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A recent report by Yun et al. describes the detection of RAS dimers using intact mass spectrometry and investigates the role that membrane lipids, nucleotide state, and binding partners have in their formation., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 The Author. Published by Elsevier Ltd.. All rights reserved.)

The family of Ras-like GTPases consists of over 150 different members, regulated by an even larger number of guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs) that comprise cellular switch networks that govern cell motility, growth, polarity, protein trafficking, and gene expression. Efforts to develop selective small molecule probes and drugs for these proteins have been hampered by the high affinity of guanosine triphosphate (GTP) and lack of allosteric regulatory sites. This paradigm was recently challenged by the discovery of a cryptic allosteric pocket in the switch II region of K-Ras. Here, we ask whether similar pockets are present in GTPases beyond K-Ras. We systematically surveyed members of the Ras, Rho, and Rab family of GTPases and found that many GTPases exhibit targetable switch II pockets. Notable differences in the composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously undruggable family., Competing Interests: Declaration of interests K.M.S., J.M., and L.Z. are inventors on patents owned by University of California, San Francisco, covering GTPase-targeting small molecules. K.M.S. has consulting agreements for the following companies, which involve monetary and/or stock compensation: AperTOR, BioTheryX, BridGene Biosciences, Erasca, Exai, G Protein Therapeutics, Genentech, Initial Therapeutics, Kumquat Biosciences, Kura Oncology, Lyterian, Merck, Montara Therapeutics, Nested, Nextech, Revolution Medicines, Rezo, Totus, Type6 Therapeutics, Vevo, Vicinitas, and Wellspring Biosciences (Araxes Pharma). J.E.B. has consulting agreements for the following companies, which involve monetary and/or stock compensation: Reactive Biosciences, Scorpion Therapeutics, and Olema Oncology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of interests The authors declare no competing interests.

Signaling through the Ras-MAPK pathway can exhibit switch-like activation, which has been attributed to the underlying positive feedback and bimodality in the activation of RasGDP to RasGTP by SOS. SOS contains both catalytic and allosteric Ras binding sites, and a common assumption is that allosteric activation selectively by RasGTP provides the mechanism of positive feedback. However, recent single-molecule studies have revealed that SOS catalytic rates are independent of the nucleotide state of Ras in the allosteric binding site, raising doubt about this as a positive feedback mechanism. Here, we perform detailed kinetic analyses of receptor-mediated recruitment of full-length SOS to the membrane while simultaneously monitoring its catalytic activation of Ras. These results, along with kinetic modeling, expose the autoinhibition release step in SOS, rather than either recruitment or allosteric activation, as the underlying mechanism giving rise to positive feedback in Ras activation., Competing Interests: Declaration of interests All authors declare they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

M-RAS plays a crucial role in the RAF-MEK signaling pathway. When activated by GTP, M-RAS forms a complex with SHOC2 and PP1C, initiating downstream RAF-MEK signal transduction. In this study, the crystal structure of the GDP-bound human M-RAS protein is presented with two forms of crystal packing. Both the full-length and truncated human M-RAS structures aligned well with the high-confidence section of the AlphaFold2-predicted structure with low r.m.s.d., except for the Switch regions. Despite high sequence similarity to the available mouse M-RAS structure, the full-length human M-RAS structure exhibits unique crystal packing. This inactive human M-RAS structure could offer novel insights for the design of selective compounds targeting M-RAS.

RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOS cat ) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOS cat . These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation., Competing Interests: Competing interests statement:The authors declare no competing interest.

RAS proteins are conserved guanosine triphosphate (GTP) hydrolases (GTPases) that act as molecular binary switches and play vital roles in numerous cellular processes. Upon GTP binding, RAS GTPases adopt an active conformation and interact with specific proteins termed RAS effectors that contain a conserved ubiquitin-like domain, thereby facilitating downstream signaling. Over 50 effector proteins have been identified in the human proteome, and many have been studied as potential mediators of RAS-dependent signaling pathways. Biochemical and structural analyses have provided mechanistic insights into these effectors, and studies using model organisms have complemented our understanding of their role in physiology and disease. Yet, many critical aspects regarding the dynamics and biological function of RAS-effector complexes remain to be elucidated. In this review, we discuss the mechanisms and functions of known RAS effector proteins, provide structural perspectives on RAS-effector interactions, evaluate their significance in RAS-mediated signaling, and explore their potential as therapeutic targets., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway).

Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1•RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads., (© 2024. The Author(s).)

Self-assemblies (i.e., nanoclusters) of the RAS GTPase on the membrane act as scaffolds that activate downstream RAF kinases and drive MAPK signaling for cell proliferation and tumorigenesis. However, the mechanistic details of nanoclustering remain largely unknown. Here, size-tunable nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses revealed the structural basis of the cooperative assembly processes of fully processed KRAS, mutated in a quarter of human cancers. The cooperativity is modulated by the mutation and nucleotide states of KRAS and the lipid composition of the membrane. Notably, the oncogenic mutants assemble in nonsequential pathways with two mutually cooperative 'α/α' and 'α/β' interfaces, while α/α dimerization of wild-type KRAS promotes the secondary α/β interaction sequentially. Mutation-based interface engineering was used to selectively trap the oligomeric intermediates of KRAS and probe their favorable interface interactions. Transiently exposed interfaces were available for the assembly. Real-time NMR demonstrated that higher-order oligomers retain higher numbers of active GTP-bound protomers in KRAS GTPase cycling. These data provide a deeper understanding of the nanocluster-enhanced signaling in response to the environment. Furthermore, our methodology is applicable to assemblies of many other membrane GTPases and lipid nanoparticle-based formulations of stable protein oligomers with enhanced cooperativity., (© 2024 Wiley-VCH GmbH.)

