Conserved interactions required for inhibition of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands' interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds predicted to bind to the protease. Additional screen, using DOCKovalent was carried out on DrugBank library (11,414 experimental and approved drugs) and resulted in 6 covalent compounds. The selected compounds from both screens were tested in vitro by a protease activity inhibition assay. Two compounds showed activity at the 50 µM concentration range. Our analysis and findings can facilitate and focus the development of highly potent inhibitors against SARS-CoV-2 infection.

Blind prediction of interfacial water positions in CAPRI.

We report the first assessment of blind predictions of water positions at protein-protein interfaces, performed as part of the critical assessment of predicted interactions (CAPRI) community-wide experiment. Groups submitting docking predictions for the complex of the DNase domain of colicin E2 and Im2 immunity protein (CAPRI Target 47), were invited to predict the positions of interfacial water molecules using the method of their choice. The predictions-20 groups submitted a total of 195 models-were assessed by measuring the recall fraction of water-mediated protein contacts. Of the 176 high- or medium-quality docking models-a very good docking performance per se-only 44% had a recall fraction above 0.3, and a mere 6% above 0.5. The actual water positions were in general predicted to an accuracy level no better than 1.5 Å, and even in good models about half of the contacts represented false positives. This notwithstanding, three hotspot interface water positions were quite well predicted, and so was one of the water positions that is believed to stabilize the loop that confers specificity in these complexes. Overall the best interface water predictions was achieved by groups that also produced high-quality docking models, indicating that accurate modelling of the protein portion is a determinant factor. The use of established molecular mechanics force fields, coupled to sampling and optimization procedures also seemed to confer an advantage. Insights gained from this analysis should help improve the prediction of protein-water interactions and their role in stabilizing protein complexes.

Symmetry-based self-assembled nanotubes constructed using native protein structures: the key role of flexible linkers.

We construct nanotubes using native protein structures and their native associations from structural databases. The construction is based on a shape-guided symmetric self-assembly concept. Our strategy involves fusing judiciously-selected oligomerization domains via peptide linkers. Linkers are inherently flexible, hence their choice is critical: they should position the domains in three-dimensional space in the desired orientation while retaining their own natural conformational tendencies; however, at the same time, retain the construct stability. Here we outline a design scheme which accounts for linker flexibility considerations, and present two examples. The first is HIV-1 capsid protein, which in vitro self-assembles into nanotubes and conical capsids, and its linker exists as a short flexible loop. The second involves novel nanotubes construction based on antimicrobial homodimer Magainin 2, employing linkers of distinct lengths and flexibility levels. Our strategy utilizes the abundance of unique shapes and sizes of proteins and their building blocks which can assemble into a vast number of combinations, and consequently, nanotubes of distinct morphologies and diameters. Computational design and assessment methodologies can help reduce the number of candidates for experimental validation. This is an invited paper for a special issue on protein dynamics, here focusing on flexibility in nanotube design based on protein building blocks.

Allosteric regulation of glycogen synthase kinase 3ß: a theoretical study.

Glycogen synthase kinase 3ß (GSK-3ß) is a serine-threonine kinase belonging to the CMGC family that plays a key role in many biological processes, such as glucose metabolism, cell cycle regulation, and proliferation. Like most protein kinases, GSK-3ß is regulated via multiple pathways and sites. We performed all-atom molecular dynamics simulations on the unphosphorylated and phosphorylated unbound GSK-3ß and the phosphorylated GSK-3ß bound to a peptide substrate, its product, and a derived inhibitor. We found that GSK-3ß autophosphorylation at residue Tyr(216) results in widening of the catalytic groove, thereby facilitating substrate access. In addition, we studied the interactions of the phosphorylated GSK-3ß with a substrate and peptide inhibitor located at the active site and observed higher affinity of the inhibitor to the kinase. Furthermore, we detected a potential remote binding site which was previously identified in other kinases. In agreement with experiments we observed that binding of specific peptides at this remote site leads to stabilization of the activation loop located in the active site. We speculate that this stabilization could enhance the catalytic activity of the kinase. We point to this remote site as being structurally conserved and suggest that the allosteric phenomenon observed here may occur in the protein kinase superfamily.

Computational validation of protein nanotubes.

We present a novel generic computational method to assess protein nanotubes with variable diameter sizes at the atomic level given their low resolution protomeric structures. The method is based on the symmetrical assembly of a repeating protein subunit into a nanotube. Given the protein unit-cell, and the tube diameter and helicity, we carry out all-atom molecular dynamics simulations, combined with a unique mathematical transformation. This allows us to mimic nanotubes of even huge sizes without end or edge effects. All our simulation setups attempt to explicitly adhere to the conditions under which the experiments were conducted. Thus, we are able to obtain high resolution atomic-scale structures at reasonable computational costs. We expect that our approach would prove useful in assessing protein nanotubes, as well as in silico constructions of novel nanobiomaterials.

Mutational analysis and allosteric effects in the HIV-1 capsid protein carboxyl-terminal dimerization domain.

The carboxyl-terminal domain (CTD, residues 146-231) of the HIV-1 capsid (CA) protein plays an important role in the CA-CA dimerization and viral assembly of the human immunodeficiency virus type 1. Disrupting the native conformation of the CA is essential for blocking viral capsid formation and viral replication. Thus, it is important to identify the exact nature of the structural changes and driving forces of the CTD dimerization that take place in mutant forms. Here, we compare the structural stability, conformational dynamics, and association force of the CTD dimers for both wild-type and mutated sequences using all-atom explicit-solvent molecular dynamics (MD). The simulations show that Q155N and E159D at the major homology region (MHR) and W184A and M185A at the helix 2 region are energetically less favorable than the wild-type, imposing profound negative effects on intermolecular CA-CA dimerization. Detailed structural analysis shows that three mutants (Q155N, E159D, and W184A) display much more flexible local structures and weaker CA-CA association than the wildtype, primarily due to the loss of interactions (hydrogen bonds, side chain hydrophobic contacts, and pi-stacking) with their neighboring residues. Most interestingly, the MHR that is far from the interacting dimeric interface is more sensitive to the mutations than the helix 2 region that is located at the CA-CA dimeric interface, indicating that structural changes in the distinct motif of the CA could similarly allosterically prevent the CA capsid formation. In addition, the structural and free energy comparison of the five residues shorter CA (151-231) dimer with the CA (146-231) dimer further indicates that hydrophobic interactions, side chain packing, and hydrogen bonds are the major, dominant driving forces in stabilizing the CA interface.

Self-assembly of fused homo-oligomers to create nanotubes.

The formation of a nanostructure by self-assembly of a peptide or protein building block depends on the ability of the building block to spontaneously assemble into an ordered structure. We first describe a protocol of fusing homo-oligomer proteins with a given three-dimensional (3D) structure to create new building blocks. According to this protocol, a single monomer A that self-assembles with identical copies to create an oligomer A(1) is covalently linked, through a short linker L, to another monomer B that self-assembles with identical copies to create the oligomer B(j). The result is a fused monomer A-L-B, which has the ability to self-assemble into a nanostructure (A-L-B)(k). We control the self-assembly process of A-L-B by mapping the fused building block onto a planar sheet and wrapping the sheet around a cylinder with the target's dimensions. Finally, we validate the created nanotubes by an optimization procedure. We provide examples of two nanotubes in atomistic model details. One of these has experimental data. In principal, such a protocol should enable the creation of a wide variety of potentially useful protein-based nanotubes with control over their physical and chemical properties.