RESUMO
In three-dimensional domain swapping, two protein monomers exchange a part of their structures to form an intertwined homodimer, whose subunits resemble the monomer. Several viral proteins domain swap to increase their structural complexity or functional avidity. The main protease (Mpro) of the severe acute respiratory syndrome (SARS) coronavirus proteolyzes viral polyproteins and has been a target for anti-SARS drug design. Domain swapping in the α-helical C-terminal domain of Mpro (MproC) locks Mpro into a hyperactive octameric form that is hypothesized to promote the early stages of viral replication. However, in the absence of a complete molecular understanding of the mechanism of domain swapping, investigations into the biological relevance of this octameric Mpro have stalled. Isolated MproC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of MproC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of MproC folding. Further, we find that a contact between a tryptophan in the MproC domain-swapping hinge and an arginine elsewhere forms early during folding, modulates the folding route, and promotes domain swapping to the native structure. An examination of the sequence and the structure of the tryptophan containing hinge loop shows that it has a propensity to form multiple secondary structures and contacts, indicating that it could be stabilized into either the monomer- or dimer-promoting conformations by mutations or ligand binding. Finally, because all residues in the tryptophan loop are identical in SARS-CoV and SARS-CoV-2, mutations that modulate domain swapping may provide insights into the role of octameric Mpro in the early-stage viral replication of both viruses.
Assuntos
Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/enzimologia , Domínios Proteicos , Dobramento de ProteínaRESUMO
Computational simulations of protein folding can be used to interpret experimental folding results, to design new folding experiments, and to test the effects of mutations and small molecules on folding. However, whereas major experimental and computational progress has been made in understanding how small proteins fold, research on larger, multidomain proteins, which comprise the majority of proteins, is less advanced. Specifically, large proteins often fold via long-lived partially folded intermediates, whose structures, potentially toxic oligomerization, and interactions with cellular chaperones remain poorly understood. Molecular dynamics based folding simulations that rely on knowledge of the native structure can provide critical, detailed information on folding free energy landscapes, intermediates, and pathways. Further, increases in computational power and methodological advances have made folding simulations of large proteins practical and valuable. Here, using serpins that inhibit proteases as an example, we review native-centric methods for simulating the folding of large proteins. These synergistic approaches range from Go and related structure-based models that can predict the effects of the native structure on folding to all-atom-based methods that include side-chain chemistry and can predict how disease-associated mutations may impact folding. The application of these computational approaches to serpins and other large proteins highlights the successes and limitations of current computational methods and underscores how computational results can be used to inform experiments. These powerful simulation approaches in combination with experiments can provide unique insights into how large proteins fold and misfold, expanding our ability to predict and manipulate protein folding.
Assuntos
Simulação de Dinâmica Molecular , Dobramento de Proteína , Animais , Humanos , Serpinas/química , Serpinas/metabolismoRESUMO
Protein-folding can go wrong in vivo and in vitro, with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a "xenonucleus", which is a prefolded peptide that mimics the folding nucleus of a protein. Using stopped-flow kinetics, NMR spectroscopy, Förster resonance energy transfer, single-molecule force measurements, and molecular dynamics simulations, we demonstrate that a xenonucleus can make the refolding of ubiquitin faster by 33 ± 5%, while variants of the same peptide have little or no effect. Our approach provides a novel method for constructing specific, genetically encodable folding catalysts for suitable proteins that have a well-defined contiguous folding nucleus.
Assuntos
Ubiquitina/química , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Ubiquitina/metabolismoRESUMO
For successful protease inhibition, the reactive center loop (RCL) of the two-domain serine protease inhibitor, α1-antitrypsin (α1-AT), needs to remain exposed in a metastable active conformation. The α1-AT RCL is sequestered in a ß-sheet in the stable latent conformation. Thus, to be functional, α1-AT must always fold to a metastable conformation while avoiding folding to a stable conformation. We explore the structural basis of this choice using folding simulations of coarse-grained structure-based models of the two α1-AT conformations. Our simulations capture the key features of folding experiments performed on both conformations. The simulations also show that the free energy barrier to fold to the latent conformation is much larger than the barrier to fold to the active conformation. An entropically stabilized on-pathway intermediate lowers the barrier for folding to the active conformation. In this intermediate, the RCL is in an exposed configuration, and only one of the two α1-AT domains is folded. In contrast, early conversion of the RCL into a ß-strand increases the coupling between the two α1-AT domains in the transition state and creates a larger barrier for folding to the latent conformation. Thus, unlike what happens in several proteins, where separate regions promote folding and function, the structure of the RCL, formed early during folding, determines both the conformational and the functional fate of α1-AT. Further, the short 12-residue RCL modulates the free energy barrier and the folding cooperativity of the large 370-residue α1-AT. Finally, we suggest experiments to test the predicted folding mechanism for the latent state.