Mutations highly affect the structural flexibility of two switch domains in M-RAS considered an important target of anticancer drug design. Gaussian accelerated molecular dynamics (GaMD) simulations were applied to probe the effect of mutations P40D, D41E, and P40D/D41E/L51R on the conformational transition of the switch domains from the GTP-bound M-RAS. The analyses of free energy landscapes (FELs) not only reveal that three mutations induce less energetic states than the wild-type (WT) M-RAS but also verify that the switch domains are extremely disordered. Principal component analysis (PCA) and dynamics analysis suggest that three mutations greatly affect collective motions and structural flexibility of the switch domains that mostly overlap with binding regions of M-RAS to its effectors, which in turn disturbs the activity of M-RAS. The analyses of the interaction network between GTP and M-RAS show that the high instability in hydrogen bonding interactions (HBIs) of GTP with residue 41 and Y42 in the switch domain I drives the disordered states of the switch domains. This work is expected to provide a molecular mechanism for deeply understanding the function of M-RAS and future drug design towards the treatment of cancers.

Background: Mutations in the K-Ras gene are among the most frequent genetic alterations in various cancers, and inhibiting RAS signaling has shown promising results in treating solid tumors. However, finding effective drugs that can bind to the RAS protein remains challenging. This drove us to explore new compounds that could inhibit tumor growth, particularly in cancers that harbor K-Ras mutations., Methods: Our study used bioinformatic techniques such as E-pharmacophore virtual screening, molecular simulation, principal component analysis (PCA), extra precision (XP) docking, and ADMET analyses to identify potential inhibitors for K-Ras mutants G12C and G12D., Results: In our study, we discovered that inhibitors such as afatinib, osimertinib, and hydroxychloroquine strongly inhibit the G12C mutant. Similarly, hydroxyzine, zuclopenthixol, fluphenazine, and doxapram were potent inhibitors for the G12D mutant. Notably, all six of these molecules exhibit a high binding affinity for the H95 cryptic groove present in the mutant structure. These molecules exhibited a unique affinity mechanism at the molecular level, which was further enhanced by hydrophobic interactions. Molecular simulations and PCA revealed the formation of stable complexes within switch regions I and II. This was particularly evident in three complexes: G12C-osimertinib, G12D-fluphenazine, and G12D-zuclopenthixol. Despite the dynamic nature of switches I and II in K-Ras, the interaction of inhibitors remained stable. According to QikProp results, the properties and descriptors of the selected molecules fell within an acceptable range compared to sotorasib., Conclusions: We have successfully identified potential inhibitors of the K-Ras protein, laying the groundwork for the development of targeted therapies for cancers driven by K-Ras mutations., Competing Interests: The authors declare no conflict of interest. As C. George Priya Doss was one of guest editors of the journal, we declare that he had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Milena Georgieva., (© 2023 The Author(s). Published by IMR Press.)

K-Ras nanoclusters (NCs) concentrate all required molecules belonging to the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway in a small area where signaling events take place, increasing efficiency and specificity of signaling. Such nanostructures are characterized by controlled sizes and lifetimes distributions, but there is a poor understanding of the mechanisms involved in their dynamics of growth/decay. Here, a minimum computational model is presented to analyze the behavior of K-Ras NCs as cooperative dynamic structures that self-regulate their growth and decay according to their size. Indeed, the proposed model reveals that the growth and the local production of a K-Ras nanocluster depend positively on its actual size, whilst its lifetime is inversely proportional to the root of its size. The cooperative binding between the structural constituents of the NC (K-Ras proteins) induces oscillations in the size distributions of K-Ras NCs allowing them to range within controlled values, regulating the growth/decay dynamics of these NCs. Thereby, the size of a K-Ras NC is proposed as a key factor to regulate cell signaling, opening a range of possibilities to develop strategies for use in chronic diseases and cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Allostery, the transfer of information between distant parts of a macromolecule, is a fundamental feature of protein function and regulation. However, allosteric mechanisms are usually not explained by protein structure, requiring information on correlated fluctuations uniquely accessible to molecular simulation. Existing work to extract allosteric pathways from molecular dynamics simulations has focused on thermodynamic correlations. Here, we show how kinetic correlations encode complementary information essential to explain observed variations in allosteric regulation. We applied kinetic and thermodynamic correlation analysis on atomistic simulations of H, K, and NRas isoforms in the apo, GTP, and GDP-bound states of Ras protein, with and without complexing to its downstream effector, Raf. We show that switch I and switch II are the primary components of thermodynamic and kinetic allosteric networks, consistent with the key roles of these two motifs. These networks connect the switches to an allosteric loop recently discovered from a crystal structure of HRas. This allosteric loop is inactive in KRas, but is coupled to the hydrolysis arm switch II in NRas and HRas. We find that the mechanism in the latter two isoforms are thermodynamic and kinetic, respectively. Binding of Raf-RBD further activates thermodynamic allostery in HRas and KRas but has limited effect on NRas. These results indicate that kinetic and thermodynamic correlations are both needed to explain protein function and allostery. These two distinct channels of allosteric regulation, and their combinatorial variability, may explain how subtle mutational differences can lead to diverse regulatory profiles among enzymatic proteins., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Ras proteins in the mitogen-activated protein kinase (MAPK) signaling pathway represent one of the most frequently mutated oncogenes in cancer. Ras binds guanosine nucleotides and cycles between active (GTP) and inactive (GDP) conformations to regulate the MAPK signaling pathway. Guanosine and other nucleotides exist in cells as either 2'-hydroxy or 2'-deoxy forms, and imbalances in the deoxyribonucleotide triphosphate pool have been associated with different diseases, such as diabetes, obesity, and cancer. However, the biochemical properties of Ras bound to dGNP are not well understood. Herein, we use native mass spectrometry to monitor the intrinsic GTPase activity of H-Ras and N-Ras oncogenic mutants, revealing that the rate of 2'-deoxy guanosine triphosphate (dGTP) hydrolysis differs compared to the hydroxylated form, in some cases by seven-fold. Moreover, K-Ras expressed from HEK293 cells exhibited a higher than anticipated abundance of dGNP, despite the low abundance of dGNP in cells. Additionally, the GTPase and dGTPase activity of K-Ras G12C was found to be accelerated by 10.2- and 3.8-fold in the presence of small molecule covalent inhibitors, which may open opportunities for the development of Pan-Ras inhibitors. The molecular assemblies formed between H-Ras and N-Ras, including mutant forms, with the catalytic domain of SOS (SOS cat ) were also investigated. The results show that the different mutants of H-Ras and N-Ras not only engage SOS cat differently, but these assemblies are also dependent on the form of guanosine triphosphate bound to Ras. These findings bring to the forefront a new perspective on the nucleotide-dependent biochemical properties of Ras that may have implications for the activation of the MAPK signaling pathway and Ras-driven cancers.

