Allosteric Pathways Research Articles

Mapping N- to C-terminal allosteric coupling through disruption of a putative CD74 activation site in D-dopachrome tautomerase

The macrophage migration inhibitory factor (MIF) protein family consists of MIF and D-dopachrome tautomerase (also known as MIF-2). These homologs share 34% sequence identity while maintaining nearly indistinguishable tertiary and quaternary structure, which is likely a major contributor to their overlapping functions, including the binding and activation of the cluster of differentiation 74 (CD74) receptor to mediate inflammation. Previously, we investigated a novel allosteric site, Tyr99, that modulated N-terminal catalytic activity in MIF through a "pathway" of dynamically coupled residues. In a comparative study, we revealed an analogous allosteric pathway in MIF-2 despite its unique primary sequence. Disruptions of the MIF and MIF-2 N termini also diminished CD74 activation at the C terminus, though the receptor activation site is not fully defined in MIF-2. In this study, we use site-directed mutagenesis, NMR spectroscopy, molecular simulations, invitro and invivo biochemistry to explore the putative CD74 activation region of MIF-2 based on homology to MIF. We also confirm its reciprocal structural coupling to the MIF-2 allosteric site and N-terminal enzymatic site. Thus, we provide further insight into the CD74 activation site of MIF-2 and its allosteric coupling for immunoregulation.

Allosteric communication in the gating mechanism for controlled protein degradation by the bacterial ClpP peptidase.

Proteolysis is essential for the control of metabolic pathways and the cell cycle. Bacterial caseinolytic proteases (Clp) use peptidase components, such as ClpP, to degrade defective substrate proteins and to regulate cellular levels of stress-response proteins. To ensure selective degradation, access to the proteolytic chamber of the double-ring ClpP tetradecamer is controlled by a critical gating mechanism of the two axial pores. The binding of conserved loops of the Clp ATPase component of the protease or small molecules, such as acyldepsipeptide (ADEP), at peripheral ClpP ring sites, triggers axial pore opening through dramatic conformational transitions of flexible N-terminal loops between disordered conformations in the "closed" pore state and ordered hairpins in the "open" pore state. In this study, we probe the allosteric communication underlying these conformational changes by comparing residue-residue couplings in molecular dynamics simulations of each configuration. Both principal component and normal mode analyses highlight large-scale conformational changes in the N-terminal loop regions and smaller amplitude motions of the peptidase core. Community network analysis reveals a switch between intra- and inter-protomer coupling in the open-closed pore transition. Allosteric pathways that connect the ADEP binding sites to N-terminal loops are rewired in this transition, with shorter network paths in the open pore configuration supporting stronger intra- and inter-ring coupling. Structural perturbations, either through the removal of ADEP molecules or point mutations, alter the allosteric network to weaken the coupling.

Mapping allosteric pathway in NIa‐Pro using computational approach

BackgroundComputer simulation studies complement in vitro experiments and provide avenue to understand allosteric regulation in the absence of other molecular viewing techniques. Molecular dynamics captures internal motion within the protein and enables tracing the communication path between a catalytic site and a distal allosteric site. In this article, we have identified the communication pathway between the viral protein genome linked (VPg) binding region and catalytic active site in nuclear inclusion protein‐a protease (NIa‐Pro).MethodsMolecular dynamics followed by in silico analyses have been used to map the allosteric pathway.ResultsThis study delineates the residue interaction network involved in allosteric regulation of NIa‐Pro activity by VPg. Simulation studies indicate that point mutations in the VPg interaction interface of NIa‐Pro lead to disruption in these networks and change the orientation of catalytic residues. His142Ala and His167Ala mutations do not show a substantial change in the overall protease structure, but rather in the residue interaction network and catalytic site geometry.ConclusionOur mutagenic study delineates the allosteric pathway and facilitates the understanding of the modulation of NIa‐Pro activity on a molecular level in the absence of the structure of its complex with the known regulator VPg. Additionally, our in silico analysis explains the molecular concepts and highlights the dynamics behind the previously reported wet lab study findings.

Electrostatic networks for characterization of allosteric pathways: Allosteric paths in Cas9 apo, DNA- and RNA-bound forms.