Assuntos
Dobramento de Proteína , alfa 1-Antitripsina/química , Domínio Catalítico , Simulação por Computador , Modelos Químicos , Modelos Moleculares , Conformação ProteicaRESUMO
Coarse-grained GoÌ -like models, based on the principle of minimal frustration, provide valuable insight into fundamental questions in the field of protein folding and dynamics. In conjunction with commonly used molecular dynamics (MD) simulations, energy landscape exploration methods like discrete path sampling (DPS) with GoÌ -like models can provide quantitative details of the thermodynamics and kinetics of proteins. Here we present Go-kit, a software that facilitates the setup of MD and DPS simulations of several flavors of GoÌ -like models. Go-kit is designed for use with MD (GROMACS) and DPS (PATHSAMPLE) simulation engines that are open source. The Go-kit code is written in python2.7 and is also open source. A case study for the ribosomal protein S6 is discussed to illustrate the utility of the software, which is available at https://github.com/gokit1/gokit .
Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Software , Termodinâmica , Proteínas de Bactérias/química , Cinética , Conformação Proteica , Dobramento de Proteína , Proteína S6 Ribossômica/química , Thermus thermophilus/químicaRESUMO
One of the main barriers to accurate computational protein structure prediction is searching the vast space of protein conformations. Distance restraints or inter-residue contacts have been used to reduce this search space, easing the discovery of the correct folded state. It has been suggested that about 1 contact for every 12 residues may be sufficient to predict structure at fold level accuracy. Here, we use coarse-grained structure-based models in conjunction with molecular dynamics simulations to examine this empirical prediction. We generate sparse contact maps for 15 proteins of varying sequence lengths and topologies and find that given perfect secondary-structural information, a small fraction of the native contact map (5%-10%) suffices to fold proteins to their correct native states. We also find that different sparse maps are not equivalent and we make several observations about the type of maps that are successful at such structure prediction. Long range contacts are found to encode more information than shorter range ones, especially for α and αß-proteins. However, this distinction reduces for ß-proteins. Choosing contacts that are a consensus from successful maps gives predictive sparse maps as does choosing contacts that are well spread out over the protein structure. Additionally, the folding of proteins can also be used to choose predictive sparse maps. Overall, we conclude that structure-based models can be used to understand the efficacy of structure-prediction restraints and could, in future, be tuned to include specific force-field interactions, secondary structure errors and noise in the sparse maps.
Assuntos
Proteínas/química , Animais , Bactérias/química , Proteínas de Bactérias/química , Bacteriófagos/química , Bases de Dados de Proteínas , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Dobramento de Proteína , Proteínas Virais/químicaRESUMO
The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge.