Ras is a central cellular hub protein controlling multiple cell fates. How Ras interacts with a variety of potential effector proteins is relatively unexplored, with only some key effectors characterized in great detail. Here, we have used homology modeling based on X-ray and AlphaFold2 templates to build structural models for 54 Ras-effector complexes. These models were used to estimate binding affinities using a supervised learning regressor. Furthermore, we systematically introduced Ras "branch-pruning" (or branchegetic) mutations to identify 200 interface mutations that affect the binding energy with at least one of the model structures. The impacts of these branchegetic mutants were integrated into a mathematical model to assess the potential for rewiring interactions at the Ras hub on a systems level. These findings have provided a quantitative understanding of Ras-effector interfaces and their impact on systems properties of a key cellular hub., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Motile cells have developed a large array of molecular machineries to actively change their direction of movement in response to spatial cues from their environment. In this process, small GTPases act as molecular switches and work in tandem with regulators and sensors of their guanine nucleotide status (GAP, GEF, GDI and effectors) to dynamically polarize the cell and regulate its motility. In this review, we focus on Myxococcus xanthus as a model organism to elucidate the function of an atypical small Ras GTPase system in the control of directed cell motility. M. xanthus cells direct their motility by reversing their direction of movement through a mechanism involving the redirection of the motility apparatus to the opposite cell pole. The reversal frequency of moving M. xanthus cells is controlled by modular and interconnected protein networks linking the chemosensory-like frizzy (Frz) pathway - that transmits environmental signals - to the downstream Ras-like Mgl polarity control system - that comprises the Ras-like MglA GTPase protein and its regulators. Here, we discuss how variations in the GTPase interactome landscape underlie single-cell decisions and consequently, multicellular patterns., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Ras family GTPases (H/K/N-Ras) modulate numerous effectors, including the lipid kinase PI3K (phosphatidylinositol-3-kinase) that generates growth signal lipid PIP 3 (phosphatidylinositol-3,4,5-triphosphate). Active GTP-Ras binds PI3K with high affinity, thereby stimulating PIP 3 production. We hypothesize the affinity of this binding interaction could be significantly increased or decreased by Ras mutations at PI3K contact positions, with clinical implications since some Ras mutations at PI3K contact positions are disease-linked. To enable tests of this hypothesis, we have developed an approach combining UV spectral deconvolution, HPLC, and microscale thermophoresis to quantify the K D for binding. The approach measures the total Ras concentration, the fraction of Ras in the active state, and the affinity of active Ras binding to its docking site on PI3K Ras binding domain (RBD) in solution. The approach is illustrated by K D measurements for the binding of active H-Ras and representative mutants, each loaded with GTP or GMPPNP, to PI3Kγ RBD. The findings demonstrate that quantitation of the Ras activation state increases the precision of K D measurements, while also revealing that Ras mutations can increase (Q25L), decrease (D38E, Y40C), or have no effect (G13R) on PI3K binding affinity. Significant Ras affinity changes are predicted to alter PI3K regulation and PIP 3 growth signals., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

RAS proteins regulate most aspects of cellular physiology. They are mutated in 30% of human cancers and 4% of developmental disorders termed Rasopathies. They cycle between active GTP-bound and inactive GDP-bound states. When active, they can interact with a wide range of effectors that control fundamental biochemical and biological processes. Emerging evidence suggests that RAS proteins are not simple on/off switches but sophisticated information processing devices that compute cell fate decisions by integrating external and internal cues. A critical component of this compute function is the dynamic regulation of RAS activation and downstream signaling that allows RAS to produce a rich and nuanced spectrum of biological outputs. We discuss recent findings how the dynamics of RAS and its downstream signaling is regulated. Starting from the structural and biochemical properties of wild-type and mutant RAS proteins and their activation cycle, we examine higher molecular assemblies, effector interactions and downstream signaling outputs, all under the aspect of dynamic regulation. We also consider how computational and mathematical modeling approaches contribute to analyze and understand the pleiotropic functions of RAS in health and disease., (© 2023 The Author(s).)

