRESUMO
Predicting the quaternary structure of protein complex is an important problem. Inter-chain residue-residue contact prediction can provide useful information to guide the ab initio reconstruction of quaternary structures. However, few methods have been developed to build quaternary structures from predicted inter-chain contacts. Here, we develop the first method based on gradient descent optimization (GD) to build quaternary structures of protein dimers utilizing inter-chain contacts as distance restraints. We evaluate GD on several datasets of homodimers and heterodimers using true/predicted contacts and monomer structures as input. GD consistently performs better than both simulated annealing and Markov Chain Monte Carlo simulation. Starting from an arbitrarily quaternary structure randomly initialized from the tertiary structures of protein chains and using true inter-chain contacts as input, GD can reconstruct high-quality structural models for homodimers and heterodimers with average TM-score ranging from 0.92 to 0.99 and average interface root mean square distance from 0.72 Å to 1.64 Å. On a dataset of 115 homodimers, using predicted inter-chain contacts as restraints, the average TM-score of the structural models built by GD is 0.76. For 46% of the homodimers, high-quality structural models with TM-score ≥ 0.9 are reconstructed from predicted contacts. There is a strong correlation between the quality of the reconstructed models and the precision and recall of predicted contacts. Only a moderate precision or recall of inter-chain contact prediction is needed to build good structural models for most homodimers. Moreover, GD improves the quality of quaternary structures predicted by AlphaFold2 on a Critical Assessment of Techniques for Protein Structure Prediction-Critical Assessments of Predictions of Interactions dataset.
Assuntos
Proteínas/química , Biologia Computacional , Bases de Dados de Proteínas , Simulação de Acoplamento Molecular , Método de Monte Carlo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
In CASP14, 39 research groups submitted more than 2500 3D models on 22 protein complexes. In general, the community performed well in predicting the fold of the assemblies (for 80% of the targets), although it faced significant challenges in reproducing the native contacts. This is especially the case for the complexes without whole-assembly templates. The leading predictor, BAKER-experimental, used a methodology combining classical techniques (template-based modeling, protein docking) with deep learning-based contact predictions and a fold-and-dock approach. The Venclovas team achieved the runner-up position with template-based modeling and docking. By analyzing the target interfaces, we showed that the complexes with depleted charged contacts or dominating hydrophobic interactions were the most challenging ones to predict. We also demonstrated that if AlphaFold2 predictions were at hand, the interface prediction challenge could be alleviated for most of the targets. All in all, it is evident that new approaches are needed for the accurate prediction of assemblies, which undoubtedly will expand on the significant improvements in the tertiary structure prediction field.
Assuntos
Modelos Moleculares , Conformação Proteica , Proteínas , Software , Biologia Computacional , Bases de Dados de Proteínas , Estrutura Quaternária de Proteína , Proteínas/química , Proteínas/metabolismo , Análise de Sequência de ProteínaRESUMO
Our web server, PIZSA (http://cospi.iiserpune.ac.in/pizsa), assesses the likelihood of protein-protein interactions by assigning a Z Score computed from interface residue contacts. Our score takes into account the optimal number of atoms that mediate the interaction between pairs of residues and whether these contacts emanate from the main chain or side chain. We tested the score on 174 native interactions for which 100 decoys each were constructed using ZDOCK. The native structure scored better than any of the decoys in 146 cases and was able to rank within the 95th percentile in 162 cases. This easily outperforms a competing method, CIPS. We also benchmarked our scoring scheme on 15 targets from the CAPRI dataset and found that our method had results comparable to that of CIPS. Further, our method is able to analyse higher order protein complexes without the need to explicitly identify chains as receptors or ligands. The PIZSA server is easy to use and could be used to score any input three-dimensional structure and provide a residue pair-wise break up of the results. Attractively, our server offers a platform for users to upload their own potentials and could serve as an ideal testing ground for this class of scoring schemes.
