RESUMO
The ability of cytoskeletal motors to move unidirectionally along filamentous tracks is central to their role in cargo transport, motility and cell division. Kinesin and myosin motor families have a subclass that moves towards the opposite end of the microtubule or actin filament with respect to the rest of the motor family1,2, whereas all dynein motors that have been studied so far exclusively move towards the minus end of the microtubule3. Guided by cryo-electron microscopy and molecular dynamics simulations, we sought to understand the mechanism that underpins the directionality of dynein by engineering a Saccharomyces cerevisiae dynein that is directed towards the plus end of the microtubule. Here, using single-molecule assays, we show that elongation or shortening of the coiled-coil stalk that connects the motor to the microtubule controls the helical directionality of dynein around microtubules. By changing the length and angle of the stalk, we successfully reversed the motility towards the plus end of the microtubule. These modifications act by altering the direction in which the dynein linker swings relative to the microtubule, rather than by reversing the asymmetric unbinding of the motor from the microtubule. Because the length and angle of the dynein stalk are fully conserved among species, our findings provide an explanation for why all dyneins move towards the minus end of the microtubule.
Assuntos
Microscopia Crioeletrônica , Dineínas/química , Dineínas/metabolismo , Microtúbulos/metabolismo , Simulação de Dinâmica Molecular , Movimento , Saccharomyces cerevisiae , Dineínas/genética , Dineínas/ultraestrutura , Microtúbulos/química , Modelos Biológicos , Nucleotídeos/metabolismo , Prolina/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem Individual de MoléculaRESUMO
The Delta variant spreads more rapidly than previous variants of SARS-CoV-2. This variant comprises several mutations on the receptor-binding domain (RBDDelta) of its spike glycoprotein, which binds to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptors in host cells. The RBD-PD interaction has been targeted by antibodies and nanobodies to prevent viral infection, but their effectiveness against the Delta variant remains unclear. Here, we investigated RBDDelta-PD interactions in the presence and absence of nanobodies H11-H4, H11-D4, and Ty1 by performing 21.8 µs of all-atom molecular dynamics simulations. Unbiased simulations revealed that Delta variant mutations strengthen RBD binding to ACE2 by increasing the hydrophobic interactions and salt bridge formation, but weaken interactions with H11-H4, H11-D4, and Ty1. Among these nanobodies H11-H4 and H11-D4 bind RBD without overlapping ACE2. They were unable to dislocate ACE2 from RBDDelta when bound side by side with ACE2 on RBD. Steered molecular dynamics simulations at comparable loading rates to high-speed atomic force microscopy (AFM) experiments estimated lower rupture forces of the nanobodies from RBDDelta compared to ACE2. Our results suggest that existing nanobodies are less effective to inhibit RBDDelta-PD interactions and a new generation of nanobodies is needed to neutralize the Delta variant.
Assuntos
Tratamento Farmacológico da COVID-19 , Anticorpos de Domínio Único , Enzima de Conversão de Angiotensina 2 , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , SARS-CoV-2 , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human cells upon binding of its spike (S) glycoproteins to ACE2 receptors. Several nanobodies neutralize SARS-CoV-2 infection by binding to the receptor-binding domain (RBD) of the S protein, but how their binding antagonizes S-ACE2 interactions is not well understood. Here, we identified interactions between the RBD and nanobodies H11-H4, H11-D4, and Ty1 by performing all-atom molecular dynamics simulations. H11-H4 and H11-D4 can bind to RBD without overlapping with ACE2. H11-H4, and to a lesser extent H11-D4, binding dislocates ACE2 from its binding site due to electrostatic repulsion. In comparison, Ty1 overlaps with ACE2 on RBD and has a similar binding strength to ACE2. Mutations in the Alpha variant of SARS-CoV-2 had a minor effect in RBD binding strengths of ACE2 and nanobodies, but reduced the ability of H11-H4 and H11-D4 to dislocate ACE2 from RBD. In comparison, the Beta variant weakened the RBD binding strengths of H11-H4 and H11-D4, which were less effective to dislocate ACE2 binding. Unexpectedly, mutations in Beta strengthened Ty1 binding to RBD, suggesting that this nanobody may be more effective to neutralize the Beta variant of SARS-CoV-2.
