Mechanistic insights into ligand dissociation from the SARS-CoV-2 spike glycoprotein.

The COVID-19 pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spurred an urgent need for effective therapeutic interventions. The spike glycoprotein of the SARS-CoV-2 is crucial for infiltrating host cells, rendering it a key candidate for drug development. By interacting with the human angiotensin-converting enzyme 2 (ACE2) receptor, the spike initiates the infection of SARS-CoV-2. Linoleate is known to bind the spike glycoprotein, subsequently reducing its interaction with ACE2. However, the detailed mechanisms underlying the protein-ligand interaction remain unclear. In this study, we characterized the pathways of ligand dissociation and the conformational changes associated with the spike glycoprotein by using ligand Gaussian accelerated molecular dynamics (LiGaMD). Our simulations resulted in eight complete ligand dissociation trajectories, unveiling two distinct ligand unbinding pathways. The preference between these two pathways depends on the gate distance between two α-helices in the receptor binding domain (RBD) and the position of the N-linked glycan at N343. Our study also highlights the essential contributions of K417, N121 glycan, and N165 glycan in ligand unbinding, which are equally crucial in enhancing spike-ACE2 binding. We suggest that the presence of the ligand influences the motions of these residues and glycans, consequently reducing accessibility for spike-ACE2 binding. These findings enhance our understanding of ligand dissociation from the spike glycoprotein and offer significant implications for drug design strategies in the battle against COVID-19.

Activation of Polycystin-1 Signaling by Binding of Stalk-derived Peptide Agonists.

Polycystin-1 (PC1) is the membrane protein product of the PKD1 gene whose mutation is responsible for 85% of the cases of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is primarily characterized by the formation of renal cysts and potential kidney failure. PC1 is an atypical G protein-coupled receptor (GPCR) consisting of 11 transmembrane helices and an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal (NTF) and membrane-embedded C-terminal (CTF) fragments. Recently, signaling activation of the PC1 CTF was shown to be regulated by a stalk tethered agonist (TA), a distinct mechanism observed in the adhesion GPCR family. A novel allosteric activation pathway was elucidated for the PC1 CTF through a combination of Gaussian accelerated molecular dynamics (GaMD), mutagenesis and cellular signaling experiments. Here, we show that synthetic, soluble peptides with 7 to 21 residues derived from the stalk TA, in particular, peptides including the first 9 residues (p9), 17 residues (p17) and 21 residues (p21) exhibited the ability to re-activate signaling by a stalkless PC1 CTF mutant in cellular assays. To reveal molecular mechanisms of stalk peptide-mediated signaling activation, we have applied a novel Peptide GaMD (Pep-GaMD) algorithm to elucidate binding conformations of selected stalk peptide agonists p9, p17 and p21 to the stalkless PC1 CTF. The simulations revealed multiple specific binding regions of the stalk peptide agonists to the PC1 protein including an "intermediate" bound yet inactive state. Our Pep-GaMD simulation findings were consistent with the cellular assay experimental data. Binding of peptide agonists to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of the PC1 CTF signaling activation mechanism. Using sequence covariation analysis of PC1 homologs, we further showed that the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. Therefore, structural dynamic insights into the mechanisms of PC1 activation by stalk-derived peptide agonists have enabled an in-depth understanding of PC1 signaling. They will form a foundation for development of PC1 as a therapeutic target for the treatment of ADPKD.

Structural dynamics of chemokine receptors.

Membrane proteins such as G protein-coupled receptors (GPCRs) are involved in awide range of physiological and pathological cellular processes. Binding of extracellular signals to GPCRs, including hormones, neurotransmitters, peptides and proteins, can activate intracellular signaling cascades via G protein interaction. Chemokine receptors are key GPCRs implicated in cancers, immune responses, cell migration and inflammation. Specifically, the CCR5 and CXCR4 chemokine receptors serve as important therapeutic targets against Human Immunodeficiency virus (HIV) entry into human cells. Maraviroc and Vicriviroc, two clinically used HIV entry inhibitors, are antagonists of the CCR5 receptor. These drugs block HIV entry, but ultimately resistance develops, due to emergence of viruses that can utilize the CXCR4 co-receptor. Unfortunately, development of chemokine receptor antagonists as selective drugs of HIV infection has been greatly hindered as their target orthosteric site is conserved among different receptor subtypes. Accordingly, it is important to understand the structural dynamics of these receptors to develop more effective therapeutics. In this chapter, we describe the latest advances in studies of these two key chemokine receptors with respect to their structures, dynamics and function.