Allostery is a fundamental process by which biological macromolecules transmit the effect of a local perturbation at one site to a distal, functional site, allowing for regulation of activity. The long-range coupling between residues that gives rise to allostery in a protein is built up from short-range electrostatic and hydrophobic interactions. These are arguably the largest determinants of protein structure and are essential regulators of protein function. We introduce an effective coulombic electrostatic coupling network obtained from the analysis of molecular dynamics simulations of Cas9 in its apo, DNA- and RNA-bound forms. We characterize key electrostatic events that determine its functional activity and targeting precision. We demonstrate the locality of the electrostatic-interaction network over other connectivity matrices as validated through direct comparisons to NMR measurements. We define an electrostatic-based centrality metric that allows us to pinpoint relevant donor-acceptor pairs that promote charge displacements that modulate the cross-interaction between the PAM-interacting region and catalytic domains. We determine key amino acid residues central to the network, allowing us to identify a circular allosteric pathway that channels perturbations from the PAM-interacting domain to the HNH and RuvCII domains, and then back to the PAM-contacting region. The connectivity around HNH is important for controlling the directionality of signal transfer from and towards the PAM-interacting domain. The effective coulombic electrostatic coupling network makes it possible to elucidate allosteric pathways and provides valuable interpretations of experimental measurements.

Predicting protein-membrane interfaces using molecular simulations and ensemble machine learning.

Abnormal protein-membrane attachment is involved in deregulated cellular pathways and in disease. The possibility to modulate protein-membrane interactions represents a new promising therapeutic strategy for membrane proteins. In this talk, we explore the free energy landscape of membrane protein dimerization using parallel tempering metadynamics simulations in the well-tempered ensemble and coarse-grained force fields and reproduce the structure and energetics of the dimerization process of membrane proteins and proteins in an aqueous solution in reasonable accuracy and throughput. We propose that the use of enhanced sampling simulations with a refined coarse-grained force field and appropriately defined collective variables is a robust approach for studying the protein dimerization process, although one should be cautious of the energy minima ranking. Moreover, we study oncogenes, including the H1047R and E545K hotspot mutants of PI3Kα, and KRAS-4B to understand the basis of protein overactivation. We calculate their allosteric pathways and show residues important in delivering communication signals between functional domains of each protein. Finally, we describe an ensemble machine learning methodology to predict protein-membrane interfaces of peripheral membrane proteins and present a drug design pipeline for drugging protein-membrane interfaces using the DREAMM web-server https://dreamm.ni4os.eu. Taking into account these results, we investigate opportunities for allosteric drug design.

Selective virtual screening of hypoxia inducible factor agonists and antagonists using artificial intelligence and linear response theory.

Hypoxia inducible factor (HIF) is a heterodimeric transcription factor that mediates a signaling cascade under low oxygen concentration and prevents cell death. Under normoxia, enhanced HIF activity promotes tumorigenesis, and it is considered as a key oncogenic driver in renal cell carcinoma. Enhancing HIF activity is a therapeutic approach to treat renal anaemia, thus making both HIF activators (agonists) and inhibitors (antagonists) a potential therapeutic target under different disease conditions. While known HIF agonists and antagonists have opposing effect on the HIF activity, both binds to same cavity in the C-terminal domain of HIF (PASB domain). We identify the atomic level mechanism behind differential effect of agonists/antagonists on HIF activity using molecular dynamics simulations and linear response theory. Agonists and antagonists trigger different allosteric pathway from the PASB domain to the DNA-binding domain of HIF transcription factor. The ligand induced external forces on the protein residues explains the observed allosteric effect and used as a descriptor to predict the activation/inhibition potential of a given ligand. A large-scale structure-based virtual screening of 700 million molecules from ZINC database is performed to identify the potential new ligands of HIF. Binding energies of 10 million ligands is predicted using Autodock Vina and used to train a directed-message passing neural network (D-MPNN) to predict binding energies of the remaining molecules, hence enabling the screening of the larger portion of molecules at a reasonable computational cost. The top candidate molecules are further classified as potential inhibitors based on the forces exerted by the ligands on protein residues.

State-dependent salt bridge interaction during activation in prokaryotic cyclic nucleotide-gated channel SthK.