Assuntos
COVID-19 , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Sítios de Ligação , Humanos , Ligação Proteica , SARS-CoV-2RESUMO
In 2020, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people worldwide and caused the coronavirus disease 2019 (COVID-19). Spike (S) glycoproteins on the viral membrane bind to ACE2 receptors on the host cell membrane and initiate fusion, and S protein is currently among the primary drug target to inhibit viral entry. The S protein can be in a receptor inaccessible (closed) or accessible (open) state based on down and up positions of its receptor-binding domain (RBD), respectively. However, conformational dynamics and the transition pathway between closed to open states remain unexplored. Here, we performed all-atom molecular dynamics (MD) simulations starting from closed and open states of the S protein trimer in the presence of explicit water and ions. MD simulations showed that RBD forms a higher number of interdomain interactions and exhibits lower mobility in its down position than its up position. MD simulations starting from intermediate conformations between the open and closed states indicated that RBD switches to the up position through a semi-open intermediate that potentially reduces the free energy barrier between the closed and open states. Free energy landscapes were constructed, and a minimum energy pathway connecting the closed and open states was proposed. Because RBD-ACE2 binding is compatible with the semi-open state, but not with the closed state of the S protein, we propose that the formation of the intermediate state is a prerequisite for the host cell recognition.
Assuntos
Betacoronavirus/química , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2 , Sítios de Ligação , Ligação de Hidrogênio , Modelos Químicos , Simulação de Dinâmica Molecular , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Análise de Componente Principal , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Receptores Virais/química , Receptores Virais/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , TermodinâmicaRESUMO
Cyclophilin 40 (Cyp40) is a member of the immunophilin family that acts as a peptidyl-prolyl-isomerase enzyme and binds to the heat shock protein 90 (Hsp90). Its structure comprises an N-terminal cyclophilin domain and a C-terminal tetratricopeptide (TPR) domain. Cyp40 is overexpressed in prostate cancer and certain T-cell lymphomas. The groove for Hsp90 binding on the TPR domain includes residues Lys227 and Lys308, referred to as the carboxylate clamp, and is essential for Cyp40-Hsp90 binding. In this study, the effect of two mutations, K227A and K308A, and their combinative mutant was investigated by performing a total of 5.76 µs of all-atom molecular dynamics (MD) simulations in explicit solvent. All simulations, except the K308A mutant, were found to adopt two distinct (extended or compact) conformers defined by different cyclophilin-TPR interdomain distances. The K308A mutant was only observed in the extended form which is observed in the Cyp40 X-ray structure. The wild-type, K227A, and combined mutant also showed bimodal distributions. The experimental melting temperature, Tm, values of the mutants correlate with the degree of compactness with the K308A extended mutant having a marginally lower melting temperature. Another novel measure of compactness determined from the MD data, the "coordination shell volume," also shows a direct correlation with Tm. In addition, the MD simulations show an allosteric effect with the mutations in the remote TPR domain having a pronounced effect on the molecular motions of the enzymatic cyclophilin domain which helps rationalise the experimentally observed increase in enzyme activity measured for all three mutations.
Assuntos
Ciclofilinas/química , Mutação Puntual/genética , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos/genética , Termodinâmica , Temperatura de TransiçãoRESUMO
The bacterial sodium-coupled leucine/alanine transporter LeuT is broadly used as a model system for studying the transport mechanism of neurotransmitters because of its structural and functional homology to mammalian transporters such as serotonin, dopamine, or norepinephrine transporters, and because of the resolution of its structure in different states. Although the binding sites (S1 for substrate, and Na1 and Na2 for two co-transported sodium ions) have been resolved, we still lack a mechanistic understanding of coupled Na(+)- and substrate-binding events. We present here results from extensive (>20 µs) unbiased molecular dynamics simulations generated using the latest computing technology. Simulations show that sodium binds initially the Na1 site, but not Na2, and, consistently, sodium unbinding/escape to the extracellular (EC) region first takes place at Na2, succeeded by Na1. Na2 diffusion back to the EC medium requires prior dissociation of substrate from S1. Significantly, Na(+) binding (and unbinding) consistently involves a transient binding to a newly discovered site, Na1â³, near S1, as an intermediate state. A robust sequence of substrate uptake events coupled to sodium bindings and translocations between those sites assisted by hydration emerges from the simulations: (i) bindings of a first Na(+) to Na1â³, translocation to Na1, a second Na(+) to vacated Na1â³ and then to Na2, and substrate to S1; (ii) rotation of Phe(253) aromatic group to seclude the substrate from the EC region; and (iii) concerted tilting of TM1b and TM6a toward TM3 and TM8 to close the EC vestibule.
