Predicted structural proteome of Sphagnum divinum and proteome-scale annotation.

MOTIVATION: Sphagnum-dominated peatlands store a substantial amount of terrestrial carbon. The genus is undersampled and under-studied. No experimental crystal structure from any Sphagnum species exists in the Protein Data Bank and fewer than 200 Sphagnum-related genes have structural models available in the AlphaFold Protein Structure Database. Tools and resources are needed to help bridge these gaps, and to enable the analysis of other structural proteomes now made possible by accurate structure prediction. RESULTS: We present the predicted structural proteome (25 134 primary transcripts) of Sphagnum divinum computed using AlphaFold, structural alignment results of all high-confidence models against an annotated nonredundant crystallographic database of over 90,000 structures, a structure-based classification of putative Enzyme Commission (EC) numbers across this proteome, and the computational method to perform this proteome-scale structure-based annotation. AVAILABILITY AND IMPLEMENTATION: All data and code are available in public repositories, detailed at https://github.com/BSDExabio/SAFA. The structural models of the S. divinum proteome have been deposited in the ModelArchive repository at https://modelarchive.org/doi/10.5452/ma-ornl-sphdiv.

tinyIFD: A High-Throughput Binding Pose Refinement Workflow Through Induced-Fit Ligand Docking.

A critical step in structure-based drug discovery is predicting whether and how a candidate molecule binds to a model of a therapeutic target. However, substantial protein side chain movements prevent current screening methods, such as docking, from accurately predicting the ligand conformations and require expensive refinements to produce viable candidates. We present the development of a high-throughput and flexible ligand pose refinement workflow, called "tinyIFD". The main features of the workflow include the use of specialized high-throughput, small-system MD simulation code mdgx.cuda and an actively learning model zoo approach. We show the application of this workflow on a large test set of diverse protein targets, achieving 66% and 76% success rates for finding a crystal-like pose within the top-2 and top-5 poses, respectively. We also applied this workflow to the SARS-CoV-2 main protease (Mpro) inhibitors, where we demonstrate the benefit of the active learning aspect in this workflow.

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2.

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M-1 s-1, achieves sub-µM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

Core cysteine residues in the Plasminogen-Apple-Nematode (PAN) domain are critical for HGF/c-MET signaling.

The Plasminogen-Apple-Nematode (PAN) domain, with a core of four to six cysteine residues, is found in > 28,000 proteins across 959 genera. Still, its role in protein function is not fully understood. The PAN domain was initially characterized in numerous proteins, including HGF. Dysregulation of HGF-mediated signaling results in multiple deadly cancers. The binding of HGF to its cell surface receptor, c-MET, triggers all biological impacts. Here, we show that mutating four core cysteine residues in the HGF PAN domain reduces c-MET interaction, subsequent c-MET autophosphorylation, and phosphorylation of its downstream targets, perinuclear localization, cellular internalization of HGF, and its receptor, c-MET, and c-MET ubiquitination. Furthermore, transcriptional activation of HGF/c-MET signaling-related genes involved in cancer progression, invasion, metastasis, and cell survival were impaired. Thus, targeting the PAN domain of HGF may represent a mechanism for selectively regulating the binding and activation of the c-MET pathway.

OpenMDlr: parallel, open-source tools for general protein structure modeling and refinement from pairwise distances.

SUMMARY: Easy-to-use, open-source, general-purpose programs for modeling a protein structure from inter-atomic distances are needed for modeling from experimental data and refinement of predicted protein structures. OpenMDlr is an open-source Python package for modeling protein structures from pairwise distances between any atoms, and optionally, dihedral angles. We provide a user-friendly input format for harnessing modern biomolecular force fields in an easy-to-install package that can efficiently make use of multiple compute cores. AVAILABILITY AND IMPLEMENTATION: OpenMDlr is available at https://github.com/BSDExabio/OpenMDlr-amber. The package is written in Python (versions 3.x). All dependencies are open-source and can be installed with the Conda package management system. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Hit Expansion of a Noncovalent SARS-CoV-2 Main Protease Inhibitor.

Inhibition of the SARS-CoV-2 main protease (Mpro) is a major focus of drug discovery efforts against COVID-19. Here we report a hit expansion of non-covalent inhibitors of Mpro. Starting from a recently discovered scaffold (The COVID Moonshot Consortium. Open Science Discovery of Oral Non-Covalent SARS-CoV-2 Main Protease Inhibitor Therapeutics. bioRxiv 2020.10.29.339317) represented by an isoquinoline series, we searched a database of over a billion compounds using a cheminformatics molecular fingerprinting approach. We identified and tested 48 compounds in enzyme inhibition assays, of which 21 exhibited inhibitory activity above 50% at 20 µM. Among these, four compounds with IC50 values around 1 µM were found. Interestingly, despite the large search space, the isoquinolone motif was conserved in each of these four strongest binders. Room-temperature X-ray structures of co-crystallized protein-inhibitor complexes were determined up to 1.9 Å resolution for two of these compounds as well as one of the stronger inhibitors in the original isoquinoline series, revealing essential interactions with the binding site and water molecules. Molecular dynamics simulations and quantum chemical calculations further elucidate the binding interactions as well as electrostatic effects on ligand binding. The results help explain the strength of this new non-covalent scaffold for Mpro inhibition and inform lead optimization efforts for this series, while demonstrating the effectiveness of a high-throughput computational approach to expanding a pharmacophore library.