The cyclic nucleotide-binding domain (CNBD) family of voltage-gated ion channels are complex molecular machines regulating membrane excitability in many cell types. While the protein structures of many CNBD channel family members have provided considerable insights into channel architecture, the mechanism of how cyclic nucleotide binding modulates channel opening is still unknown. The prokaryotic cyclic nucleotide-gated (CNG) channel, SthK, from Spirochaeta thermophila, has considerable structural and functional similarity to eukaryotic CNG channels. SthK can be readily expressed in E. coli spheroplasts and its response to ligand activation recorded with inside-out patch-clamp electrophysiology to test the functionality of various mutations. Using thermodynamic mutant cycle analysis, we have shown that there is a stabilizing interaction between arginine R125 at the end of the S4 voltage sensor helix and aspartate D261 in the B’ helix of the C-linker when the channel is in the presence of cyclic adenosine monophosphate (cAMP). Furthermore, when these residues (R125 and D261) were both mutated to cysteines, we were able to create a spontaneous disulfide bridge between these residues and lock the channel in the open, activated state, even in the absence of cAMP. These results indicate that we have identified an important state-dependent salt bridge between the C-linker domain and voltage sensor domain of this channel. Both charged residues are conserved in the eukaryotic CNGA1 channel structure, where it seems likely they play a similar role in state-dependent stabilization of the open state of these channels. Additionally, the aspartate residue is conserved across all CNG and HCN channels, further highlighting the potential significance of this C-linker residue. These new results help to better characterize the ligand-activated allosteric pathway in CNBD channels.

Reaction coordinate dependent fibrillar formation of human glutamine synthetase tunes activity.

Metabolic enzymes must adapt to changing environments to meet the energetic needs of the cell. Frequently, enzymes form supramolecular fibrils under different metabolic states to regulate function. Here, we show that human glutamine synthetase (GS) is capable of reversibly forming fibril structures along its forward reaction coordinate. In addition to fibril formation, high-resolution cryoEM maps demonstrate coordinated conformational changes that facilitate catalysis. Detailed enzymology and characterization of disease mutations of GS demonstrate an allosteric pathway connecting the active site to the overall enzyme compositional state. Together with a human cell model demonstrating correlated growth phenotype with GS enzymatic activity, we show that GS compositional state changes are a mechanism of enzyme regulation.

Revealing the allosteric mechanism of pH-dependence in the proton-activated chloride channel.

The ability of the Proton-activated chloride (PAC) channel to perform its chloride transfer function across the membrane was shown to be dependent on the pH in the extracellular environment. Thus, an increase of extracellular pH from pH 4.5 to pH 8 results in a conformational change in transmembrane domain (TMD) of PAC and abolishes chloride conductivity. Mutagenesis of various extracellular domain (ECD) regions of PAC, including the Acidic Pocket, the beta 3 sheet of the “finger region” and the distal end of the ECD, altered the pH-sensitivity of PAC without revealing how such changes affect the pH sensitivity and its effect on the structure and dynamics of the distal end of the TMD. To understand this mechanism we used molecular pH-specific molecular dynamics (MD) simulations of PAC protein with the ECpH-MD method, capturing the pH-dependent conformational transitions between the active and inactive forms of PAC protein. The effect of changes in protonation states of a number of experimentally determined pH-sensor residues revealed their specific structural roles in the mechanism of conformational change underlying the changes in PAC activity. The allosteric pathways connecting these pH-sensors in the ECD to the TMD at different pH values revealed and quantified from analyses of the various MD trajectories with the rigorous N-body information theory (NbIT) method. Together with the structural data determined for various states of PAC (inactive, active, desensitized), the atomistic level of the dynamics driving the allosteric communication offers a comprehensive mechanistic understanding of the pH-dependence in PAC function.

Identification of an Evolutionarily Conserved Allosteric Network in Steroid Receptors.

Allosteric pathways in proteins describe networks comprising amino acid residues which may facilitate the propagation of signals between distant sites. Through inter-residue interactions, dynamic and conformational changes can be transmitted from the site of perturbation to an allosteric site. While sophisticated computational methods have been developed to characterize such allosteric pathways linking specific sites on proteins, few attempts have been made to apply these approaches toward identifying new allosteric sites. Here, we use molecular dynamics simulations and suboptimal path analysis to discover new allosteric networks in steroid receptors with a focus on evolutionarily conserved pathways. Using modern receptors and a reconstructed ancestral receptor, we identify networks connecting several sites to the activation function surface 2 (AF-2), the site of coregulator recruitment. One of these networks is conserved across the entire family, connecting a predicted allosteric site located between helices 9 and 10 of the ligand-binding domain. We investigate the basis of this conserved network as well as the importance of this site, discovering that the site lies in a region of the ligand-binding domain characterized by conserved inter-residue contacts. This study suggests an evolutionarily importance of the helix 9-helix 10 site in steroid receptors and identifies an approach that may be applied to discover previously unknown allosteric sites in proteins.

Study on the allosteric activation mechanism of SHP2 via elastic network models and neural relational inference molecular dynamics simulation.

