The crystal structure of RAS was first solved 25 years ago. In spite of tremendous and sustained efforts, there are still no drugs in the clinic that directly target this major driver of human cancers. Recent success in the discovery of compounds that bind RAS and inhibit signaling has fueled renewed enthusiasm, and in-depth understanding of the structure and function of RAS has opened new avenues for direct targeting. To succeed, we must focus on the molecular details of the RAS structure and understand at a high-resolution level how the oncogenic mutants impair function. Structural networks of intramolecular communication between the RAS active site and membraneinteracting regions on the G-domain are disrupted in oncogenic mutants. Although conserved across the isoforms, these networks are near hot spots of protein-ligand interactions with amino acid composition that varies among RAS proteins. These differences could have an effect on stabilization of conformational states of interest in attenuating signaling through RAS. The development of strategies to target these novel sites will add a fresh direction in the quest to conquer RAS-driven cancers.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.

Highlights d CaM exhibits multiple modes of interaction in complex with FME KRas4B 1-185 -GTP d KRas4B-CaM yields a highly populated L-shaped molecular topology with extended CaM d CaM mainly targets the polybasic HVR, sequestering the farnesyl group d The signaling lipids can prevent the departure of KRas4B from the membrane

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

Water and ligand binding play critical roles in the structure and function of proteins, yet their binding sites and significance are difficult to predict a priori. Multiple solvent crystal structures (MSCS) is a method where several X‐ray crystal structures are solved, each in a unique solvent environment, with organic molecules that serve as probes of the protein surface for sites evolved to bind ligands, while the first hydration shell is essentially maintained. When superimposed, these structures contain a vast amount of information regarding hot spots of protein‐protein or protein‐ligand interactions, as well as conserved water‐binding sites retained with the change in solvent properties. Optimized mining of this information requires reliable structural data and a consistent, objective analysis tool. Detection of related solvent positions (DRoP) was developed to automatically organize and rank the water or small organic molecule binding sites within a given set of structures. It is a flexible tool that can also be used in conserved water analysis given multiple structures of any protein independent of the MSCS method. The DRoP output is an HTML format list of the solvent sites ordered by conservation rank in its population within the set of structures, along with renumbered and recolored PDB files for visualization and facile analysis. Here, we present a previously unpublished set of MSCS structures of bovine pancreatic ribonuclease A (RNase A) and use it together with published structures to illustrate the capabilities of DRoP.

The Ras GTPase superfamily of proteins coordinates a diverse set of cellular outcomes, including cell morphology, vesicle transport, and cell proliferation. Primary amino acid sequence analysis has identified Specificity determinant positions (SDPs) that drive diversified functions specific to the Ras, Rho, Rab, and Arf subfamilies (Rojas et al. 2012, J Cell Biol 196:189–201). The inclusion of water molecules in structural and functional adaptation is likely to be a major response to the selection pressures that drive evolution, yet hydration patterns are not included in phylogenetic analysis. This article shows that conserved crystallographic water molecules coevolved with SDP residues in the differentiation of proteins within the Ras superfamily of small GTPases. The patterns of water conservation between protein subfamilies parallel those of sequence‐based evolutionary trees. Thus, hydration patterns have the potential to help elucidate functional significance in the evolution of amino acid residues observed in phylogenetic analysis of homologous proteins. © 2019 Wiley Periodicals, Inc.

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