Assuntos
Algoritmos , Hemoglobinas/química , Simulação de Acoplamento Molecular/métodos , Proteínas/química , Software , Sequência de Aminoácidos , Benchmarking , Sítios de Ligação , Cristalografia por Raios X , Hemoglobinas/metabolismo , Humanos , Internet , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas/metabolismo , Homologia Estrutural de Proteína , TermodinâmicaRESUMO
Myosin X is an unconventional actin-based molecular motor involved in filopodial formation, microtubule-actin filament interaction, and cell migration. Myosin X is an important component of filopodia regulation, localizing to tips of growing filopodia by an unclear targeting mechanism. The native α-helical dimerization domain of myosin X is thought to associate with antiparallel polarity of the two amino acid chains, making myosin X the only myosin that is currently considered to form antiparallel dimers. This study aims to determine if antiparallel dimerization of myosin X imparts selectivity toward actin bundles by comparing the motility of parallel and antiparallel dimers of myosin X on single and fascin-bundled actin filaments. Antiparallel myosin X dimers exhibit selective processivity on fascin-bundled actin and are only weakly processive on single actin filaments below saturating [ATP]. Artificial forced parallel dimers of myosin X are robustly processive on both single and bundled actin, exhibiting no selectivity. To determine the relationship between gating of the reaction steps and observed differences in motility, a mathematical model was developed to correlate the parameters of motility with the biochemical and mechanical kinetics of the dimer. Results from the model, constrained by experimental data, suggest that the probability of binding forward, toward the barbed end of the actin filament, is lower in antiparallel myosin X on single actin filaments compared to fascin-actin bundles and compared to constructs of myosin X with parallel dimerization.
Assuntos
Citoesqueleto de Actina/metabolismo , Miosinas/química , Miosinas/metabolismo , Multimerização Proteica , Animais , Bovinos , Cinética , Modelos Moleculares , Método de Monte Carlo , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
Rotational diffusion measurement is predicted as an important method in cell biology because the rotational properties directly reflect molecular interactions and environment in the cell. To prove this concept, polarization-dependent fluorescence correlation spectroscopy (pol-FCS) measurements of purified fluorescent proteins were conducted in viscous solution. With the comparison between the translational and rotational diffusion coefficients obtained from pol-FCS measurements, the hydrodynamic radius of an enhanced green fluorescent protein (EGFP) was estimated as a control measurement. The orientation of oligomer EGFP in living cells was also estimated by pol-FCS and compared with Monte Carlo simulations. The results of this pol-FCS experiment indicate that this method allows an estimation of the molecular orientation using the characteristics of rotational diffusion. Further, it can be applied to analyze the degree of molecular orientation and multimerization or detection of tiny aggregation of aggregate-prone proteins.
Assuntos
Proteínas de Fluorescência Verde/química , Espectrometria de Fluorescência/métodos , Animais , Células COS , Chlorocebus aethiops , Polarização de Fluorescência , Hidrodinâmica , Simulação de Dinâmica Molecular , Método de Monte Carlo , Agregados Proteicos , Estrutura Quaternária de Proteína , Proteínas Recombinantes/químicaRESUMO
The spread of ß-lactamases that hydrolyze penicillins, cephalosporins and carbapenems among Gram-negative bacteria has limited options for treating bacterial infections. Initially, Klebsiella pneumoniae carbapenemase-2 (KPC-2) emerged as a widespread carbapenem hydrolyzing ß-lactamase that also hydrolyzes penicillins and cephalosporins but not cephamycins and ceftazidime. In recent years, single and double amino acid substitution variants of KPC-2 have emerged among clinical isolates that show increased resistance to ceftazidime. Because it confers multi-drug resistance, KPC ß-lactamase is a threat to public health. In this study, the evolution of KPC-2 function was determined in nine clinically isolated variants by examining the effects of the substitutions on enzyme kinetic parameters, protein stability and antibiotic resistance profile. The results indicate that the amino acid substitutions associated with KPC-2 natural variants lead to increased catalytic efficiency for ceftazidime hydrolysis and a consequent increase in ceftazidime resistance. Single substitutions lead to modest increases in catalytic activity while the double mutants exhibit significantly increased ceftazidime hydrolysis and resistance levels. The P104R, V240G and H274Y substitutions in single and double mutant combinations lead to the largest increases in ceftazidime hydrolysis and resistance. Molecular modeling suggests that the P104R and H274Y mutations could facilitate ceftazidime hydrolysis through increased hydrogen bonding interactions with the substrate while the V240G substitution may enhance backbone flexibility so that larger substrates might be accommodated in the active site. Additionally, we observed a strong correlation between gain of catalytic function for ceftazidime hydrolysis and loss of enzyme stability, which is in agreement with the 'stability-function tradeoff' phenomenon. The high Tm of KPC-2 (66.5°C) provides an evolutionary advantage as compared to other class A enzymes such as TEM (51.5°C) and CTX-M (51°C) in that it can acquire multiple destabilizing substitutions without losing the ability to fold into a functional enzyme.