As a ubiquitous protein tyrosine phosphatase, SHP2 is involved in PD-1/PD-L1 mediated tumor immune escape and undergoes substantial conformational changes. Therefore, it is considered an ideal target for tumor intervention. However, the allosteric mechanisms of SHP2 binding PD-1 intracellular ITIM/ITSM phosphopeptides remain unclear, which greatly hinders the development of novel structure-based anticancer allosteric inhibitors. In this work, the open and closed structural models of SHP2 are first constructed based on this knowledge; next their motion modes are investigated via elastic network models such as the Gaussian network model (GNM), anisotropic network model (ANM) and adaptive anisotropic network model (aANM); and finally, a possible allosteric signaling pathway is proposed using a neural relational inference molecular dynamics (NRI-MD) simulation embedded with an artificial intelligence (AI) strategy. In GNM and ANM, the N-SH2, C-SH2 and PTP domains all exhibit distinct dynamics partitions, and the N-SH2/C-SH2 regions show a rigid rotation relative to PTP. According to a series of intermediate snapshots given by aANM, N-SH2 is first identified with pY223 specifically, inducing a D'E-loop to change from β-sheets to random coils, and then, C-SH2 serves as a fulcrum to drive N-SH2 to rotate 110° completely away from the original active sites of PTP. Finally, a possible allosteric signaling-transfer path for SHP2, namely R220-R138-T108-R32, is proposed based on NRI-MD sampling. This work provides a possible allosteric mechanism of SHP2, which is helpful for the following design of novel allosteric inhibitors and is expected to be used in clinical synergies with PD-1 monoclonal antibody.

Analysis of coordinated NMR chemical shifts to map allosteric regulatory networks in proteins.

The exquisite sensitivity of the NMR chemical shift to local environment makes it an ideal probe to assess atomic level perturbations in proteins of all sizes and structural compositions. Recent advances in solution and solid-state NMR spectroscopy of biomolecules have leveraged the chemical shift to report on short- and long-range couplings between individual amino acids to establish "networks" of residues that form the basis of allosteric pathways that transmit chemical signals through the protein matrix to induce functional responses. The simple premise that thermodynamically and functionally coupled regions of a protein (i.e. active and allosteric sites) should be reciprocally sensitive to structural or dynamic perturbations has enabled NMR spectroscopy, the premier method for molecular resolution of protein structural fluctuations, to occupy a place at the forefront of investigations into protein allostery. Here, we detail several key methods of NMR chemical shift analysis to extract mechanistic information about long-range chemical signaling in a protein, focusing on practical methodological aspects and the circumstances under which a given approach would be relevant. We also detail some of the experimental considerations that should be made when applying these methods to specific protein systems.

Functional insight into Cordyceps militaris sugar transporters by structure modeling, network analysis and allosteric regulation.

Insights into the structures, functions and dynamics of Cordyceps militaris (C. militaris) sugar transporters are necessary for understanding their versatile metabolic capability for fungal growth. The sequence-function relationship study of 85 C. militaris sugar transporters showed that there is a gap between phylogenetic-based subfamily classification and their functions. Beyond protein sequences, structural modeling and principal component analysis of the structural ensemble revealed the different folds of the Car and Org subfamilies. Performing channel detection and network analysis found that the Alp and Hex subfamilies can be specifically distinguished from others by the betweenness of channel residues. Signature dynamics analysis further suggested that the Hex subfamily demonstrates different dynamics, with high flexibility at the H1 region in TM11. Furthermore, the H1 region as an allosteric site was examined by network parameter calculations that guided allosteric pathways between this region and the channel cavity. Together with gene expression data of C. militaris, e.g., Hex06741 in the Hex subfamily, it was promisingly expressed when sugar utilization was altered. This work demonstrates an in silico framework for investigating C. militaris sugar transporters as an example case study of the allosteric activity of the Hex subfamily and can facilitate sugar transporter engineering design that can further optimize the preferable sugar utilization and fermentation process of C. militaris.

Allosteric regulation in STAT3 interdomains is mediated by a rigid core: SH2 domain regulation by CCD in D170A variant.

