Two hearts beat as one: the debate over RAS dimers continues.

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.

KRas4b-Calmodulin Interaction with Membrane Surfaces: Role of Headgroup, Acyl Chain, and Electrostatics.

KRas4b is a small plasma membrane-bound G-protein that regulates signal transduction pathways. The interaction of KRas4b with the plasma membrane is governed by both its basic C-terminus, which is farnesylated and methylated, and the lipid composition of the membrane itself. The signaling activity of KRas4b is intricately related to its interaction with various binding partners at the plasma membrane, underlining the critical role played by the lipid environment. The calcium-binding protein calmodulin binds farnesylated KRas4b and plays an important role in the dynamic spatial cycle of KRas4b trafficking in the cell. We utilize Biolayer Interferometry to assay the role of lipid headgroup, chain length, and electrostatics in the dissociation kinetics of fully post-translationally modified KRas4b from Nanodisc bilayers with defined lipid compositions. Our results suggest that calmodulin promotes the dissociation of KRas4b from an anionic membrane, with a comparatively slower displacement of KRas4b from PIP2 relative to PS containing bilayers. In addition to this headgroup dependence, KRas4b dissociation appears to be slower from Nanodiscs wherein the lipid composition contains mismatched, unsaturated acyl chains as compared to lipids with a matched acyl chain length. These findings contribute to understanding the role of the lipid composition in the binding of KRas4b and release from lipid bilayers, showing that the overall charge of the bilayer, the identity of the headgroups present, and the length and saturation of the acyl chains play key roles in KRas4b release from the membrane, potentially providing insights in targeting Ras-membrane interactions for therapeutic interventions.

Beyond KRAS(G12C): Biochemical and Computational Characterization of Sotorasib and Adagrasib Binding Specificity and the Critical Role of H95 and Y96.

Mutated KRAS proteins are frequently expressed in some of the most lethal human cancers and thus have been a target of intensive drug discovery efforts for decades. Lately, KRAS(G12C) switch-II pocket (SII-P)-targeting covalent small molecule inhibitors have finally reached clinical practice. Sotorasib (AMG-510) was the first FDA-approved covalent inhibitor to treat KRAS(G12C)-positive nonsmall cell lung cancer (NSCLC), followed soon by adagrasib (MRTX849). Both drugs target the GDP-bound state of KRAS(G12C), exploiting the strong nucleophilicity of acquired cysteine. Here, we evaluate the similarities and differences between sotorasib and adagrasib in their RAS SII-P binding by applying biochemical, cellular, and computational methods. Exact knowledge of SII-P engagement can enable targeting this site by reversible inhibitors for KRAS mutants beyond G12C. We show that adagrasib is strictly KRAS- but not KRAS(G12C)-specific due to its strong and unreplaceable interaction with H95. Unlike adagrasib, sotorasib is less dependent on H95 for its binding, making it a RAS isoform-agnostic compound, having a similar functionality also with NRAS and HRAS G12C mutants. Our results emphasize the accessibility of SII-P beyond oncogenic G12C and aid in understanding the molecular mechanism behind the clinically observed drug resistance, associated especially with secondary mutations on KRAS H95 and Y96.

Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket.

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.

Determining KRAS4B-Targeting Compound Specificity by Top-Down Mass Spectrometry.

We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform. This method allows us to comprehensively evaluate compound specificity, clearly detect nonspecific binding events, and determine the order and frequency with which they occur. We provide two proof-of-concept examples of our method employing publicly available compounds, along with detailed protocols for sample preparation, top-down MS data acquisition, targeted proteoform MS2 fragmentation, and analysis of the resulting data. Our results demonstrate the concentration dependence of KRAS4B-compound engagement and highlight the ability of top-down MS to directly map compound binding location(s) without disrupting the KRAS4B primary structure. Our hope is that this novel method may help accelerate the identification of new successful targeted inhibitors for KRAS4B and other RAS isoforms.

Nucleotide Exchange on RAS Proteins Using Hydrolysable and Non-hydrolysable Nucleotides.