Assuntos
Proteínas de Bactérias/química , Simulação de Dinâmica Molecular , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/química , Sódio/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cinética , Mutação , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Biomolecular conformational transitions are essential to biological functions. Most experimental methods report on the long-lived functional states of biomolecules, but information about the transition pathways between these stable states is generally scarce. Such transitions involve short-lived conformational states that are difficult to detect experimentally. For this reason, computational methods are needed to produce plausible hypothetical transition pathways that can then be probed experimentally. Here we propose a simple and computationally efficient method, called ANMPathway, for constructing a physically reasonable pathway between two endpoints of a conformational transition. We adopt a coarse-grained representation of the protein and construct a two-state potential by combining two elastic network models (ENMs) representative of the experimental structures resolved for the endpoints. The two-state potential has a cusp hypersurface in the configuration space where the energies from both the ENMs are equal. We first search for the minimum energy structure on the cusp hypersurface and then treat it as the transition state. The continuous pathway is subsequently constructed by following the steepest descent energy minimization trajectories starting from the transition state on each side of the cusp hypersurface. Application to several systems of broad biological interest such as adenylate kinase, ATP-driven calcium pump SERCA, leucine transporter and glutamate transporter shows that ANMPathway yields results in good agreement with those from other similar methods and with data obtained from all-atom molecular dynamics simulations, in support of the utility of this simple and efficient approach. Notably the method provides experimentally testable predictions, including the formation of non-native contacts during the transition which we were able to detect in two of the systems we studied. An open-access web server has been created to deliver ANMPathway results.
Assuntos
Modelos Moleculares , Conformação Proteica , Adenilato Quinase/química , Sistema X-AG de Transporte de Aminoácidos/química , Proteínas de Transporte/química , Leucina/química , Retículo Sarcoplasmático/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/químicaRESUMO
The bacterial sodium-coupled leucine transporter (LeuT) has been broadly used as a structural model for understanding the structure-dynamics-function of mammalian neurotransmitter transporters as well as other solute carriers that share the same fold (LeuT fold), as the first member of the family crystallographically resolved in multiple states: outward-facing open, outward-facing occluded, and inward-facing open. Yet, a complete picture of the energy landscape of (sub)states visited along the LeuT transport cycle has been elusive. In an attempt to visualize the conformational spectrum of LeuT, we performed extensive simulations of LeuT dimer dynamics in the presence of substrate (Ala or Leu) and co-transported Na(+) ions, in explicit membrane and water. We used both conventional molecular dynamics (MD) simulations (with Anton supercomputing machine) and a recently introduced method, collective MD, that takes advantage of collective modes of motions predicted by the anisotropic network model. Free energy landscapes constructed based on â¼40 µs trajectories reveal multiple substates occluded to the extracellular (EC) and/or intracellular (IC) media, varying in the levels of exposure of LeuT to EC or IC vestibules. The IC-facing transmembrane (TM) helical segment TM1a shows an opening, albeit to a smaller extent and in a slightly different direction than that observed in the inward-facing open crystal structure. The study provides insights into the spectrum of conformational substates and paths accessible to LeuT and highlights the differences between Ala- and Leu-bound substates.