Signal Transducer and Activator of Transcription 3 (STAT3) plays a crucial role in cancer development and thus is a viable target for cancer treatment. STAT3 functions as a dimer mediated by phosphorylation of the SRC-homology 2 (SH2) domain, a key target for therapeutic drugs. While great efforts have been employed towards the development of compounds that directly target the SH2 domain, no compound has yet been approved by the FDA due to a lack of specificity and pharmacologic efficacy. Studies have shown that allosteric regulation of SH2 via the coiled-coil domain (CCD) is an alternative drug design strategy. Several CCD effectors have been shown to modulate SH2 binding and affinity, and at the time of writing at least one drug candidate has entered phase I clinical trials. However, the mechanism for SH2 regulation via CCD is poorly understood. Here, we investigate structural and dynamic features of STAT3 and compare the wild type to the reduced function variant D170A in order to delineate mechanistic differences and propose allosteric pathways. Molecular dynamics simulations were employed to explore conformational space of STAT3 and the variant, followed by structural, conformation, and dynamic analysis. The trajectories explored show distinctive conformational changes in the SH2 domain for the D170A variant, indicating long range allosteric effects. Multiple analyses provide evidence for long range communication pathways between the two STAT3 domains, which seem to be mediated by a rigid core which connects the CCD and SH2 domains via the linker domain (LD) and transmits conformational changes through a network of short-range interactions. The proposed allosteric mechanism provides new insight into the understanding of intramolecular signaling in STAT3 and potential pharmaceutical control of STAT3 specificity and activity.

Post-translational Modifications of Cyclophilin D Fine-Tune Its Conformational Dynamics and Activity: Implications for Its Mitochondrial Function.

Mitochondria are the powerhouse of a cell, whose disruption due to mitochondrial pore opening can cause cell death, leading to necrosis and many other diseases. The peptidyl-prolyl cis-trans isomerase cyclophilin D (CypD) is a key player in the regulation of the mitochondrial pore. The activity of CypD can be modulated by the post-translational modification (PTM). However, the detailed mechanism of this functional modulation is not well understood. Here, we investigate the catalytic mechanism of unmodified and modified CypD by calculating the reaction free energy profiles and characterizing the function-related conformational dynamics using molecular dynamics simulations and associated analyses. Our results show that unmodified and modified CypD considerably lower the isomerization free energy barrier compared to a free peptide substrate, supporting the catalytic activity of CypD in the simulation systems. The unmodified CypD reduces the free energy difference between the cis and trans states of the peptide substrate, suggesting a stronger binding affinity of CypD toward cis, consistent with experiments. In contrast, phosphorylated CypD further stabilizes trans, leading to a lower catalytic rate in the trans-to-cis direction. The differential catalytic activities of the unmodified and phosphorylated CypD are due to a significant shift of the conformational ensemble upon phosphorylation under different functional states. Interestingly, the local flexibility is both reduced and enhanced at distinct regions by phosphorylation, which is explained by a "seesaw" model of flexibility modulation. The allosteric pathway between the phosphorylation site and a distal site displaying substantial conformational changes upon phosphorylation is also identified, which is influenced by the presence of the substrate or the substrate conformation. Similar conclusions are obtained for the acetylation of CypD using the same peptide substrate and the influence of substrate sequence is also examined. Our work may serve as the basis for the understanding of other PTMs and PTM-initiated allosteric regulations in CypD.

Dynamics and Allosteric Information Pathways of Unphosphorylated c-Cbl.

Human c-Cbl is a RING-type ligase and plays a central role in the protein degradation cascade. To elucidate its conformational changes related to substrate binding, we performed molecular dynamics simulations of different variants/states of c-Cbl for a cumulative time of 68 μs. Our simulations demonstrate that before the substrate binds, the RING domain samples a broad set of conformational states at a biologically relevant salt concentration, including the closed, partially open, and fully open states, whereas substrate binding leads to a restricted conformational sampling. Phe378 and the C-terminal region play an essential role in stabilizing the partially open state. To visualize the allosteric signal transmission pathways from the substrate-binding site to the 40 Å apart RING domain and identify the critical residues for allostery, we have created a subgraph from the optimal and suboptimal paths. Redundant paths are seen in the SH2 domain where the substrate binds, while the major bottlenecks are found at the junction between the SH2 domain and the linker helix region as well as that between the SH2 domain and the 4H bundle. These bottlenecks separate the paths into two overall routes. The nodes/residues at the bottlenecks on the subgraph are considered allosteric hot spots. This subgraph approach provides a general tool for network visualization and determination of critical residues for allostery. The structurally and allosterically critical residues identified in our work are testable and would provide valuable insights into the emerging strategies for drug discovery, such as targeted protein degradation.

In Silico Study of the Acquired Resistance Caused by the Secondary Mutations of KRAS G12C Protein Using Long Time Molecular Dynamics Simulation and Markov State Model Analysis.