Biochemical and biophysical assays using recombinant RAS require the protein to be in either the active or inactive state. Here we describe methods to exchange the nucleotide present in the purified RAS protein with either GDPßS, GppNHp, or GTP depending on the assay requirement. In addition, we also describe the HPLC method used to validate the exchange process and provide information on the efficiency of the nucleotide exchange.

Progress in Targeting KRAS Directly.

RAS research has entered the world of translational and clinical science. Progress has been based on our appreciation of the role of RAS mutations in different types of cancer and the effects of these mutations on the biochemical, structural, and biophysical properties of the RAS proteins themselves, particularly KRAS, on which most attention has been focused. This knowledge base, while still growing, has enabled creative chemical approaches to targeting KRAS directly. Our understanding of RAS signaling pathways in normal and cancer cells plays an important role for developing RAS inhibitors but also continues to reveal new approaches to targeting RAS through disruption of signaling complexes and downstream pathways.

Affinity Measurement of Non-covalent Interactions of the Covalent KRAS G12C GDP Inhibitor MRTX849 to RAS Isoforms Using Surface Plasmon Resonance.

Surface plasmon resonance (SPR) is an optical effect at an electron-rich surface that enables affinity measurements of biomolecules in real time. It is label free and versatile, not limited to proteins, nucleic acids, and small molecules. SPR is a widely accepted method to measure not only affinity of molecular interactions but also association and dissociation rates of such interactions. In this chapter, we describe a general method to measure the affinity of a small molecule drug, MRTX849, to GDP bound HRAS, KRAS, and NRAS.

Measurement of KRAS-GTPase Activity.

Oncogenic mutations in KRAS typically impact the GAP-mediated and intrinsic GTP hydrolysis activity resulting in elevated levels of cellular KRAS-GTP. The development of biochemical assays for GTPase activity provides an opportunity to quantitatively measure the impact of these mutations on GTP hydrolysis. Here we describe a biochemical assay that measures the release of free phosphate upon hydrolysis of the GTP nucleotide and allows the measurement of intrinsic or GAP-stimulated GTP hydrolysis by KRAS. This assay can be used to measure GTPase activity under single turnover conditions.

KRAS4b:RAF-1 Homogenous Time-Resolved Fluorescence Resonance Energy Transfer Assay for Drug Discovery.

Homogenous time-resolved FRET (HTRF) assays have become one of the most popular tools for pharmaceutical drug screening efforts over the last two decades. Large Stokes shifts and long fluorescent lifetimes of lanthanide chelates lead to robust signal to noise, as well as decreased false positive rates compared to traditional assay techniques. In this chapter, we describe an HTRF protein-protein interaction (PPI) assay for the KRAS4b G-domain in the GppNHp-bound state and the RAF-1-RBD currently used for drug screens. Application of this assay contributes to the identification of lead compounds targeting the GTP-bound active state of K-RAS.

A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement.

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4BG12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site.

Membrane lipids drive formation of KRAS4b-RAF1 RBDCRD nanoclusters on the membrane.

The oncogene RAS, extensively studied for decades, presents persistent gaps in understanding, hindering the development of effective therapeutic strategies due to a lack of precise details on how RAS initiates MAPK signaling with RAF effector proteins at the plasma membrane. Recent advances in X-ray crystallography, cryo-EM, and super-resolution fluorescence microscopy offer structural and spatial insights, yet the molecular mechanisms involving protein-protein and protein-lipid interactions in RAS-mediated signaling require further characterization. This study utilizes single-molecule experimental techniques, nuclear magnetic resonance spectroscopy, and the computational Machine-Learned Modeling Infrastructure (MuMMI) to examine KRAS4b and RAF1 on a biologically relevant lipid bilayer. MuMMI captures long-timescale events while preserving detailed atomic descriptions, providing testable models for experimental validation. Both in vitro and computational studies reveal that RBDCRD binding alters KRAS lateral diffusion on the lipid bilayer, increasing cluster size and decreasing diffusion. RAS and membrane binding cause hydrophobic residues in the CRD region to penetrate the bilayer, stabilizing complexes through ß-strand elongation. These cooperative interactions among lipids, KRAS4b, and RAF1 are proposed as essential for forming nanoclusters, potentially a critical step in MAP kinase signal activation.