Assuntos
Engenharia de Proteínas/métodos , Proteínas/química , Sítios de Ligação , Simulação por Computador , Detergentes/farmacologia , Cinética , Ligantes , Modelos Moleculares , Peptídeo Hidrolases/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , TermodinâmicaRESUMO
Comparative studies of proteins from a family have been used to understand the factors that determine the folding routes of proteins. It has been conjectured that the folding mechanism of ribonuclease-H (RNase-H) proteins is determined by the topology of their fold. To test this hypothesis, we computationally studied the folding of four proteins from the RNase-H family, which have the overall RNase-H fold, but whose topologies differ in the region termed CORE in E. coli RNase-H. We simulated the folding of these proteins using molecular dynamics (MD) simulations of a coarse-grained structure-based model (SBM) which captures the effects of topology and found that the four proteins had similar folding routes. However, these simulated folding routes do not agree with the folding routes of those RNase-H proteins that have been experimentally characterized. We next simulated the proteins using an SBM which specifically accounts for packing energetics and found that these routes not only vary substantially across the simulated RNase-H proteins but also agree with experiments. Thus, the packing energetics determine the folding mechanism of the RNase-H proteins. By comparing the differing folding routes calculated from the two models, we isolated packing interactions that promote these differences. We find that the balance of packing energetics between CORE and the rest of the protein is different across the different RNase-Hs. This balance determines the folding route. Our studies suggest that proteins from the RNase-H family should be used for experimentally detecting structurally distinct folding routes.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , Ribonuclease H/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de ProteínaRESUMO
Having multiple domains in proteins can lead to partial folding and increased aggregation. Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity. In agreement with bulk experiments, a coarse-grained structure-based model of the three-domain protein, E. coli Adenylate kinase (AKE), folds cooperatively. Domain interfaces have previously been implicated in the cooperative folding of multi-domain proteins. To understand their role in AKE folding, we computationally create mutants with deleted inter-domain interfaces and simulate their folding. We find that inter-domain interfaces play a minor role in the folding cooperativity of AKE. On further analysis, we find that unlike other multi-domain proteins whose folding has been studied, the domains of AKE are not singly-linked. Two of its domains have two linkers to the third one, i.e., they are inserted into the third one. We use circular permutation to modify AKE chain-connectivity and convert inserted-domains into singly-linked domains. We find that domain insertion in AKE achieves the following: (1) It facilitates folding cooperativity even when domains have different stabilities. Insertion constrains the N- and C-termini of inserted domains and stabilizes their folded states. Therefore, domains that perform conformational transitions can be smaller with fewer stabilizing interactions. (2) Inter-domain interactions are not needed to promote folding cooperativity and can be tuned for function. In AKE, these interactions help promote conformational dynamics limited catalysis. Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins.
Assuntos
Adenilato Quinase/química , Adenilato Quinase/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Adenilato Quinase/genética , Biologia Computacional , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutação , Dobramento de Proteína , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Proteins fold into three-dimensional structures in a funneled energy landscape. This landscape is also used for functional activity. Frustration in this landscape can arise from the competing evolutionary pressures of biological function and reliable folding. Thus, the ensemble of partially folded states can populate multiple routes on this journey to the native state. Although protein folding kinetics experiments have shown the presence of such routes for several proteins, there has been sparse information about the structural diversity of these routes. In addition, why a given protein populates a particular route more often than another protein of similar structure and sequence is not clear. Whereas multiple routes are observed in theoretical studies on the folding of interleukin-1ß (IL-1ß), experimental results indicate one dominant route where the central portion of the protein folds first, and is then followed by closure of the barrel in this ß-trefoil fold. Here we show, using a combination of computation and experiment, that the presence of functionally important regions like the ß-bulge in the signaling protein IL-1ß strongly influences the choice of folding routes. By deleting the ß-bulge, we directly observe the presence of route-switching. This route-switching provides a direct link between route selection and the folding and functional landscapes of a protein.
Assuntos
Interleucina-1beta/fisiologia , Interleucina-1beta/química , Modelos Teóricos , Dobramento de ProteínaRESUMO
The topology of the designed protein Top7 is not found in natural proteins. Top7 shows signatures of non-cooperative folding in both experimental studies and computer simulations. In particular, molecular dynamics of coarse-grained structure-based models of Top7 show a well-populated C-terminal folding-intermediate. Since most similarly sized globular proteins are cooperative folders, the non-natural topology of Top7 has been suggested as a reason for its non-cooperative folding. Here, we computationally examine the folding of Top7 with the intent of making it cooperative. We find that its folding cooperativity can be increased in two ways: (a) Optimization of packing interactions in the N-terminal half of the protein enables further folding of the C-terminal intermediate. (b) Reduction in the packing density of the C-terminal region destabilizes the intermediate. In practice, these strategies are implemented in our Top7 model through modifications to the contact-map. These modifications do not alter the topology of Top7 but result in cooperative folding. Amino-acid mutations that mimic these modifications also lead to a significant increase in folding cooperativity. Finally, we devise a method to randomize the sizes of amino-acids within the same topology, and confirm that the structure of Top7 makes its folding sensitive to packing interactions. In contrast, the ribosomal protein S6, which has secondary structure similar to Top7, has folding which is much less sensitive to packing perturbations. Thus, it should be possible to make a sequence fold cooperatively to the structure of Top7, but to do so its side-chain packing needs to be carefully designed.