Assuntos
Evolução Biológica , Modelos Moleculares , beta-Lactamases/química , beta-Lactamases/metabolismo , Antibacterianos/metabolismo , Ceftazidima/metabolismo , Estabilidade Enzimática/fisiologia , Hidrólise , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Mutação , Estrutura Quaternária de ProteínaRESUMO
Capsid protein (CA) is the building block of virus coats. To help understand how the HIV CA proteins self-organize into large assemblies of various shapes, we aim to computationally evaluate the binding affinity and interfaces in a CA homodimer. We model the N- and C-terminal domains (NTD and CTD) of the CA as rigid bodies and treat the five-residue loop between the two domains as a flexible linker. We adopt a transferrable residue-level coarse-grained energy function to describe the interactions between the protein domains. In seven extensive Monte Carlo simulations with different volumes, a large number of binding/unbinding transitions between the two CA proteins are observed, thus allowing a reliable estimation of the equilibrium probabilities for the dimeric vs monomeric forms. The obtained dissociation constant for the CA homodimer from our simulations, 20-25 µM, is in reasonable agreement with experimental measurement. A wide range of binding interfaces, primarily between the NTDs, are identified in the simulations. Although some observed bound structures here closely resemble the major binding interfaces in the capsid assembly, they are statistically insignificant in our simulation trajectories. Our results suggest that although the general purpose energy functions adopted here could reasonably reproduce the overall binding affinity for the CA homodimer, further adjustment would be needed to accurately represent the relative strength of individual binding interfaces.
Assuntos
Proteínas do Capsídeo/química , HIV , Método de Monte Carlo , Multimerização Proteica , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Heterochromatin protein 1 (HP1) participates in establishing and maintaining heterochromatin via its histone-modification-dependent chromatin interactions. In recent papers HP1 binding to nucleosomal arrays was measured in vitro and interpreted in terms of nearest-neighbour cooperative binding. This mode of chromatin interaction could lead to the spreading of HP1 along the nucleosome chain. Here, we reanalysed previous data by representing the nucleosome chain as a 1D binding lattice and showed how the experimental HP1 binding isotherms can be explained by a simpler model without cooperative interactions between neighboring HP1 dimers. Based on these calculations and spatial models of dinucleosomes and nucleosome chains, we propose that binding stoichiometry depends on the nucleosome repeat length (NRL) rather than protein interactions between HP1 dimers. According to our calculations, more open nucleosome arrays with long DNA linkers are characterized by a larger number of binding sites in comparison to chains with a short NRL. Furthermore, we demonstrate by Monte Carlo simulations that the NRL dependent folding of the nucleosome chain can induce allosteric changes of HP1 binding sites. Thus, HP1 chromatin interactions can be modulated by the change of binding stoichiometry and the type of binding to condensed (methylated) and non-condensed (unmethylated) nucleosome arrays in the absence of direct interactions between HP1 dimers.
Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Modelos Moleculares , Nucleossomos/metabolismo , Sítios de Ligação , Homólogo 5 da Proteína Cromobox , DNA/química , DNA/metabolismo , Método de Monte Carlo , Conformação de Ácido Nucleico , Nucleossomos/química , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
The use of in vivo Förster resonance energy transfer (FRET) data to determine the molecular architecture of a protein complex in living cells is challenging due to data sparseness, sample heterogeneity, signal contributions from multiple donors and acceptors, unequal fluorophore brightness, photobleaching, flexibility of the linker connecting the fluorophore to the tagged protein, and spectral cross-talk. We addressed these challenges by using a Bayesian approach that produces the posterior probability of a model, given the input data. The posterior probability is defined as a function of the dependence of our FRET metric FRETR on a structure (forward model), a model of noise in the data, as well as prior information about the structure, relative populations of distinct states in the sample, forward model parameters, and data noise. The forward model was validated against kinetic Monte Carlo simulations and in vivo experimental data collected on nine systems of known structure. In addition, our Bayesian approach was validated by a benchmark of 16 protein complexes of known structure. Given the structures of each subunit of the complexes, models were computed from synthetic FRETR data with a distance root-mean-squared deviation error of 14 to 17 Å. The approach is implemented in the open-source Integrative Modeling Platform, allowing us to determine macromolecular structures through a combination of in vivo FRETR data and data from other sources, such as electron microscopy and chemical cross-linking.