Assuntos
Sistemas de Transporte de Aminoácidos/química , Proteínas de Bactérias/química , Simulação de Dinâmica Molecular , Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Conformação Proteica , Sódio/metabolismo , TermodinâmicaRESUMO
Dyneins are an AAA+ motor responsible for motility and force generation toward the minus end of microtubules. Dynein motility is powered by nucleotide-dependent transitions of its linker domain, which transitions between straight (post-powerstroke) and bent (pre-powerstroke) conformations. To understand the dynamics and energetics of the linker, we performed all-atom molecular dynamics simulations of human dynein-2 primed for its power stroke. Simulations revealed that the linker can adopt either a bent conformation or a semi-bent conformation, separated by a 5.7 kT energy barrier. The linker cannot switch back to its straight conformation in the pre-powerstroke state due to a steric clash with the AAA+ ring. Simulations also showed that an isolated linker has a free energy minimum near the semi-bent conformation in the absence of the AAA+ ring, indicating that the linker stores energy as it bends and releases this energy during the powerstroke.
Assuntos
Dineínas , Simulação de Dinâmica Molecular , Humanos , Dineínas/metabolismo , Dineínas/química , Termodinâmica , Ligação Proteica , Conformação ProteicaRESUMO
The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.
Assuntos
Anáfase , Proteínas de Drosophila , Drosophila melanogaster , Dineínas , Microtúbulos , Animais , Dineínas/metabolismo , Dineínas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Microtúbulos/metabolismo , Microtúbulos/genética , Simulação de Dinâmica Molecular , Mutação/genética , Fuso Acromático/metabolismo , Fuso Acromático/genética , Humanos , Mutação de Sentido IncorretoRESUMO
PR65 is the HEAT repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem repeat protein. Its conformational mechanics plays a crucial role in PP2A function by opening/closing substrate binding/catalysis interface. Using in silico saturation mutagenesis, we identified PR65 "hinge" residues whose substitutions could alter its conformational adaptability and thereby PP2A function, and selected six mutations that were verified to be expressed and soluble. Molecular simulations and nanoaperture optical tweezers revealed consistent results on the specific effects of the mutations on the structure and dynamics of PR65. Two mutants observed in simulations to stabilize extended/open conformations exhibited higher corner frequencies and lower translational scattering in experiments, indicating a shift toward extended conformations, whereas another displayed the opposite features, confirmed by both simulations and experiments. The study highlights the power of single-molecule nanoaperture-based tweezers integrated with in silico approaches for exploring the effect of mutations on protein structure and dynamics.
Assuntos
Simulação de Dinâmica Molecular , Pinças Ópticas , Mutação Puntual , Conformação Proteica , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , HumanosRESUMO
Efficient and accurate mapping of transition pathways is a challenging problem in allosteric proteins. We propose here a to our knowledge new methodology called collective molecular dynamics (coMD). coMD takes advantage of the collective modes of motions encoded by the fold, simultaneously evaluating the interactions and energetics via a full-atomic MD simulation protocol. The basic approach is to deform the structure collectively along the modes predicted by the anisotropic network model, upon selecting them via a Monte Carlo/Metropolis algorithm from among the complete pool of all accessible modes. Application to adenylate kinase, an allosteric enzyme composed of three domains, CORE, LID, and NMP, shows that both open-to-closed and closed-to-open transitions are readily sampled by coMD, with large-scale motions of the LID dominating. An energy-barrier crossing occurs during the NMP movements. The energy barrier originates from a switch between the salt bridges K136-D118 at the LID-CORE interface and K57-E170 and D33-R156 at the CORE-NMP and LID-NMP interfaces, respectively. Despite its simplicity and computing efficiency, coMD yields ensembles of transition pathways in close accord with detailed full atomic simulations, lending support to its utility as a multiscale hybrid method for efficiently exploring the allosteric transitions of multidomain or multimeric proteins.
Assuntos
Adenilato Quinase/química , Algoritmos , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Animais , Anisotropia , Dados de Sequência MolecularRESUMO
PR65 is the HEAT-repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem-repeat protein, forming a spring-like architecture. PR65 conformational mechanics play a crucial role in PP2A function by opening/closing the substrate-binding/catalysis interface. Using in-silico saturation mutagenesis we identified "hinge" residues of PR65, whose substitutions are predicted to restrict its conformational adaptability and thereby disrupt PP2A function. Molecular simulations revealed that a subset of hinge mutations stabilized the extended/open conformation, whereas another had the opposite effect. By trapping in nanoaperture optical tweezer, we characterized PR65 motion and showed that the former mutants exhibited higher corner frequencies and lower translational scattering, indicating a shift towards extended conformations, whereas the latter showed the opposite behavior. Thus, experiments confirm the conformations predicted computationally. The study highlights the utility of nanoaperture-based tweezers for exploring structure and dynamics, and the power of integrating this single-molecule method with in silico approaches.