Critical Non-Covalent Binding Intermediate for an Allosteric Covalent Inhibitor of SUMO E1.

Post-translational modifications by small ubiquitin-like modifiers (SUMOs) are dysregulated in many types of cancers. The SUMO E1 enzyme has recently been suggested as a new immuno-oncology target. COH000 was recently identified as a highly specific allosteric covalent inhibitor of SUMO E1. However, a marked discrepancy was found between the X-ray structure of the covalent COH000-bound SUMO E1 complex and the available structure-activity relationship (SAR) data of inhibitor analogues due to unresolved noncovalent protein-ligand interactions. Here, we have investigated noncovalent interactions between COH000 and SUMO E1 during inhibitor dissociation through novel Ligand Gaussian accelerated molecular dynamics (LiGaMD) simulations. Our simulations have identified a critical low-energy non-covalent binding intermediate conformation of COH000 that agreed excellently with published and new SAR data of the COH000 analogues, which were otherwise inconsistent with the X-ray structure. Altogether, our biochemical experiments and LiGaMD simulations have uncovered a critical non-covalent binding intermediate during allosteric inhibition of the SUMO E1 complex.

Mechanism of tethered agonist-mediated signaling by polycystin-1.

Polycystin-1 (PC1) is an important unusual G protein-coupled receptor (GPCR) with 11 transmembrane domains, and its mutations account for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 shares multiple characteristics with Adhesion GPCRs. These include a GPCR proteolysis site that autocatalytically divides these proteins into extracellular, N-terminal, and membrane-embedded, C-terminal fragments (CTF), and a tethered agonist (TA) within the N-terminal stalk of the CTF that is suggested to activate signaling. However, the mechanism by which a TA can activate PC1 is not known. Here, we have combined functional cellular signaling experiments of PC1 CTF expression constructs encoding wild type, stalkless, and three different ADPKD stalk variants with all-atom Gaussian accelerated molecular dynamics (GaMD) simulations to investigate TA-mediated signaling activation. Correlations of residue motions and free-energy profiles calculated from the GaMD simulations correlated with the differential signaling abilities of wild type and stalk variants of PC1 CTF. They suggested an allosteric mechanism involving residue interactions connecting the stalk, Tetragonal Opening for Polycystins (TOP) domain, and putative pore loop in TA-mediated activation of PC1 CTF. Key interacting residues such as N3074­S3585 and R3848­E4078 predicted from the GaMD simulations were validated by mutagenesis experiments. Together, these complementary analyses have provided insights into a TA-mediated activation mechanism of PC1 CTF signaling, which will be important for future rational drug design targeting PC1.

Mechanism of Peptide Agonist Binding in CXCR4 Chemokine Receptor.

Chemokine receptors are key G-protein-coupled receptors (GPCRs) that control cell migration in immune system responses, development of cardiovascular and central nervous systems, and numerous diseases. In particular, the CXCR4 chemokine receptor promotes metastasis, tumor growth and angiogenesis in cancers. CXCR4 is also used as one of the two co-receptors for T-tropic HIV-1 entry into host cells. Therefore, CXCR4 serves as an important therapeutic target for treating cancers and HIV infection. Apart from the CXCL12 endogenous peptide agonist, previous studies suggested that the first 17 amino acids of CXCL12 are sufficient to activate CXCR4. Two 17-residue peptides with positions 1-4 mutated to RSVM and ASLW functioned as super and partial agonists of CXCR4, respectively. However, the mechanism of peptide agonist binding in CXCR4 remains unclear. Here, we have investigated this mechanism through all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method. The Pep-GaMD simulations have allowed us to explore representative binding conformations of each peptide and identify critical low-energy states of CXCR4 activated by the super versus partial peptide agonists. Our simulations have provided important mechanistic insights into peptide agonist binding in CXCR4, which are expected to facilitate rational design of new peptide modulators of CXCR4 and other chemokine receptors.