Assuntos
Proteínas de Bactérias/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas Luminescentes/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo , Algoritmos , Teorema de Bayes , Simulação por Computador , Estrutura Molecular , Método de Monte Carlo , Mapeamento de Interação de Proteínas , Estrutura Quaternária de Proteína , Saccharomyces cerevisiaeRESUMO
To consider biological significance of glycosylation of proteins, it is necessary to evaluate the importance of sugar-recognition processes mediated by lectins. Though the interaction between sugars and proteins, especially animal lectins, is quite weak with K d approximately 10(-4) M, cellular and molecular recognitions mediated via sugar-protein interaction increase their avidity by 1-3 orders of magnitude by the self-association of both receptors and their ligands on cell surfaces. To assess the weak interaction between lectins and their sugar ligands, we established lectin tetramer binding to cell surface glycans using flow cytometry. This strategy is highly sensitive, and useful to determine whether or not a putative lectin domain may have sugar-binding ability.
Assuntos
Membrana Celular/metabolismo , Citometria de Fluxo/métodos , Lectinas/química , Lectinas/metabolismo , Polissacarídeos/metabolismo , Multimerização Proteica , Biotinilação , Células HeLa , Humanos , Ficoeritrina/química , Ligação Proteica , Estrutura Quaternária de Proteína , SolubilidadeRESUMO
Hepatitis B virus core-antigen (capsid protein) and e-antigen (an immune regulator) have almost complete sequence identity, yet the dimeric proteins (termed Cp149d and Cp(-10)149d , respectively) adopt quite distinct quaternary structures. Here we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) to study their structural properties. We detect many regions that differ substantially in their HDX dynamics. Significantly, whilst all regions in Cp(-10)149d exchange by EX2-type kinetics, a number of regions in Cp149d were shown to exhibit a mixture of EX2- and EX1-type kinetics, hinting at conformational heterogeneity in these regions. Comparison of the HDX of the free Cp149d with that in assembled capsids (Cp149c ) indicated increased resistance to exchange at the C-terminus where the inter-dimer contacts occur. Furthermore, evidence of mixed exchange kinetics were not observed in Cp149c , implying a reduction in flexibility upon capsid formation. Cp(-10)149d undergoes a drastic structural change when the intermolecular disulphide bridge is reduced, adopting a Cp149d -like structure, as evidenced by the detected HDX dynamics being more consistent with Cp149d in many, albeit not all, regions. These results demonstrate the highly dynamic nature of these similar proteins. To probe the effect of these structural differences on the resulting antigenicity, we investigated binding of the antibody fragment (Fab E1) that is known to bind a conformational epitope on the four-helix bundle. Whilst Fab E1 binds to Cp149c and Cp149d , it does not bind non-reduced and reduced Cp(-10)149d , despite unhindered access to the epitope. These results imply a remarkable sensitivity of this epitope to its structural context.
Assuntos
Medição da Troca de Deutério/métodos , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos E da Hepatite B/química , Vírus da Hepatite B/química , Espectrometria de Massas/métodos , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Cinética , Conformação Proteica , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
Globular proteins contain cavities/voids that play specific roles in controlling protein function. Elongated cavities provide migration channels for the transport of ions and small molecules to the active center of a protein or enzyme. Using Monte Carlo and Molecular Dynamics on fully atomistic protein/water models, a new computational methodology is introduced that takes into account the protein's dynamic structure and maps all the cavities in and on the surface. To demonstrate its utility, the methodology is applied to study cavity structure in myoglobin and five of its mutants. Computed cavity and channel size distributions reveal significant differences relative to the wild type myoglobin. Computer visualization of the channels leading to the heme center indicates restricted ligand access for the mutants consistent with the existing interpretations. The new methodology provides a quantitative measure of cavity structure and distributions and can become a valuable tool for the structural characterization of proteins.