Kirsten rat sarcoma viral oncogene homolog (KRAS) is a small GTPase protein which plays an important role in the treatment of KRAS mutant cancers. The FDA-approved AMG510 and MRTX849 (phase III clinical trials) are two potent KRASG12C-selective inhibitors that target KRAS G12C. However, the drug resistance caused by the second-site mutation in KRAS has emerged, and the mechanisms of drug resistance at atom level are still unclear. To clarify the mechanisms of drug resistance, we conducted long time molecular dynamics simulations (75 μs in total) to study the structural and energetic features of KRAS G12C and its four drug resistant variants to inhibitors. The combined binding free energy calculation and protein-ligand interaction fingerprint revealed that these second-site mutations indeed caused KRAS to produce different degrees of resistance to AMG510 and MRTX849. Furthermore, Markov State Models and 2D-free energy landscapes analysis revealed the difference in conformational changes of mutated KRAS bound with and without inhibitors. Furthermore, the comparative analysis of these systems showed that there were differences in their allosteric signal pathways. These findings provide the molecular mechanism of drug resistance, which helps to guide novel KRAS G12C inhibitor design to overcome drug resistance.

Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism.

Cytochrome P450 3A4 (CYP3A4) is the main P450 enzyme for drug metabolism and drug-drug interactions (DDIs), as it is involved in the metabolic process of approximately 50% of drugs. A detailed mechanistic elucidation of DDIs mediated by CYP3A4 is commonly believed to be critical for drug optimization and rational use. Here, two typical probes, midazolam (MDZ, substrate) and testosterone (TST, allosteric effector), are used to investigate the molecular mechanism of CYP3A4-mediated heterotropic allosteric interactions, through conventional molecular dynamics (cMD) and well-tempered metadynamics (WT-MTD) simulations. Distance monitoring shows that TST can stably bind in two potential peripheral sites (Site 1 and Site 2) of CYP3A4. The binding of TST at these two sites can induce conformational changes in CYP3A4 flexible loops on the basis of conformational analysis, thereby promoting the transition of the MDZ binding mode and affecting the ratio of MDZ metabolites. According to the results of the residue interaction network, multiple allosteric communication pathways are identified that can provide vivid and applicable insights into the heterotropic allostery of TST on MDZ metabolism. Comparing the regulatory effects and the communication pathways, the allosteric effect caused by TST binding in Site 2 seems to be more pronounced than in Site 1. Our findings could provide a deeper understanding of CYP3A4-mediated heterotropic allostery at the atomic level and would be helpful for rational drug use as well as the design of new allosteric modulators.

Elucidation of Binding Features and Dissociation Pathways of Inhibitors and Modulators in SARS-CoV-2 Main Protease by Multiple Molecular Dynamics Simulations.

COVID-19 can cause different neurological symptoms in some people, including smell, inability to taste, dizziness, confusion, delirium, seizures, stroke, etc. Owing to the issue of vaccine effectiveness, update and coverage, we still need one or more diversified strategies as the backstop to manage illness. Characterizing the structural basis of ligand recognition in the main protease (Mpro) of SARS-CoV-2 will facilitate its rational design and development of potential drug candidates with high affinity and selectivity against COVID-19. Up to date, covalent-, non-covalent inhibitors and allosteric modulators have been reported to bind to different active sites of Mpro. In the present work, we applied the molecular dynamics (MD) simulations to systematically characterize the potential binding features of catalytic active site and allosteric binding sites in Mpro using a dataset of 163 3D structures of Mpro-inhibitor complexes, in which our results are consistent with the current studies. In addition, umbrella sampling (US) simulations were used to explore the dissociation processes of substrate pathway and allosteric pathway. All the information provided new insights into the protein features of Mpro and will facilitate its rational drug design for COVID-19.

A new approach for extracting information from protein dynamics.

Increased ability to predict protein structures is moving research focus towards understanding protein dynamics. A promising approach is to represent protein dynamics through networks and take advantage of well-developed methods from network science. Most studies build protein dynamics networks from correlation measures, an approach that only works under very specific conditions, instead of the more robust inverse approach. Thus, we apply the inverse approach to the dynamics of protein dihedral angles, a system of internal coordinates, to avoid structural alignment. Using the well-characterized adhesion protein, FimH, we show that our method identifies networks that are physically interpretable, robust, and relevant to the allosteric pathway sites. We further use our approach to detect dynamical differences, despite structural similarity, for Siglec-8 in the immune system, and the SARS-CoV-2 spike protein. Our study demonstrates that using the inverse approach to extract a network from protein dynamics yields important biophysical insights.