Binding Analysis Using Accelerated Molecular Dynamics Simulations and Future Perspectives.

Biomolecular recognition such as binding of small molecules, nucleic acids, peptides and proteins to their target receptors plays key roles in cellular function and has been targeted for therapeutic drug design. Molecular dynamics (MD) is a computational approach to analyze these binding processes at an atomistic level, which provides valuable understandings of the mechanisms of biomolecular recognition. However, the rather slow biomolecular binding events often present challenges for conventional MD (cMD), due to limited simulation timescales (typically over hundreds of nanoseconds to tens of microseconds). In this regard, enhanced sampling methods, particularly accelerated MD (aMD), have proven useful to bridge the gap and enable all-atom simulations of biomolecular binding events. Here, we will review the recent method developments of Gaussian aMD (GaMD), ligand GaMD (LiGaMD) and peptide GaMD (Pep-GaMD), which have greatly expanded our capabilities to simulate biomolecular binding processes. Spontaneous binding of various biomolecules to their receptors has been successfully simulated by GaMD. Microsecond LiGaMD and Pep-GaMD simulations have captured repetitive binding and dissociation of small-molecule ligands and highly flexible peptides, and thus enabled ligand/peptide binding thermodynamics and kinetics calculations. We will also present relevant application studies in simulations of important drug targets and future perspectives for rational computer-aided drug design.

Gaussian accelerated molecular dynamics (GaMD): principles and applications.

Gaussian accelerated molecular dynamics (GaMD) is a robust computational method for simultaneous unconstrained enhanced sampling and free energy calculations of biomolecules. It works by adding a harmonic boost potential to smooth biomolecular potential energy surface and reduce energy barriers. GaMD greatly accelerates biomolecular simulations by orders of magnitude. Without the need to set predefined reaction coordinates or collective variables, GaMD provides unconstrained enhanced sampling and is advantageous for simulating complex biological processes. The GaMD boost potential exhibits a Gaussian distribution, thereby allowing for energetic reweighting via cumulant expansion to the second order (i.e., "Gaussian approximation"). This leads to accurate reconstruction of free energy landscapes of biomolecules. Hybrid schemes with other enhanced sampling methods, such as the replica exchange GaMD (rex-GaMD) and replica exchange umbrella sampling GaMD (GaREUS), have also been introduced, further improving sampling and free energy calculations. Recently, new "selective GaMD" algorithms including the ligand GaMD (LiGaMD) and peptide GaMD (Pep-GaMD) enabled microsecond simulations to capture repetitive dissociation and binding of small-molecule ligands and highly flexible peptides. The simulations then allowed highly efficient quantitative characterization of the ligand/peptide binding thermodynamics and kinetics. Taken together, GaMD and its innovative variants are applicable to simulate a wide variety of biomolecular dynamics, including protein folding, conformational changes and allostery, ligand binding, peptide binding, protein-protein/nucleic acid/carbohydrate interactions, and carbohydrate/nucleic acid interactions. In this review, we present principles of the GaMD algorithms and recent applications in biomolecular simulations and drug design.

Mechanism of Ligand Recognition by Human ACE2 Receptor.

Angiotensin converting enzyme 2 (ACE2) plays a key role in renin-angiotensin system regulation and amino acid homeostasis. Human ACE2 acts as the receptor for severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2. ACE2 is also widely expressed in epithelial cells of the lungs, heart, kidney, and pancreas. It is considered an important drug target for treating SARS-CoV-2 as well as pulmonary diseases, heart failure, hypertension, renal diseases, and diabetes. Despite the critical importance, the mechanism of ligand binding to the human ACE2 receptor remains unknown. Here, we have addressed this challenge through all-atom simulations using a novel ligand Gaussian accelerated molecular dynamics (LiGaMD) method. Microsecond time scale LiGaMD simulations have unprecedentedly captured multiple times of spontaneous binding and unbinding of a potent inhibitor MLN-4760 in the ACE2 receptor. With ligand far away in the unbound state, the ACE2 receptor samples distinct Open, Partially Open, Closed, and Fully Closed conformations. Upon ligand binding to the active site, conformational ensemble of the ACE2 receptor is biased toward the Closed state as observed in the X-ray experimental structure. The LiGaMD simulations thus suggest a conformational selection mechanism for ligand recognition by the highly flexible ACE2 receptor, which is expected to facilitate rational drug design targeting human ACE2 against coronaviruses and other related human diseases.