Assuntos
Simulação de Dinâmica Molecular , Mioglobina/ultraestrutura , Estrutura Quaternária de Proteína , Biologia Computacional , Cristalografia por Raios X , Método de Monte Carlo , Mutação , Mioglobina/química , Mioglobina/genética , Propriedades de Superfície , TermodinâmicaRESUMO
Coarse-grained simulations with charge hopping were performed for a positively charged tetrameric transthyretin (TTR) protein complex with a total charge of +20. Charges were allowed to move among basic amino acid sites as well as N-termini. Charge distributions and radii of gyration were calculated for complexes simulated at two temperatures, 300 and 600 K, under different scenarios. One scenario treated the complex in its normal state allowing charge to move to any basic site. Another scenario blocked protonation of all the N-termini except one. A final scenario used the complex in its normal state but added a basic-site containing tether (charge tag) near the N-terminus of one chain. The differences in monomer unfolding and charging were monitored in all three scenarios and compared. The simulation results show the importance of the N-terminus in leading the unfolding of the monomer units; a process that follows a zipper-like mechanism. Overall, experimentally modifying the complex by adding a tether or blocking the protonation of N-termini may give the potential for controlling the unraveling and subsequent dissociation of protein complexes.
Assuntos
Modelos Moleculares , Pré-Albumina/química , Substituição de Aminoácidos , Temperatura Alta , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Cinética , Simulação de Dinâmica Molecular , Método de Monte Carlo , Proteínas Mutantes/química , Pré-Albumina/genética , Sinais Direcionadores de Proteínas , Estrutura Quaternária de Proteína , Desdobramento de Proteína , Propriedades de Superfície , VolatilizaçãoRESUMO
Partial sequences of 110 type 2 poliovirus strains isolated from sewage in Slovakia in 2003-2005, and most probably originating from a single dose of oral poliovirus vaccine, were subjected to a detailed genetic analysis. Evolutionary patterns of these vaccine derived poliovirus strains (SVK-aVDPV2) were compared to those of type 1 and type 3 wild poliovirus (WPV) lineages considered to have a single seed strain origin, respectively. The 102 unique SVK-aVDPV VP1 sequences were monophyletic differing from that of the most likely parental poliovirus type 2/Sabin (PV2 Sabin) by 12.5-15.6%. Judging from this difference and from the rate of accumulation of synonymous transversions during the 22 month observation period, the relevant oral poliovirus vaccine dose had been administered to an unknown recipient more than 12 years earlier. The patterns of nucleotide substitution during the observation period differed from those found in the studied lineages of WPV1 or 3, including a lower transition/transversion (Ts/Tv) bias and strikingly lower Ts/Tv rate ratios at the 2(nd) codon position for both purines and pyrimidines. A relatively low preference of transitions at the 2(nd) codon position was also found in the large set of VP1 sequences of Nigerian circulating (c)VDPV2, as well as in the smaller sets from the Hispaniola cVDPV1 and Egypt cVDPV2 outbreaks, and among aVDPV1and aVDPV2 strains recently isolated from sewage in Finland. Codon-wise analysis of synonymous versus non-synonymous substitution rates in the VP1 sequences suggested that in five codons, those coding for amino acids at sites 24, 144, 147, 221 and 222, there may have been positive selection during the observation period. We conclude that pattern of poliovirus VP1 evolution in prolonged infection may differ from that found in WPV epidemics. Further studies on sufficiently large independent datasets are needed to confirm this suggestion and to reveal its potential significance.