Allosteric mechanisms are pervasive in nature, but human-designed allosteric perturbagens are rare. The history of KRAS G12C inhibitor development suggests that covalent chemistry may be a key to expanding the armamentarium of allosteric inhibitors. In that effort, irreversible targeting of a cysteine converted a non-deal allosteric binding pocket and low affinity ligands into a tractable drugging strategy. Here we examine the feasibility of expanding this approach to other allosteric pockets of RAS and kinase family members, given that both protein families are regulators of vital cellular processes that are often dysregulated in cancer and other human diseases. Moreover, these heavily studied families are the subject of numerous drug development campaigns that have resulted, sometimes serendipitously, in the discovery of allosteric inhibitors. We consequently conducted a comprehensive search for cysteines, a commonly targeted amino acid for covalent drugs, using AlphaFold-generated structures of those families. This new analysis presents potential opportunities for allosteric targeting of validated and understudied drug targets, with an emphasis on cancer therapy., Competing Interests: Competing Interests Statement Kenneth Westover is a member of the Scientific Advisory Board for Vibliome Therapeutics and on the Advosory Panel for Reactive Biosciences. The Westover lab receives or has received research funding from Astellas and Revolution Medicines. None of these relationships are in conflict with the content of this manuscript., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The RAS-RAF pathway is one of the most commonly dysregulated in human cancers 1-3 . Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation 4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer 5 and active RAF dimer 5-8 bound to 14-3-3 9,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF 11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS 11-13 , but the structure of SHOC2 and the architecture of the presumptive SHOC2-PP1C-RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2-PP1C-MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway., (© 2022. The Author(s).)

RAS-MAPK signalling is fundamental for cell proliferation and is altered in most human cancers 1-3 . However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF-MEK1-14-3-3 complexes 4 , the intermediate steps that lead to RAF activation remain unclear. The MRAS-SHOC2-PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF-RAS association 3,5,6 . MRAS, SHOC2 and PP1C are mutated in rasopathies-developmental syndromes caused by aberrant MAPK pathway activation 6-14 -and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)-RAS-driven tumours 15-18 . Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS-SHOC2-PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS-GTP molecules recruit RAF-14-3-3 and SHOC2-PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein-protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS-MAPK signalling, therefore providing the structural basis for therapeutic interventions., (© 2022. The Author(s).)

Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling 1 . SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes 2-5 . However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development ., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

As the hub of major signaling pathways, Ras proteins are implicated in 19% of tumor-caused cancers due to perturbations in their conformational and/or catalytic properties. Despite numerous studies, the functions of the conformational substates for the most important isoform, KRas, remain elusive. In this work, we perform an extensive simulation analysis on the conformational landscape of KRas in its various chemical states during the GTP hydrolysis cycle: the reactant state KRasGTP·Mg 2+ , the intermediate state KRasGDP·Pi·Mg 2+ and the product state KRasGDP·Mg 2+ . The results from enhanced sampling simulations reveal that State 1 of KRasGTP·Mg 2+ has multiple stable substates in solution, one of which might account for interacting with GEFs. State 2 of KRasGTP·Mg 2+ features two substates "Tyr32 in " and "Tyr32 out ", which are poised to interact with effectors and GAPs, respectively. For the intermediate state KRasGDP·Pi·Mg 2+ , Gln61 and Pi are found to assume a broad set of conformations, which might account for the weak oncogenic effect of Gln61 mutations in KRas in contrast to the situation in HRas and NRas. Finally, the product state KRasGDP·Mg 2+ has more than two stable substates in solution, pointing to a conformation-selection mechanism for complexation with GEFs. Based on these results, some specific inhibition strategies for targeting the binding sites of the high-energy substates of KRas during GTP hydrolysis are discussed.

KRAS is the most frequently mutated RAS protein in cancer patients, and it is estimated that about 20% of the cancer patients in the United States carried mutant RAS proteins. To accelerate therapeutic development, structures and dynamics of RAS proteins had been extensively studied by various biophysical techniques for decades. Although 31 P NMR studies revealed population equilibrium of the two major states in the active GMPPNP-bound form, more complex conformational dynamics in RAS proteins and oncogenic mutants subtly modulate the interactions with their downstream effectors. We established a set of customized NMR relaxation dispersion techniques to efficiently and systematically examine the ms-μs conformational dynamics of RAS proteins. This method allowed us to observe varying synchronized motions that connect the effector and allosteric lobes in KRAS. We demonstrated the role of conformational dynamics of KRAS in controlling its interaction with the Ras-binding domain of the downstream effector RAF1, the first kinase in the MAPK pathway. This allows one to explain, as well as to predict, the altered binding affinities of various KRAS mutants, which was neither previously reported nor apparent from the structural perspective.

S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics., (Copyright © 2021. Published by Elsevier Ltd.)