Efficient purification and assembly of ribonucleoprotein complex for interaction analysis by MST assay coupled with GaMD simulations.

Here, we describe a generic protocol for monitoring protein-RNA interaction using a cleavable GFP fusion of a recombinant RNA-binding protein. We detail each expression and purification step, including high salt and heparin column for contaminant RNA removal. After the assembly of RNA into the ribonucleoprotein complex, the MicroScale Thermophoresis assay enables the binding affinity to be obtained quickly with a small amount of sample. Further Gaussian accelerated molecular dynamics simulations allow us to analyze protein:RNA interactions in detail. For complete details on the use and execution of this protocol, please refer to Gao et al. (2020).

Mechanism of ligand recognition by human ACE2 receptor.

Angiotensin converting enzyme 2 (ACE2) plays a key role in renin-angiotensin system regulation and amino acid homeostasis. Human ACE2 acts as the receptor for severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2. ACE2 is also widely expressed in epithelial cells of lungs, heart, kidney and pancreas. It is considered an important drug target for treating SARS-CoV-2, as well as pulmonary diseases, heart failure, hypertension, renal diseases and diabetes. Despite the critical importance, the mechanism of ligand binding to the human ACE2 receptor remains unknown. Here, we address this challenge through all-atom simulations using a novel ligand Gaussian accelerated molecular dynamics (LiGaMD) method. Microsecond LiGaMD simulations have successfully captured both binding and unbinding of the MLN-4760 inhibitor in the ACE2 receptor. In the ligand unbound state, the ACE2 receptor samples distinct Open, Partially Open and Closed conformations. Ligand binding biases the receptor conformational ensemble towards the Closed state. The LiGaMD simulations thus suggest a conformational selection mechanism for ligand recognition by the ACE2 receptor. Our simulation findings are expected to facilitate rational drug design of ACE2 against coronaviruses and other related human diseases.

Structure of the Human Respiratory Syncytial Virus M2-1 Protein in Complex with a Short Positive-Sense Gene-End RNA.

The M2-1 protein of human respiratory syncytial virus (HRSV) is a transcription anti-terminator that regulates the processivity of the HRSV RNA-dependent RNA polymerase (RdRP). Here, we report a crystal structure of HRSV M2-1 bound to a short positive-sense gene-end RNA (SH7) at 2.7 Å resolution. We identified multiple critical residues of M2-1 involved in RNA interaction and examined their roles using mutagenesis and MicroScale Thermophoresis (MST) assay. We found that hydrophobic residue Phe23 is indispensable for M2-1 to recognize the base of RNA. We also captured spontaneous binding of RNA (SH7) to M2-1 in all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method. Both experiments and simulations revealed that the interactions of RNA with two separate domains of M2-1, the zinc-binding domain (ZBD) and the core domain (CD), are independent of each other. Collectively, our results provided a structural basis for RNA recognition by HRSV M2-1.

Pathway and mechanism of drug binding to chemokine receptors revealed by accelerated molecular simulations.

Background: Chemokine GPCRs play key roles in biology and medicine. Particularly, CXCR4 promotes cancer metastasis and facilitate HIV entry into host cells. Plerixafor (PLX) is a CXCR4 drug, but the pathway and binding site of PLX in CXCR4 remain unknown. Results & methodology: We have performed molecular docking and all-atom simulations using Gaussian accelerated molecular dynamics (GaMD), which are consistent with previous mutation experiments, suggesting that PLX binds to the orthosteric site of CXCR4 as an antagonist. The GaMD simulations further revealed an intermediate allosteric binding site at the extracellular mouth of CXCR4. Conclusion: The newly identified allosteric site can be targeted for novel drug design targeting CXCR4 and other chemokine receptors.