Assuntos
Conformação Proteica , Engenharia de Proteínas/métodos , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Biologia Computacional , Simulação por Computador , Mutação , Estabilidade Proteica , TermodinâmicaRESUMO
The SARS-CoV-2 main protease (Mpro) plays an essential role in viral replication, cleaving viral polyproteins into functional proteins. This makes Mpro an important drug target. Mpro consists of an N-terminal catalytic domain and a C-terminal α-helical domain (MproC). Previous studies have shown that peptides derived from a given protein sequence (self-peptides) can affect the folding and, in turn, the function of that protein. Since the SARS-CoV-1 MproC is known to stabilize its Mpro and regulate its function, we hypothesized that SARS-CoV-2 MproC-derived self-peptides may modulate the folding and the function of SARS-CoV-2 Mpro. To test this, we studied the folding of MproC in the presence of various self-peptides using coarse-grained structure-based models and molecular dynamics simulations. In these simulations of MproC and one self-peptide, we found that two self-peptides, the α1-helix and the loop between α4 and α5 (loop4), could replace the equivalent native sequences in the MproC structure. Replacement of either sequence in full-length Mpro should, in principle, be able to perturb Mpro function albeit through different mechanisms. Some general principles for the rational design of self-peptide inhibitors emerge: The simulations show that prefolded self-peptides are more likely to replace native sequences than those which do not possess structure. Additionally, the α1-helix self-peptide is kinetically stable and once inserted rarely exchanges with the native α1-helix, while the loop4 self-peptide is easily replaced by the native loop4, making it less useful for modulating function. In summary, a prefolded α1-derived peptide should be able to inhibit SARS-CoV-2 Mpro function.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Cisteína Endopeptidases/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Simulação de Dinâmica Molecular , Simulação de Acoplamento Molecular , Antivirais/químicaRESUMO
Kinetic stability, defined as the rate of protein unfolding, is central to determining the functional lifetime of proteins, both in nature and in wide-ranging medical and biotechnological applications. Further, high kinetic stability is generally correlated with high resistance against chemical and thermal denaturation, as well as proteolytic degradation. Despite its significance, specific mechanisms governing kinetic stability remain largely unknown, and few studies address the rational design of kinetic stability. Here, we describe a method for designing protein kinetic stability that uses protein long-range order, absolute contact order, and simulated free energy barriers of unfolding to quantitatively analyze and predict unfolding kinetics. We analyze two ß-trefoil proteins: hisactophilin, a quasi-three-fold symmetric natural protein with moderate stability, and ThreeFoil, a designed three-fold symmetric protein with extremely high kinetic stability. The quantitative analysis identifies marked differences in long-range interactions across the protein hydrophobic cores that partially account for the differences in kinetic stability. Swapping the core interactions of ThreeFoil into hisactophilin increases kinetic stability with close agreement between predicted and experimentally measured unfolding rates. These results demonstrate the predictive power of readily applied measures of protein topology for altering kinetic stability and recommend core engineering as a tractable target for rationally designing kinetic stability that may be widely applicable.
RESUMO
Some non-enveloped virus capsids assemble from multiple copies of a single type of coat-protein (CP). The comparative energetics of the diverse CP-CP interfaces present in such capsids likely govern virus assembly-disassembly mechanisms. The T = 3 icosahedral capsids comprise 180 CP copies arranged about two-, three-, five- and six-fold axes of (quasi-)rotation symmetry. Structurally diverse CPs can assemble into T = 3 capsids. Specifically, the Leviviridae CPs are structurally distinct from the Bromoviridae, Tombusviridae and Tymoviridae CPs which fold into the classic "jelly-roll" fold. However, capsids from across the four families are known to disassemble into dimers. To understand whether the overall symmetry of the capsid or the structural details of the CP determine virus assembly-disassembly mechanisms, we analyze the different CP-CP interfaces that occur in the four virus families. Previous work studied protein homodimer interfaces using interface size (relative to the monomer) and hydrophobicity. Here, we analyze all CP-CP interfaces using these two parameters and find that the dimerization interface (present between two CPs congruent through a two-fold axis of rotation) has a larger relative size in the Leviviridae than in the other viruses. The relative sizes of the other Leviviridae interfaces and all the jelly-roll interfaces are similar. However, the dimerization interfaces across families have slightly higher hydrophobicity, potentially making them stronger than other interfaces. Finally, although the CP-monomers of the jelly-roll viruses are structurally similar, differences in their dimerization interfaces leads to varied dimer flexibility. Overall, differences in CP-structures may induce different modes of swelling and assembly-disassembly in the T = 3 viruses.