RESUMO
The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate in vivo studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.
RESUMO
Binding of two short peptides of sequences ASN-ASP-MET-PHE-ARG-LEU and LEU-LEU-PHE-MET-GLN-HIS and their bound complex structures is studied. Molecular dynamic simulations of the three structures around their respective minimum energy conformations are performed and a quasi-harmonic analysis is performed over the trajectories generated. The fluctuation correlation matrix is constructed for all C(α) -atoms of the peptides for the full trajectory. The spring constant matrix between peptide C(α) -atoms is obtained from the correlation matrix. Statistical thermodynamics of fluctuations, the energies, entropies, and the free energies of binding are discussed in terms of the quasi-harmonic model. Sites contributing to the stability of the system and presenting high affinity for binding are determined. Contribution of hydrophobic forces to binding is discussed. Quasi-harmonic approximation identifies the essential subspace of motions, the important interactions, and binding sites, gives the energetic contribution of each individual interaction, and filters out noise observed in molecular dynamics owing to uncorrelated motions. Comparison of the molecular dynamics results with those of the quasi-harmonic model shows the importance of entropy change, resulting from water molecules being liberated from the surfaces of the two peptides upon binding.
Assuntos
Modelos Químicos , Oligopeptídeos/química , Simulação de Dinâmica Molecular , Oligopeptídeos/metabolismo , Fenômenos Físicos , TermodinâmicaRESUMO
SARS-CoV-2 infection is initiated by binding of the receptor-binding domain (RBD) of its spike glycoprotein to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptors in host cells. Recently detected Omicron variant of SARS-CoV-2 (B.1.1.529) is heavily mutated on RBD. First the BA.1 and later the BA.2 variant became the most dominant strains of the Omicron variant. To investigate how the mutations of these strains affect RBD-PD interactions, we performed all-atom molecular dynamics simulations of the BA.1 and BA.2 RBD-PD in the presence of full-length glycans, explicit water, and ions. Simulations revealed that RBDs of BA.1 and BA.2 variants exhibit a more dispersed interaction network and make an increased number of salt bridges and hydrophobic interactions with PD compared to wild-type RBD. Although BA.1 and BA.2 differ in two residues at the RBD-ACE2 interface, no major difference in RBD-PD interactions and binding strengths were observed between these variants. Using the conformations sampled in each trajectory, the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) method estimated â¼34% and â¼51% stronger binding free energies to PD for BA.1 and BA.2 RBD, respectively, than wild-type RBD, which may result in higher binding efficiency of the Omicron variant to infect host cells.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/química , Humanos , Mutação , Ligação Proteica , Receptores Virais/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Microtubule (MT)-associated protein 7 (MAP7) is a required cofactor for kinesin-1-driven transport of intracellular cargoes. Using cryo-electron microscopy and single-molecule imaging, we investigated how MAP7 binds MTs and facilitates kinesin-1 motility. The MT-binding domain (MTBD) of MAP7 bound MTs as an extended α helix between the protofilament ridge and the site of lateral contact. Unexpectedly, the MTBD partially overlapped with the binding site of kinesin-1 and inhibited its motility. However, by tethering kinesin-1 to the MT, the projection domain of MAP7 prevented dissociation of the motor and facilitated its binding to available neighboring sites. The inhibitory effect of the MTBD dominated as MTs became saturated with MAP7. Our results reveal biphasic regulation of kinesin-1 by MAP7 in the context of their competitive binding to MTs.