Residue-Level Allostery Propagates through the Effective Coarse-Grained Hessian.

The long-ranged coupling between residues that gives rise to allostery in a protein is built up from short-ranged physical interactions. Computational tools used to predict this coupling and its functional relevance have relied on the application of graph theoretical metrics to residue-level correlations measured from all-atom molecular dynamics simulations. The short-ranged interactions that yield these long-ranged residue-level correlations are quantified by the effective coarse-grained Hessian. Here we compute an effective harmonic coarse-grained Hessian from simulations of a benchmark allosteric protein, IGPS, and demonstrate the improved locality of this graph Laplacian over two other connectivity matrices. Additionally, two centrality metrics are developed that indicate the direct and indirect importance of each residue at producing the covariance between the effector binding pocket and the active site. The residue importance indicated by these two metrics is corroborated by previous mutagenesis experiments and leads to unique functional insights; in contrast to previous computational analyses, our results suggest that fP76-hK181 is the most important contact for conveying direct allosteric paths across the HisF-HisH interface. The connectivity around fD98 is found to be important at affecting allostery through indirect means.

RNA-Dependent Structures of the RNA-Binding Loop in the Flavivirus NS3 Helicase.

The flavivirus NS3 protein is a helicase that has pivotal functions during the viral genome replication process, where it unwinds double-stranded RNA and translocates along the nucleic acid polymer in a nucleoside triphosphate hydrolysis-dependent mechanism. Crystallographic and computational studies of the flavivirus NS3 helicase have identified the RNA-binding loop as an interesting structural element that may function as a component of the RNA-enhanced NTPase activity observed for this family of helicases. Microsecond-long unbiased molecular dynamics and extensive replica exchange umbrella sampling simulations of the Zika NS3 helicase have been performed to investigate the RNA dependence of this loop's structural conformations. Specifically, the effect of the bound single-stranded RNA (ssRNA) oligomer on the putative "open" and "closed" conformations of this loop is studied. In the Apo substrate state, the two loop conformations are nearly isoergonic (ΔAO→C = -0.22 kcal mol-1), explaining the structural ambiguity observed in Apo NS3h crystal structures. The bound ssRNA is seen to stabilize the "open" conformation (ΔAO→C = 1.97 kcal mol-1) through direct protein-RNA interactions at the top of the loop. Interestingly, a small ssRNA oligomer bound over 13 Å away from the loop is seen to affect the free energy surface to favor the "open" structure, while minimizing barriers between the two states. Both the mechanism of the "open" to "closed" transition and important residues of the RNA-binding loop structures are characterized. From these results, point mutations that are hypothesized to stabilize the "closed" RNA-binding loop and negatively impact RNA-binding and the RNA-enhanced NTPase activity are posited.

Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft.

The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential "communication hub" for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

Allostery in the dengue virus NS3 helicase: Insights into the NTPase cycle from molecular simulations.

The C-terminus domain of non-structural 3 (NS3) protein of the Flaviviridae viruses (e.g. HCV, dengue, West Nile, Zika) is a nucleotide triphosphatase (NTPase) -dependent superfamily 2 (SF2) helicase that unwinds double-stranded RNA while translocating along the nucleic polymer. Due to these functions, NS3 is an important target for antiviral development yet the biophysics of this enzyme are poorly understood. Microsecond-long molecular dynamic simulations of the dengue NS3 helicase domain are reported from which allosteric effects of RNA and NTPase substrates are observed. The presence of a bound single-stranded RNA catalytically enhances the phosphate hydrolysis reaction by affecting the dynamics and positioning of waters within the hydrolysis active site. Coupled with results from the simulations, electronic structure calculations of the reaction are used to quantify this enhancement to be a 150-fold increase, in qualitative agreement with the experimental enhancement factor of 10-100. Additionally, protein-RNA interactions exhibit NTPase substrate-induced allostery, where the presence of a nucleotide (e.g. ATP or ADP) structurally perturbs residues in direct contact with the phosphodiester backbone of the RNA. Residue-residue network analyses highlight pathways of short ranged interactions that connect the two active sites. These analyses identify motif V as a highly connected region of protein structure through which energy released from either active site is hypothesized to move, thereby inducing the observed allosteric effects. These results lay the foundation for the design of novel allosteric inhibitors of NS3.

Facile hydrolysis and alcoholysis of palladium acetate.

Palladium(II) acetate is readily converted into [Pd3 (µ(2) -OH)(OAc)5 ] (1) in the presence of water in a range of organic solvents and is also slowly converted in the solid state. Complex 1 can also be formed in nominally anhydrous solvents. Similarly, the analogous alkoxide complexes [Pd3 (µ(2) -OR)(OAc)5 ] (3) are easily formed in solutions of palladium(II) acetate containing a range of alcohols. An examination of a representative Wacker-type oxidation shows that the Pd-OH complex 1 and a related Pd-oxo complex 4 can be excluded as potential catalytic intermediates in the absence of exogenous water.