RESUMO
Many single-domain proteins are not only stable and water-soluble, but they also populate few to no intermediates during folding. This reduces interactions between partially folded proteins, misfolding, and aggregation, and makes the proteins tractable in biotechnological applications. Natural proteins fold thus, not necessarily only because their structures are well-suited for folding, but because their sequences optimize packing and fit their structures well. In contrast, folding experiments on the de novo designed Top7 suggest that it populates several intermediates. Additionally, in de novo protein design, where sequences are designed for natural and new non-natural structures, tens of sequences still need to be tested before success is achieved. Both these issues may be caused by the specific scaffolds used in design, i.e., some protein scaffolds may be more tolerant to packing perturbations and varied sequences. Here, we report a computational method for assessing the response of protein structures to packing perturbations. We then benchmark this method using designed proteins and find that it can identify scaffolds whose folding gets disrupted upon perturbing packing, leading to the population of intermediates. The method can also isolate regions of both natural and designed scaffolds that are sensitive to such perturbations and identify contacts which when present can rescue folding. Overall, this method can be used to identify protein scaffolds that are more amenable to whole protein design as well as to identify protein regions which are sensitive to perturbations and where further mutations should be avoided during protein engineering.
RESUMO
Despite having remarkably similar three-dimensional structures and stabilities, IL-1beta promotes signaling, whereas IL-1Ra inhibits it. Their energy landscapes are similar and have coevolved to facilitate competitive binding to the IL-1 receptor. Nevertheless, we find that IL-1Ra folds faster than IL-1beta. A structural alignment of the proteins shows differences mainly in two loops, a beta-bulge of IL-1beta and a loop in IL-1Ra that interacts with residue K145 and connects beta-strands 11 and 12. Bioassays indicate that inserting the beta-bulge from IL-1beta confers partial signaling capability onto a K145D mutant of IL-1Ra. Based on the alignment, mutational assays and our computational folding results, we hypothesize that functional regions are not central to the beta-trefoil motif and cause slow folding. The IL-1beta beta-bulge facilitates activity and replacing it by the IL-1Ra beta-turn results in a hybrid protein that folds faster than IL-1beta. Inserting the beta11-beta12 connecting-loop, which aids inhibition, into either IL-1beta or the hybrid protein slows folding. Thus, regions that aid function (either through activity or inhibition) can be inferred from folding traps via structural differences. Mapping functional properties onto the numerous folds determined in structural genomics efforts is an area of intense interest. Our studies provide a systematic approach to mapping the functional genomics of a fold family.
Assuntos
Dobramento de Proteína , Receptores de Interleucina-1/química , Algoritmos , Motivos de Aminoácidos , Sítios de Ligação , Análise Mutacional de DNA , Genômica , Humanos , Interleucina-1beta/química , Cinética , Conformação Molecular , Conformação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , SoftwareRESUMO
Recent experimental studies suggest that the mature GFP has an unconventional landscape composed of an early folding event with a typical funneled landscape, followed by a very slow search and rearrangement step into the locked, active chromophore-containing structure. As we have shown previously, the substantial difference in time scales is what generates the observed hysteresis in thermodynamic folding. The interconversion between locked and the soft folding structures at intermediate denaturant concentrations is so slow that it is not observed under the typical experimental observation time. Simulations of a coarse-grained model were used to describe the fast folding event as well as identify native-like intermediates on energy landscapes enroute to the fluorescent native fold. Interestingly, these simulations reveal structural features of the slow dynamic transition to chromophore activation. Experimental evidence presented here shows that the trapped, native-like intermediate has structural heterogeneity in residues previously linked to chromophore formation. We propose that the final step of GFP folding is a "locking" mechanism leading to chromophore formation and high stability. The combination of previous experimental work and current simulation work is explained in the context of a dual-basin folding mechanism described above.