RAS proteins (HRAS, KRAS, NRAS) participate in many physiological signal transduction processes related to cell growth, division, and survival. The RAS proteins are small (188/189 amino acid residues) and they function as GTPases. These proteins toggle between inactive and functional forms; the conversion of inactive RAS-GDP to active RAS-GTP as mediated by guanine nucleotide exchange factors (GEFs) turns the switch on and the intrinsic RAS-GTPase activity stimulated by the GTPase activating proteins (GAPs) turns the switch off. RAS is upstream to the RAS-RAF-MEK-ERK and the PI3-kinase-AKT signaling modules. Importantly, the overall incidence of RAS mutations in all cancers is about 19% and RAS mutants have been a pharmacological target for more than three decades. About 84% of all RAS mutations involve KRAS. Except for the GTP/GDP binding site, the RAS proteins lack other deep surface pockets thereby hindering efforts to identify high-affinity antagonists; thus, they have been considered to be undruggable. KRAS mutations frequently occur in lung, colorectal, and pancreatic cancers, the three most deadly cancers in the United States. Studies within the last decade demonstrated that the covalent modification of KRAS C12, which accounts for about 10% of all RAS mutations, led to the discovery of an adjacent pocket (called the switch II pocket) that accommodated a portion of the drug. This led to the development of sotorasib as a second-line treatment of KRAS G12C -mutant non-small cell lung cancer. Considerable effort also has been expended to develop MAP kinase and PI3-kinase pathway inhibitors as indirect RAS antagonists., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The lipid-anchored RAS (Rat sarcoma) small GTPases (guanosine triphosphate hydrolases) are highly prevalent in human cancer. Traditional strategies of targeting the enzymatic activities of RAS have been shown to be difficult. Alternatively, RAS function and pathology are mostly restricted to nanoclusters on the plasma membrane (PM). Lipids are important structural components of these signaling platforms on the PM. However, how RAS nanoclusters selectively enrich distinct lipids in the PM, how different lipids contribute to RAS signaling and oncogenesis and whether the selective lipid sorting of RAS nanoclusters can be targeted have not been well-understood. Latest advances in quantitative super-resolution imaging and molecular dynamic simulations have allowed detailed characterization RAS/lipid interactions. In this review, we discuss the latest findings on the select lipid composition (with headgroup and acyl chain specificities) within RAS nanoclusters, the specific mechanisms for the select lipid sorting of RAS nanoclusters on the PM and how perturbing lipid compositions within RAS nanoclusters impacts RAS function and pathology. We also describe different strategies of manipulating lipid composition within RAS nanoclusters on the PM.

Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses., (© 2021. The Author(s).)

Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras-Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix-loop-helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations., Competing Interests: The authors declare no competing interest.

To acquire extremely thermostable proteins of given functions is challenging for conventional protein engineering. Here we applied ABACUS, a statistical energy function we developed for de novo amino acid sequence design, to globally redesign a Ras-binding domain (RBD), and obtained an extremely thermostable RBD that unfolds reversibly at above 110°C, the redesigned RBD experimentally confirmed to have expected structure and Ras-binding interface. Directed evolution of the redesigned RBD improved its Ras-binding affinity to the native protein level without excessive loss of thermostability. The designed amino acid substitutions were mostly at the protein surface. For many substitutions, strong epistasis or significantly differentiated effects on thermostability in the native sequence context relative to the redesigned sequence context were observed, suggesting the globally redesigned sequence to be unreachable through combining beneficial mutations of the native sequence. Further analyses revealed that by replacing 38 of a total of 48 non-interfacial surface residues at once, ABACUS redesign was able to globally "invert" the protein's charge distribution pattern in an optimized way. Our study demonstrates that computational protein design provides powerful new tools to solve challenging protein engineering problems., (© 2021 Wiley Periodicals LLC.)

The binding free energy calculation of protein-ligand complexes is necessary for research into virus-host interactions and the relevant applications in drug discovery. However, many current computational methods of such calculations are either inefficient or inaccurate in practice. Utilizing implicit solvent models in the molecular mechanics generalized Born surface area (MM/GBSA) framework allows for efficient calculations without significant loss of accuracy. Here, GBNSR6, a new flavor of the generalized Born model, is employed in the MM/GBSA framework for measuring the binding affinity between SARS-CoV-2 spike protein and the human ACE2 receptor. A computational protocol is developed based on the widely studied Ras-Raf complex, which has similar binding free energy to SARS-CoV-2/ACE2. Two options for representing the dielectric boundary of the complexes are evaluated: one based on the standard Bondi radii and the other based on a newly developed set of atomic radii (OPT1), optimized specifically for protein-ligand binding. Predictions based on the two radii sets provide upper and lower bounds on the experimental references: -14.7(ΔGbindBondi)<-10.6(ΔGbindExp.)<-4.1(ΔGbindOPT1) kcal/mol. The consensus estimates of the two bounds show quantitative agreement with the experiment values. This work also presents a novel truncation method and computational strategies for efficient entropy calculations with normal mode analysis. Interestingly, it is observed that a significant decrease in the number of snapshots does not affect the accuracy of entropy calculation, while it does lower computation time appreciably. The proposed MM/GBSA protocol can be used to study the binding mechanism of new variants of SARS-CoV-2, as well as other relevant structures.