Comparative analysis of KRAS4a and KRAS4b splice variants reveals distinctive structural and functional properties.

KRAS, the most frequently mutated oncogene in human cancer, produces two isoforms, KRAS4a and KRAS4b, through alternative splicing. These isoforms differ in exon 4, which encodes the final 15 residues of the G-domain and hypervariable regions (HVRs), vital for trafficking and membrane localization. While KRAS4b has been extensively studied, KRAS4a has been largely overlooked. Our multidisciplinary study compared the structural and functional characteristics of KRAS4a and KRAS4b, revealing distinct structural properties and thermal stability. Position 151 influences KRAS4a's thermal stability, while position 153 affects binding to RAF1 CRD protein. Nuclear magnetic resonance analysis identified localized structural differences near sequence variations and provided a solution-state conformational ensemble. Notably, KRAS4a exhibits substantial transcript abundance in bile ducts, liver, and stomach, with transcript levels approaching KRAS4b in the colon and rectum. Functional disparities were observed in full-length KRAS variants, highlighting the impact of HVR variations on interaction with trafficking proteins and downstream effectors like RAF and PI3K within cells.

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR.

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.

Allosteric Regulation of Switch-II Domain Controls KRAS Oncogenicity.

RAS proteins are GTPases that regulate a wide range of cellular processes. RAS activity is dependent on its nucleotide-binding status, which is modulated by guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP). KRAS can be acetylated at lysine 104 (K104), and an acetylation-mimetic mutation of K104 to glutamine (K104Q) attenuates the in vitro-transforming capacity of oncogenic KRAS by interrupting GEF-induced nucleotide exchange. To assess the effect of this mutation in vivo, we used CRISPR-Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional KrasLSL-G12D alleles. Homozygous animals for K104Q were viable, fertile, and arose at the expected Mendelian frequency, indicating that K104Q is not a complete loss-of-function mutation. Consistent with our previous findings from in vitro studies, however, the oncogenic activity of KRASG12D was significantly attenuated by mutation at K104. Biochemical and structural analysis indicated that the G12D and K104Q mutations cooperate to suppress GEF-mediated nucleotide exchange, explaining the preferential effect of K104Q on oncogenic KRAS. Furthermore, K104 functioned in an allosteric network with M72, R73, and G75 on the α2 helix of the switch-II region. Intriguingly, point mutation of glycine 75 to alanine (G75A) also showed a strong negative regulatory effect on KRASG12D. These data demonstrate that lysine at position 104 is critical for the full oncogenic activity of mutant KRAS and suggest that modulating the sites in its allosteric network may provide a unique therapeutic approach in cancers expressing mutant KRAS. SIGNIFICANCE: An allosteric network formed by interaction between lysine 104 and residues in the switch-II domain is required for KRAS oncogenicity, which could be exploited for developing inhibitors of the activated oncoprotein.

Direct Modulators of K-Ras-Membrane Interactions.

Protein-membrane interactions (PMIs) are ubiquitous in cellular signaling. Initial steps of signal transduction cascades often rely on transient and dynamic interactions with the inner plasma membrane leaflet to populate and regulate signaling hotspots. Methods to target and modulate these interactions could yield attractive tool compounds and drug candidates. Here, we demonstrate that the conjugation of a medium-chain lipid tail to the covalent K-Ras(G12C) binder MRTX849 at a solvent-exposed site enables such direct modulation of PMIs. The conjugated lipid tail interacts with the tethered membrane and changes the relative membrane orientation and conformation of K-Ras(G12C), as shown by molecular dynamics (MD) simulation-supported NMR studies. In cells, this PMI modulation restricts the lateral mobility of K-Ras(G12C) and disrupts nanoclusters. The described strategy could be broadly applicable to selectively modulate transient PMIs.

Revealing KRas4b topology on the membrane surface.