Known compounds and new lessons: structural and electronic basis of flavonoid-based bioactivities.

Flavonoids correspond to a major class of polyphenolic phytochemicals with flavone as major parent scaffold. This class of compounds is attributed with very rich nutritional as well as therapeutic values. The present study focuses on a panel of 16 flavonoid molecules that are demonstrated to exhibit various bioactivities like anti-angiogenic, anti-inflammatory as well as possess antioxidant potential. The electronic basis of these bioactivities is rarely explored, and structural basis of flavonoid-induced cyclooxygenase (COX) inhibition has still remained an uncharted area. The current report thus focuses on providing an electronic explanation of these bioactivities using density functional theory-based quantum chemical descriptors. We also attempt to provide a structure-activity relation model for COX by inhibition of these 16 flavonoids using molecular docking. Here, we report molecular dynamics data from 16 flavonoid-COX-2 complexes performed for 50 nanoseconds each that demonstrates key structural and dynamic aspects of flavonoid-based COX inhibition in light of observed experimental facts. Interaction analysis and evaluation of side-chain dynamics presented in current study are well in agreement with the empirical study and is hoped to pave new avenues towards design and development of COX-2 selective chemical agents. Abbreviations2'HFN-2'hydroxy flavonone2D2 dimension3D3 dimension3H7MF3-hydroxy-7-methoxy flavone4'HFN-4'hydroxy flavonone4'MF- 4'methoxy flavone7HFN7-hydroxy flavononeCHARMMChemistry at Harvard Macromolecular MechanicsCOXcyclooxygenaseCOX-1cyclooxygenase-1COX-2cyclooxygenase-2DMdipole momentDPPH- 2, 2diphenyl-1-picryl hydrazineEAelectron affinitiesEGFRepidermal growth factor receptorE-HOMOHighest occupied molecular orbital energyE-LUMOLowest unoccupied molecular orbital energyEPAeicosapentaenoic acidFROG2FRee Online druG conformation generationGAGenetic AlgorithmGROMACSGROningen MAchine for Chemical SimulationsHOMOHighest occupied molecular orbitalIPIonization potentialLOMOLowest unoccupied molecular orbitalMDMolecular dynamicsMOMolecular orbitalNAMDNanoscale Molecular DynamicsNSAIDsNon-Steroidal Anti Inflammatory DrugsNsnanosecondsNVEEnsemble-constant-energy, constant-volume, Constant particle ensemblePDB-IDProtein Data Bank IdentifierPMEParticle Mesh EwaldPyRXPython PrescriptionRMSDRoot-Mean-Square DeviationRMSFRoot-Mean-Square FluctuationRLSreactive lipid speciesROSReactive Oxygen SpeciesSASAsolvent accessible surface areaSMILESsimplified molecular-input line-entry systemSORsuperoxide anion radicalUFFUniversal force fieldVEGFvascular endothelial growth factorVEGFRvascular endothelial growth factor receptorVMDVisual molecular dynamicsCommunicated by Ramaswamy H. Sarma.

FlavoDb: a web-based chemical repository of flavonoid compounds.

There are many online resources that focus on chemical diversity of natural compounds, but only handful of resources exist that focus solely on flavonoid compounds and integrate structural and functional properties; however, extensive collated flavonoid literature is still unavailable to scientific community. Here we present an open access database 'FlavoDb' that is focused on providing physicochemical properties as well as topological descriptors that can be effectively implemented in deducing large scale quantitative structure property models of flavonoid compounds. In the current version of database, we present data on 1, 19,400 flavonoid compounds, thereby covering most of the known structural space of flavonoid class of compounds. Moreover, effective structure searching tool presented here is expected to provide an interactive and easy-to-use tool for obtaining flavonoid-based literature and allied information. Data from FlavoDb can be freely accessed via its intuitive graphical user interface made available at following web address: http://bioinfo.net.in/flavodb/home.html.