Assuntos
Proteínas de Fluorescência Verde/química , Dobramento de Proteína , Simulação por Computador , TermodinâmicaRESUMO
The capsids of RNA viruses such as MS2 are great models for studying protein self-assembly because they are made almost entirely of multiple copies of a single coat protein (CP). Although CP is the minimal repeating unit of the capsid, previous studies have shown that CP exists as a homodimer (CP2) even in an acid-disassembled system, indicating that CP2 is an obligate dimer. Here, we investigate the molecular basis of this obligate dimerization using coarse-grained structure-based models and molecular dynamics simulations. We find that, unlike monomeric proteins of similar size, CP populates a single partially folded ensemble whose "foldedness" is sensitive to denaturing conditions. In contrast, CP2 folds similarly to single-domain proteins populating only the folded and the unfolded ensembles, separated by a prominent folding free energy barrier. Several intramonomer contacts form early, but the CP2 folding barrier is crossed only when the intermonomer contacts are made. A dissection of the structure of CP2 through mutant folding simulations shows that the folding barrier arises both from the topology of CP and the interface contacts of CP2. Together, our results show that CP2 is an obligate dimer because of kinetic stability, that is, dimerization induces a folding barrier and that makes it difficult for proteins in the dimer minimum to partially unfold and access the monomeric state without completely unfolding. We discuss the advantages of this obligate dimerization in the context of dimer design and virus stability.
Assuntos
Levivirus , Dobramento de Proteína , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Dimerização , Levivirus/genéticaRESUMO
Two mechanisms, induced fit (IF) and conformational selection (CS), have been proposed to explain ligand recognition coupled conformational changes. The histidine binding protein (HisJ) adopts the CS mechanism, in which a pre-equilibrium is established between the open and the closed states with the ligand binding to the closed state. Despite being structurally similar to HisJ, the maltose binding protein (MBP) adopts the IF mechanism, in which the ligand binds the open state and induces a transition to the closed state. To understand the molecular determinants of this difference, we performed molecular dynamics (MD) simulations of coarse-grained dual structure based models. We find that intra-protein contacts unique to the closed state are sufficient to promote the conformational transition in HisJ, indicating a CS-like mechanism. In contrast, additional ligand-mimicking contacts are required to "induce" the conformational transition in MBP suggesting an IF-like mechanism. In agreement with experiments, destabilizing modifications to two structural features, the spine helix (SH) and the balancing interface (BI), present in MBP but absent in HisJ, reduce the need for ligand-mimicking contacts indicating that SH and BI act as structural restraints that keep MBP in the open state. We introduce an SH like element into HisJ and observe that this can impede the conformational transition increasing the importance of ligand-mimicking contacts. Similarly, simultaneous mutations to BI and SH in MBP reduce the barrier to conformational transitions significantly and promote a CS-like mechanism. Together, our results show that structural restraints present in the protein structure can determine the mechanism of conformational transitions and even simple models that correctly capture such structural features can predict their positions. MD simulations of such models can thus be used, in conjunction with mutational experiments, to regulate protein ligand interactions, and modulate ligand binding affinities.
RESUMO
Exploring the multi-dimensional energy landscape of a large protein in detail is a computational challenge. Such investigations may include analysis of multiple folding pathways, rate constants for important conformational transitions, locating intermediate states populated during folding, estimating energetic and entropic barriers that separate populated basins, and visualising a high-dimensional surface. The complexity of the landscape can be simplified through coarse-grained structure-based models (SBMs). These widely used coarse-grained representations of proteins provide a minimalist approximation to the free energy landscape, which subsumes the folding behaviour of many single-domain proteins. Here we describe the combination of SBMs with discrete path sampling (DPS), and show how this approach can provide details of the landscape and folding pathways. Combining SBMs and DPS provides an efficient framework for sampling the protein free energy landscape and for calculating various kinetic and thermodynamic quantities.