KRas4b is a membrane-bound regulatory protein belonging to the family of small GTPases that function as a molecular switch, facilitating signal transduction from activated membrane receptors to intracellular pathways controlling cell growth and proliferation. Oncogenic mutations locking KRas4b in the active GTP state are responsible for nearly 85% of all Ras-driven cancers. Understanding the membrane-bound state of KRas4b is crucial for designing new therapeutic approaches targeting oncogenic KRas-driven signaling pathways. Extensive research demonstrates the significant involvement of the membrane bilayer in Ras-effector interactions, with anionic lipids playing a critical role in determining protein conformations The preferred topology of KRas4b for interacting with signaling partners has been a long-time question. Computational studies suggest a membrane-proximal conformation, while other biophysical methods like neutron reflectivity propose a membrane-distal conformation. To address these gaps, we employed FRET measurements to investigate the conformation of KRas4b. Using fully post-translationally modified KRas4b, we designed a Nanodisc based FRET assay to study KRas4b-membrane interactions. We suggest an extended conformation of KRas4b relative to the membrane surface. Measurement of FRET donor - acceptor distances reveal that a negatively charged membrane surface weakly favors closer association with the membrane surface. Our findings provide insights into the role of anionic lipids in determining the dynamic conformations of KRas4b and shed light on the predominant conformation of its topology on lipid headgroups.

RAS-dependent RAF-MAPK hyperactivation by pathogenic RIT1 is a therapeutic target in Noonan syndrome-associated cardiac hypertrophy.

RIT1 is a RAS guanosine triphosphatase (GTPase) that regulates different aspects of signal transduction and is mutated in lung cancer, leukemia, and in the germline of individuals with Noonan syndrome. Pathogenic RIT1 proteins promote mitogen-activated protein kinase (MAPK) hyperactivation; however, this mechanism remains poorly understood. Here, we show that RAF kinases are direct effectors of membrane-bound mutant RIT1 necessary for MAPK activation. We identify critical residues in RIT1 that facilitate interaction with membrane lipids and show that these are necessary for association with RAF kinases and MAPK activation. Although mutant RIT1 binds to RAF kinases directly, it fails to activate MAPK signaling in the absence of classical RAS proteins. Consistent with aberrant RAF/MAPK activation as a driver of disease, we show that pathway inhibition alleviates cardiac hypertrophy in a mouse model of RIT1 mutant Noonan syndrome. These data shed light on the function of pathogenic RIT1 and identify avenues for therapeutic intervention.

Machine Learning-Driven Multiscale Modeling: Bridging the Scales with a Next-Generation Simulation Infrastructure.

Interdependence across time and length scales is common in biology, where atomic interactions can impact larger-scale phenomenon. Such dependence is especially true for a well-known cancer signaling pathway, where the membrane-bound RAS protein binds an effector protein called RAF. To capture the driving forces that bring RAS and RAF (represented as two domains, RBD and CRD) together on the plasma membrane, simulations with the ability to calculate atomic detail while having long time and large length- scales are needed. The Multiscale Machine-Learned Modeling Infrastructure (MuMMI) is able to resolve RAS/RAF protein-membrane interactions that identify specific lipid-protein fingerprints that enhance protein orientations viable for effector binding. MuMMI is a fully automated, ensemble-based multiscale approach connecting three resolution scales: (1) the coarsest scale is a continuum model able to simulate milliseconds of time for a 1 µm2 membrane, (2) the middle scale is a coarse-grained (CG) Martini bead model to explore protein-lipid interactions, and (3) the finest scale is an all-atom (AA) model capturing specific interactions between lipids and proteins. MuMMI dynamically couples adjacent scales in a pairwise manner using machine learning (ML). The dynamic coupling allows for better sampling of the refined scale from the adjacent coarse scale (forward) and on-the-fly feedback to improve the fidelity of the coarser scale from the adjacent refined scale (backward). MuMMI operates efficiently at any scale, from a few compute nodes to the largest supercomputers in the world, and is generalizable to simulate different systems. As computing resources continue to increase and multiscale methods continue to advance, fully automated multiscale simulations (like MuMMI) will be commonly used to address complex science questions.