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate to generate a key lipid second messenger, phosphatidylinositol 3,4,5-bisphosphate. PI3Kα and PI3Kγ require activation by RAS proteins to stimulate signaling pathways that control cellular growth, differentiation, motility and survival. Intriguingly, RAS binding to PI3K isoforms likely differ, as RAS mutations have been identified that discriminate between PI3Kα and PI3Kγ, consistent with low sequence homology (23%) between their RAS binding domains (RBDs). As disruption of the RAS/PI3Kα interaction reduces tumor growth in mice with RAS- and epidermal growth factor receptor driven skin and lung cancers, compounds that interfere with this key interaction may prove useful as anti-cancer agents. However, a structure of PI3Kα bound to RAS is lacking, limiting drug discovery efforts. Expression of full-length PI3K isoforms in insect cells has resulted in low yield and variable activity, limiting biophysical and structural studies of RAS/PI3K interactions. This led us to generate the first RBDs from PI3Kα and PI3Kγ that can be expressed at high yield in bacteria and bind to RAS with similar affinity to full-length PI3K. We also solved a 2.31 Å X-ray crystal structure of the PI3Kα-RBD, which aligns well to full-length PI3Kα. Structural differences between the PI3Kα and PI3Kγ RBDs are consistent with differences in thermal stability and may underly differential RAS recognition and RAS-mediated PI3K activation. These high expression, functional PI3K RBDs will aid in interrogating RAS interactions and could aid in identifying inhibitors of this key interaction., Competing Interests: Conflict of Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The many members of the Ras superfamily are small GTPases that serve as molecular switches. These proteins bind the guanine nucleotides GTP and GDP with picomolar affinities, thereby stabilizing on- and off-signaling states, respectively. Quantitative in vitro Ras studies require accurate determination of total protein, its fractional occupancy with guanine nucleotide, and spectroscopic purity. Yet the high nucleotide affinity of Ras and the overlapping UV spectra of the protein and bound nucleotide make such determinations challenging. Here we describe a generalizable UV spectral deconvolution method to analyze the total protein concentration, total nucleotide stoichiometry, and purity of Ras complexes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

RIT1 is a member of the Ras superfamily of small GTPases involved in regulation of cellular signaling. Mutations to RIT1 are involved in cancer and developmental disorders. Like many Ras subfamily members, RIT1 is localized to the plasma membrane. However, RIT1 lacks the C-terminal prenylation that helps many other subfamily members adhere to cellular membranes. We used molecular dynamics simulations to examine the mechanisms by which the C-terminal peptide (CTP) of RIT1 associates with lipid bilayers. We show that the CTP is unstructured and that its membrane interactions depend on lipid composition. While a 12-residue region of the CTP binds strongly to anionic bilayers containing phosphatidylserine lipids, the CTP termini fray from the membrane allowing for accommodation of the RIT1 globular domain at the membrane-water interface., (Published by Elsevier Ltd.)

Cyclorasins 9A5 and 9A54 are 11-mer cyclic peptides that inhibit the Ras-Raf protein interaction. The peptides share a cell-penetrating peptide (CPP)-like motif; however, only cyclorasin 9A5 can permeabilize cells to exhibit strong cell-based activity. To unveil the structural origin underlying their distinct cellular permeabilization activities, we compared the three-dimensional structures of cyclorasins 9A5 and 9A54 in water and in the less polar solvent dimethyl sulfoxide (DMSO) by solution NMR. We found that cyclorasin 9A5 changes its extended conformation in water to a compact amphipathic structure with converged aromatic residues surrounded by Arg residues in DMSO, which might contribute to its cell permeabilization activity. However, cyclorasin 9A54 cannot adopt this amphipathic structure, due to the steric hindrance between two neighboring bulky amino-acid sidechains, Tle-2 and dVal-3. We also found that the bulkiness of the sidechains at positions 2 and 3 negatively affects the cell permeabilization activities, indicating that the conformational plasticity that allows the peptides to form the amphipathic structure is important for their cell permeabilization activities., (© 2021 Wiley-VCH GmbH.)

Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS -derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

KRAS interacts with the inner leaflet of the plasma membrane (PM) using a hybrid anchor that comprises a lysine-rich polybasic domain (PBD) and a C-terminal farnesyl chain. Electrostatic interactions have been envisaged as the primary determinant of interactions between KRAS and membranes. Here, we integrated molecular dynamics (MD) simulations and superresolution spatial analysis in mammalian cells and systematically compared four equally charged KRAS anchors: the wild-type farnesyl hexa-lysine and engineered mutants comprising farnesyl hexa-arginine, geranylgeranyl hexa-lysine, and geranylgeranyl hexa-arginine. MD simulations show that these equally charged KRAS mutant anchors exhibit distinct interactions and packing patterns with different phosphatidylserine (PtdSer) species, indicating that prenylated PBD-bilayer interactions extend beyond electrostatics. Similar observations were apparent in intact cells, where each anchor exhibited binding specificities for PtdSer species with distinct acyl chain compositions. Acyl chain composition determined responsiveness of the spatial organization of different PtdSer species to diverse PM perturbations, including transmembrane potential, cholesterol depletion, and PM curvature. In consequence, the spatial organization and PM binding of each KRAS anchor precisely reflected the behavior of its preferred PtdSer ligand to these same PM perturbations. Taken together these results show that small GTPase PBD-prenyl anchors, such as that of KRAS, have the capacity to encode binding specificity for specific acyl chains as well as lipid headgroups, which allow differential responses to biophysical perturbations that may have biological and signaling consequences for the anchored GTPase., Competing Interests: The authors declare no competing interest.