Assuntos
Proteínas do Capsídeo/genética , Poliovirus/genética , Seleção Genética , Substituição de Aminoácidos , Sequência de Bases , Teorema de Bayes , Proteínas do Capsídeo/química , Códon , Sequência Conservada , Evolução Molecular , Genes Virais , Especiação Genética , Genótipo , Humanos , Modelos Genéticos , Modelos Moleculares , Método de Monte Carlo , Filogenia , Mutação Puntual , Poliovirus/imunologia , Vacina Antipólio Oral , Estrutura Quaternária de Proteína , Análise de Sequência de DNA , Esgotos/virologiaRESUMO
The equilibrium constants of trans and cis dimerization of membrane bound (2D) and freely moving (3D) adhesion receptors are expressed and compared using elementary statistical-thermodynamics. Both processes are mediated by the binding of extracellular subdomains whose range of motion in the 2D environment is reduced upon dimerization, defining a thin reaction shell where dimer formation and dissociation take place. We show that the ratio between the 2D and 3D equilibrium constants can be expressed as a product of individual factors describing, respectively, the spatial ranges of motions of the adhesive domains, and their rotational freedom within the reaction shell. The results predicted by the theory are compared to those obtained from a novel, to our knowledge, dynamical simulations methodology, whereby pairs of receptors perform realistic translational, internal, and rotational motions in 2D and 3D. We use cadherins as our model system. The theory and simulations explain how the strength of cis and trans interactions of adhesive receptors are affected both by their presence in the constrained intermembrane space and by the 2D environment of membrane surfaces. Our work provides fundamental insights as to the mechanism of lateral clustering of adhesion receptors after cell-cell contact and, more generally, to the formation of lateral microclusters of proteins on cell surfaces.
Assuntos
Membrana Celular/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Método de Monte Carlo , Multimerização Proteica , Movimento , Complexo Glicoproteico GPIb-IX de Plaquetas , Estrutura Quaternária de Proteína , TermodinâmicaRESUMO
The low frequency of T cells specific for given antigens makes the study of antigen-specific T cell responses difficult. The development of MHC class I and II tetramer staining techniques allows precise quantification and tracking of antigen-specific CD8(+) and CD4(+) T cell responses. Here, we describe a protocol for MHC class I and II tetramer staining of mouse T cells isolated from various tissues of mice infected with lymphocytic choriomeningitis virus (LCMV) or with murine cytomegalovirus (MCMV).
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe I/química , Multimerização Proteica , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Separação Celular , Chlorocebus aethiops , Reações Cruzadas , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Muromegalovirus/fisiologia , Estrutura Quaternária de Proteína , Baço/citologia , Coloração e Rotulagem , Células VeroRESUMO
The HIV-1 accessory protein Vif hijacks a cellular Cullin-RING ubiquitin ligase, CRL5, to promote degradation of the APOBEC3 (A3) family of restriction factors. Recently, the cellular transcription cofactor CBFß was shown to form a complex with CRL5-Vif and to be essential for A3 degradation and viral infectivity. We now demonstrate that CBFß is required for assembling a well-ordered CRL5-Vif complex by inhibiting Vif oligomerization and by activating CRL5-Vif via direct interaction. The CRL5-Vif-CBFß holoenzyme forms a well-defined heterohexamer, indicating that Vif simultaneously hijacks CRL5 and CBFß. Heterodimers of CBFß and RUNX transcription factors contribute toward the regulation of genes, including those with immune system functions. We show that binding of Vif to CBFß is mutually exclusive with RUNX heterodimerization and impacts the expression of genes whose regulatory domains are associated with RUNX1. Our results provide a mechanism by which a pathogen with limited coding capacity uses one factor to hijack multiple host pathways.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Citosina Desaminase/metabolismo , Regulação da Expressão Gênica , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Desaminases APOBEC , Sequência de Aminoácidos , Sequência de Bases , Fator de Ligação a CCAAT/química , Fator de Ligação a CCAAT/fisiologia , Sequência Consenso , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Citidina Desaminase , Citosina Desaminase/química , Citosina Desaminase/fisiologia , Expressão Gênica , Genes Reporter , Células HEK293 , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Estrutura Quaternária de Proteína , Linfócitos T/metabolismo , Linfócitos T/virologia , Ubiquitinação , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
Structure determination of macromolecular protein assemblies remains a challenge for well-established methods. Here, we provide an assessment of an emerging structural technique, ion mobility-mass spectrometry (IM-MS), and examine the use of collision cross-sections (CCSs), derived from IM-MS, as restraints for structure characterization of heteromeric protein assemblies. Using 15 complexes selected from the Protein Data Bank, we validate the use of low-resolution models by comparing their CCSs with those calculated for all-atom structures. We then select six heteromeric complexes, disrupting them in solution to form subcomplexes. Experimental and calculated CCSs reveal close similarity for 18 of the 21 (sub)complexes. Exploring the use of CCS as a restraint, we incorporate it into a scoring function and show good correlation between the score and similarity to the native structure for heteromers, especially when an additional symmetry restraint was introduced.