Assuntos
Cinesinas , Proteínas Associadas aos Microtúbulos , Microtúbulos , Humanos , Sítios de Ligação , Ligação Competitiva , Microscopia Crioeletrônica , Dineínas/química , Dineínas/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects human cells by binding its spike (S) glycoproteins to angiotensin-converting enzyme 2 (ACE2) receptors and causes the coronavirus disease 2019 (COVID-19). Therapeutic approaches to prevent SARS-CoV-2 infection are mostly focused on blocking S-ACE2 binding, but critical residues that stabilize this interaction are not well understood. By performing all-atom molecular dynamics (MD) simulations, we identified an extended network of salt bridges, hydrophobic and electrostatic interactions, and hydrogen bonds between the receptor-binding domain (RBD) of the S protein and ACE2. Mutagenesis of these residues on the RBD was not sufficient to destabilize binding but reduced the average work to unbind the S protein from ACE2. In particular, the hydrophobic end of RBD serves as the main anchor site and is the last to unbind from ACE2 under force. We propose that blocking the hydrophobic surface of RBD via neutralizing antibodies could prove to be an effective strategy to inhibit S-ACE2 interactions.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Ligação ProteicaRESUMO
INTRODUCTION/OBJECTIVES: The aim of this study was to evaluate the relationships among the disease activity, illness perception, daily life performance, anxiety and depression status as potential mediators in Takayasu's arteritis (TAK) patients. METHOD: In this cross-sectional study, 77 TAK patients were included. Data were collected by a clinical examination and a structured questionnaire regarding patient reported outcome measures (PROMs). Indian Takayasu's Arteritis Activity Score2010 (ITAS2010) was used to assess the disease activity (0: inactive vs ≥ 1: active). Illness Perception Questionnaire-Revise (IPQ-R), Work Productivity and Activity Impairment (WPAI) and Hospital Anxiety and Depression Scale (HADS) as PROMs were used to understand for the patient' perspective. After preliminary analysis, complex relationships among these variables were evaluated by mediation analyses in the study. RESULTS: WPAI-Daily impairment score, HADS-A and HADS-D scores as well as IPQ-R Consequence score were found be high in active TAK patients (p = 0.008; p = 0.001; p = 0.031; p = 0.001). HADS-D score was also correlated with WPAI-Daily impairment score and IPQ-R Consequence score (p < 0.05). In mediation analysis, IPQ-R Consequence score was directly mediated by disease activity (ITAS2010) (p = 0.0173) and indirectly mediated through HADS-D score (p = 0.0003). Similarly, HADS-D score was associated with poor WPAI-Daily impairment score in the mediation analysis in the indirect path (p = 0.0069). Disease activity (ITAS2010) also increased WPAI-Daily impairment score in direct path (p = 0.043). CONCLUSIONS: Active TAK patients perceived their illness more seriously and experienced more impairment in their daily life. Depression status as the mediator influenced them poorly. These interactions could give clues to improve PROMs in the clinical practice. Key Points â¢IIness perception, disease activity, mental status and daily life performance, assessed as patient-reported outcome measures, have a complex relationship in Takayasu's arteritis. â¢IPQ-R Consequence score, WPAI-Daily impairment score, HADS-Depression and HADS-Anxiety scores were found be high in active TAK patients. â¢In mediation analysis, IPQ-R Consequence score was directly mediated by disease activity and indirectly mediated through HADS-D score. Similarly, disease activity increased WPAI-Daily impairment score in direct and HADS-D in indirect paths.
Assuntos
Arterite de Takayasu , Ansiedade , Estudos Transversais , Depressão , Humanos , PercepçãoRESUMO
Mode coupling and anharmonicity in a native fluctuating protein are investigated in modal space by projecting the motion along the eigenvectors of the fluctuation correlation matrix. The probability distribution of mode fluctuations is expressed in terms of tensorial Hermite polynomials. Molecular dynamics trajectories of Crambin are generated and used to evaluate the terms of the polynomials and to obtain the modal energies. The energies of a few modes exhibit large deviations from the harmonic energy of kT/2 per mode, resulting from coupling to the surroundings, or to another specific mode or to several other modes. Slowest modes have energies that are below that of the harmonic, and a few fast modes have energies significantly larger than the harmonic. Detailed analysis of the coupling of these modes to others is presented in terms of the lowest order two-mode coupling terms. Finally, the effects of mode coupling on conformational properties of the protein are investigated.