Intracellular antibodies are valuable tools for target validation studies for clinical situations such as cancer. Recently we have shown that antibodies can be used for drug discovery in screening for chemical compounds surrogates by showing that compounds could be developed to the so-called undruggable RAS protein family. This method, called Antibody-derived compound (Abd) technology, employed intracellular antibodies binding to RAS in a competitive surface plasmon resonance chemical library screen. Success with this method requires a high affinity interaction between the antibody and the target. We now show that reduction in the affinity (dematuration) of the anti-active RAS antibody facilitates the screening of a chemical library using an in vitro AlphaScreen method. This identified active RAS specific-binding Abd compounds that inhibit the RAS-antibody interaction. One compound is shown to be a pan-RAS binder to KRAS, HRAS and NRAS-GTP proteins with a Kd of average 37 mM, offering the possibility of a new chemical series that interacts with RAS in the switch region where the intracellular antibody binds. This simple approach shows the druggability of RAS and is generally applicable to antibody-derived chemical library screening by affording flexibility through simple antibody affinity variation. This approach can be applied to find Abd compounds as surrogates of antibody-combining sites for novel drug development in a range of human diseases.

Identifying the proteins that associate with RAS oncoproteins has great potential, not only to elucidate how these mutant proteins are regulated and signal but also to identify potential therapeutic targets. Here we describe a detailed protocol to employ proximity labeling by the BioID methodology, which has the advantage of capturing weak or transient interactions, to identify in an unbiased manner those proteins within the immediate vicinity of oncogenic RAS proteins.

Small GTPases cycle between active GTP bound and inactive GDP bound forms in live cells. They act as molecular switches and regulate diverse cellular processes at different times and locations in the cell. Spatiotemporal visualization of their activity provides important insights into dynamics of cellular signaling. Conformational sensors for GTPase activity (COSGAs) are based on the conserved GTPase fold and have been used as a versatile approach for imaging small GTPase activity in the cell. Conformational changes upon GDP/GTP binding can be visualized directly in solution, on beads, or in live cells using COSGA by fluorescence lifetime imaging microscopy (FLIM) technique. Herein, we describe the construction of COSGA for imaging K-Ras GTPase activity in live cells.

Zinc oxide (ZnO) NP is considered as a nanoscale chemotherapeutic. Thus, the drug delivery of this inorganic NP is of considerable importance. Ras mutations are common in cancer and the activation of this signaling pathway is a hallmark in carcinoma, melanoma and many other aggressive malignancies. Thus, here we examined the binding and delivery of Ras binding domain (RBD), a model cancer-relevant protein and effector of Ras by ZnO NP. Shifts in zeta potential in water, PBS, DMEM and DMEM supplemented with FBS supported NP interaction to RBD. Fluorescence quenching of the NP was concentration-dependent for RBD, Stern-Volmer analysis of this data was used to estimate binding strength which was significant for ZnO-RBD (Kd < 10-5). ZnO NP interaction to RBD was further confirmed by pull-down assay demonstrated by SDS-PAGE analysis. The ability of ZnO NP to inhibit 3-D tumor spheroid was demonstrated in HeLa cell spheroids-the ZnO NP breaking apart these structures revealing a significant (>50%) zone of killing as shown by light and fluorescence microscopy after intra-vital staining. ZnO 100 nm was superior to ZnO 14 nm in terms of anticancer activity. When bound to ZnO NP, the anticancer activity of RBD was enhanced. These data indicate the potential diagnostic application or therapeutic activity of RBD-NP complexes in vivo which demands further investigation., Competing Interests: The authors have declared that no competing interests exist.

Ras is the most frequently mutated oncogene and recent drug development efforts have spurred significant new research interest. Here we review progress toward understanding how Ras functions in nanoscale, proteo-lipid signaling complexes on the plasma membrane, called nanoclusters. We discuss how G-domain reorientation is plausibly linked to Ras-nanoclustering and -dimerization. We then look at how these mechanistic features could cooperate in the engagement and activation of RAF by Ras. Moreover, we show how this structural information can be integrated with microscopy data that provide nanoscale resolution in cell biological experiments. Synthesizing the available data, we propose to distinguish between two types of Ras nanoclusters, an active, immobile RAF-dependent type and an inactive/neutral membrane anchor-dependent. We conclude that it is possible that Ras reorientation enables dynamic Ras dimerization while the whole Ras/RAF complex transits into an active state. These transient di/oligomer interfaces of Ras may be amenable to pharmacological intervention. We close by highlighting a number of open questions including whether all effectors form active nanoclusters and whether there is an isoform specific composition of Ras nanocluster.

A hallmark of the catalytically essential Walker B motif of P-loop NTPases is the presence of an acidic residue (aspartate/glutamate) for efficient Mg 2+ coordination. Although the Walker B motif has been identified in well-studied examples of P-loop NTPases, its identity is ambiguous in many families, for example, in the prokaryotic small Ras-like GTPase family of MglA. MglA, belonging to TRAFAC class of P-loop NTPases, possesses a threonine at the position equivalent to Walker B aspartate in eukaryotic Ras-like GTPases. To resolve the identity of the Walker B residue in MglA, we carried out a comprehensive analysis of Mg 2+ coordination on P-loop NTPase structures. Atoms in the octahedral coordination of Mg 2+ and their interactions comprise a network including water molecules, Walker A, Walker B and switch motifs of P-loop NTPases. Based on the conserved geometry of Mg 2+ coordination, we confirm that a conserved aspartate functions as the Walker B residue of MglA, and validate it through mutagenesis and biochemical characterization. Location of the newly identified aspartate is spatially equivalent to the Walker B residue of the ASCE division of P-loop NTPases. Furthermore, similar to the allosteric regulation of the Walker B aspartate conformation in MglA, we identify protein families in which large conformational changes involving Walker B motif potentially function as allosteric regulators. The study unravels conserved features of Mg 2+ coordination among divergent families of P-loop NTPases, especially between ancient Ras-like GTPases and ASCE family of ATPases. The conserved geometric features provide a foundation for design of nucleotide-hydrolyzing enzymes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The three human RAS proteins are mutated and constitutively activated in ∼20% of cancers leading to cell growth and proliferation. For the past three decades, many attempts have been made to inhibit these proteins with little success. Recently; however, multiple methods have emerged to inhibit KRAS, the most prevalently mutated isoform. These methods and the underlying biology will be discussed in this review with a special focus on KRAS-plasma membrane interactions., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

RIT1 is a member of the Ras family of GTPases that direct broad cellular physiological responses through tightly controlled signaling networks. The canonical Ras GTPases are well-defined regulators of the RAF/MEK/ERK pathway and mutations in these are pathogenic in cancer and a class of developmental disorders termed RASopathies. Emerging clinical evidences have now demonstrated a role for RIT1 in RASopathies, namely Noonan syndrome, and various cancers including lung adenocarcinoma and myeloid malignancies. While RIT1 has been mostly described in the context of neuronal differentiation and survival, the mechanisms underlying aberrant RIT1-mediated signaling remain elusive. Here, we will review efforts undertaken to characterize the biochemical and functional properties of the RIT1 GTPase at the molecular, cellular, and organismal level, as well as provide a phenotypic overview of different human conditions caused by RIT1 mutations. Deeper understanding of RIT1 biological function and insight to its pathogenic mechanisms are imperative to developing effective therapeutic interventions for patients with RIT1-mutant Noonan syndrome and cancer., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Dysregulation of the Ras oncogene in development causes developmental disorders, "Rasopathies," whereas mutational activation or amplification of Ras in differentiated tissues causes cancer. Rabex-5 (also called RabGEF1) inhibits Ras by promoting Ras mono- and di-ubiquitination. We report here that Rabex-5-mediated Ras ubiquitination requires Ras Tyrosine 4 (Y4), a site of known phosphorylation. Ras substitution mutants insensitive to Y4 phosphorylation did not undergo Rabex-5-mediated ubiquitination in cells and exhibited Ras gain-of-function phenotypes in vivo. Ras Y4 phosphomimic substitution increased Rabex-5-mediated ubiquitination in cells. Y4 phosphomimic substitution in oncogenic Ras blocked the morphological phenotypes associated with oncogenic Ras in vivo dependent on the presence of Rabex-5. We developed polyclonal antibodies raised against an N-terminal Ras peptide phosphorylated at Y4. These anti-phospho-Y4 antibodies showed dramatic recognition of recombinant wild-type Ras and RasG12V proteins when incubated with JAK2 or SRC kinases but not of RasY4F or RasY4F,G12V recombinant proteins suggesting that JAK2 and SRC could promote phosphorylation of Ras proteins at Y4 in vitro. Anti-phospho-Y4 antibodies also showed recognition of RasG12V protein, but not wild-type Ras, when incubated with EGFR. A role for JAK2, SRC, and EGFR (kinases with well-known roles to activate signaling through Ras), to promote Ras Y4 phosphorylation could represent a feedback mechanism to limit Ras activation and thus establish Ras homeostasis. Notably, rare variants of Ras at Y4 have been found in cerebellar glioblastomas. Therefore, our work identifies a physiologically relevant Ras ubiquitination signal and highlights a requirement for Y4 for Ras inhibition by Rabex-5 to maintain Ras pathway homeostasis and to prevent tissue transformation., Competing Interests: The authors have declared that no competing interests exist.

The lack of apparent pockets in the ground conformation of Ras has long challenged the rational design of inhibitors against this oncogenic protein. The sparsely populated, transiently formed state 1 of activated Ras, on the other hand, shows appreciable surface roughness and is increasingly recognized as a potential target for drug discovery. State 1, however, is extremely flexible, and a static structure cannot fully unveil its conformational space that can be exploited for drug design. Here, we present a conformational ensemble of state 1 that was derived using chemical shift-based modeling. The ensemble reveals the intrinsic plasticity of a druggable pocket in state 1 and demonstrates the mechanism of conformational selection for inhibitor recognition. The large set of structural templates in the ensemble, providing a comprehensive description of thermally accessible pocket conformations, is expected to significantly aid the rational design of anti-Ras drugs.

Mutations in the Parkinson's disease (PD)-associated protein leucine-rich repeat kinase 2 (LRRK2) commonly lead to a reduction of GTPase activity and increase in kinase activity. Therefore, strategies for drug development have mainly been focusing on the design of LRRK2 kinase inhibitors. We recently showed that the central RocCOR domains (Roc: Ras of complex proteins; COR: C-terminal of Roc) of a bacterial LRRK2 homolog cycle between a dimeric and monomeric form concomitant with GTP binding and hydrolysis. PD-associated mutations can slow down GTP hydrolysis by stabilizing the protein in its dimeric form. Here, we report the identification of two Nanobodies (NbRoco1 and NbRoco2) that bind the bacterial Roco protein (CtRoco) in a conformation-specific way, with a preference for the GTP-bound state. NbRoco1 considerably increases the GTP turnover rate of CtRoco and reverts the decrease in GTPase activity caused by a PD-analogous mutation. We show that NbRoco1 exerts its effect by allosterically interfering with the CtRoco dimer-monomer cycle through the destabilization of the dimeric form. Hence, we provide the first proof of principle that allosteric modulation of the RocCOR dimer-monomer cycle can alter its GTPase activity, which might present a potential novel strategy to overcome the effect of LRRK2 PD mutations., (© 2020 The Author(s).)