Abstract Activating mutations in KRAS occur at high frequency in colorectal, lung, and pancreatic cancers, which together account for a substantial proportion of global cancer mortality. Mutant KRAS is constitutively biased toward the GTP-bound state, driving persistent proliferative signaling, but simultaneously imposes oncogenic stresses that threaten cellular homeostasis. To sustain transformation, KRAS-mutant cells engage adaptive stress-response mechanisms, many of which converge on translational control mediated by the eIF2-eIF2B axis. While eIF2B is classically known as a guanine nucleotide exchange factor (GEF) for eIF2 during translation initiation, its potential role in directly regulating oncogenic signaling pathways has remained unexplored. Here, we identify a non-canonical function of eIF2B as a direct activator of mutant KRAS signaling. We demonstrate that eIF2B forms a tripartite complex with SOS and mutant KRAS at the plasma membrane (PM), thereby enhancing KRAS activation and tumorigenic signaling. Biochemical assays and structural modeling support an interaction between the catalytic ε subunit of eIF2B and the allosteric Ras-binding site of SOS, stabilizing SOS in an active conformation. This interaction potentiates SOS-mediated GDP/GTP exchange on mutant KRAS and promotes KRAS nanoclustering at the PM. Importantly, eIF2B exhibits marked specificity for mutant KRAS4B, but not KRAS4A, HRAS, or NRAS. This selectivity arises from KRAS4B’s unique polybasic membrane-anchoring domain and from eIF2B-dependent remodeling of plasma membrane lipid composition. eIF2B enhances glycosphingolipid (GSL) biosynthesis, particularly GM3 and SM4, through translational upregulation of B4GALT5, generating a membrane environment that preferentially supports mutant KRAS4B anchoring and signaling. Disruption of GSL synthesis impairs formation of the eIF2B:SOS:KRAS complex and selectively reduces mutant KRAS activation. Notably, eIF2B’s stimulation of mutant KRAS signaling occurs independently of eIF2α phosphorylation, separating its translational stress-response function from its oncogenic signaling role. Functionally, eIF2B promotes tumor growth specifically in KRAS-mutant cancer models, including human xenografts and an autochthonous KRAS G12C-driven lung adenocarcinoma model. Clinically, high expression of eIF2Bε correlates with poorer outcomes in patients with KRAS-mutant tumors. Our findings identify eIF2B as a previously unrecognized regulator of mutant KRAS-driven tumorigenesis that links translational control, membrane lipid remodeling, and oncogenic signaling. By coordinating SOS activation, KRAS membrane nanoclustering, and selective translation, eIF2B emerges as a central modulator of KRAS oncogenic output and a potential therapeutic and prognostic target. In vivo targeting of eIF2B supports its use as a combinatorial strategy with KRAS inhibition to broaden the therapeutic window in KRAS-mutant cancers. This abstract was edited and refined with the assistance of generative artificial intelligence to improve clarity and conciseness. Citation Format: Hyungdong Kim, Shiqi Diao, Kwang-Jin Cho, Hyun-Ro Lee, Junchen Liu, Pascal Egea, Tatu Pantsar, Milla Kurki, Nour Ghaddar, Shuo Wang, Jia Yi Zou, Mehdi Amiri, Ritchel Gannaban, John F. Hancock, Kylie M. Rice, Atsuo Sasaki, John Asara, Brajendra Tripathi, Douglas Lowy, Rosalie Lawrence, Maria Hatzoglou, Carlos R. Azpilcueta-Nicolas, Jean-Philip Lumb, John Columbus, Thomas J. Turbyville, Christopher B. Marshall, Mitsuhiko Ikura, Jay T. Groves, Nahum Sonenberg, Peter Walter, Antonis E. Koromilas. eIF2B Selectively Anchors and Activates Mutant KRAS4B [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr B001.

Acute myeloid leukemia (AML) is a clinically heterogeneous disease. Although the genetic abnormalities associated with poor prognosis are well defined, how they drive unfavorable outcomes remains unclear. Using published gene-expression and dependency datasets, we searched for cell-surface protein-coding genes associated with poor survival and required for AML growth, prioritizing this class of proteins for its accessibility to biologics. This search identified CD59, a GPI-anchored protein with a canonical role in complement regulation, whose high mRNA expression correlates with adverse-risk genetics and stemness signatures. CD59 silencing impaired proliferation across genetically diverse AML cell lines, reduced leukemic burden, and extended survival in cell xenograft models. Moreover, CD59 expression was enriched on leukemic stem cells (LSCs), and its depletion impaired LSC self-renewal and primary AML engraftment in vivo while sparing normal hematopoiesis. Mechanistically, these effects reflected a non-canonical role for CD59 in sustaining Ras-MAPK signaling, whereby its loss depleted inner-leaflet phosphatidylserine and impaired Ras and c-Raf membrane recruitment and activation. rILYd4, a recombinant fragment of the bacterial toxin intermedilysin that binds and degrades CD59, recapitulated these effects and sensitized cells to venetoclax in vivo. These findings reveal CD59 as a critical regulator of Ras-MAPK signaling required for AML growth and nominate its rILYd4-mediated degradation as a therapeutic strategy.

KRAS is frequently mutated in multiple cancers, with the most common mutation being G12D. The recently developed KRASG12D inhibitor MRTX1133 binds a cryptic allosteric pocket near switch II (SII-P), similar to covalent G12C inhibitors, with remarkable picoM non-covalent affinity. Despite its advancement to clinical trials, some aspects of the molecular mechanisms-of-action remain unclear, indicating a need to uncover the mechanisms underlying MRTX1133 efficacy and potential acquired resistance, thus we characterized the biochemical and biophysical outcomes of MRTX1133 binding KRAS. Hydrogen/deuterium exchange experiments showed that MRTX1133 binding to the induced SII-P reduces the overall conformational plasticity of KRASG12D. This extends well beyond SII-P, with the nucleotide-binding regions (P-loop and G-3/4/5-box motifs) particularly exhibiting stabilization. This conformational rigidification by MRTX1133 is coupled with complete arrest of the GTPase cycle: When the compound engages KRASG12D-GDP, both intrinsic and GEF-mediated nucleotide exchange are blocked while engagement of KRASG12D-GTP blocks both intrinsic and GAP-mediated hydrolysis. MRTX1133 attenuates the interaction between activated KRASG12D and the RAS-binding domain of the effector BRAF. The binding site in Switch I remains flexible, which enables binding, albeit with ∼10-fold lower affinity, and remarkably, this interaction with BRAF reverses the compound's blockage of intrinsic GTP hydrolysis. Unlike KRASWT, GDP-loaded KRASG12D surprisingly maintains a low-affinity interaction with BRAF-RBD, but MRTX1133 can circumvent this mutant-specific abnormal interaction. Taken together, MRTX1133 allosterically 'freezes' the KRASG12D nucleotide-binding site conformation, arresting the canonical GTPase cycle of this oncogenic mutant. This provides a framework for understanding the mechanisms-of-action of SII-P-directed inhibitors and how tumours may acquire resistance.

Much is known about how RAS oncoproteins regulate mRNA translation factors, but the reverse relationship, how translation factors influence RAS activity, has remained largely unexplored. At the plasma membrane (PM), Son of Sevenless (SOS) acts as the canonical guanine nucleotide exchange factor (GEF) for RAS proteins, yet mechanisms governing its specificity for individual RAS isoforms remain unknown. Here, we show that the translation initiation factor eIF2B, best known for its GEF function in translation initiation, forms a distinct complex with SOS and mutant KRAS at the PM, but not with other oncogenic RAS variants. Mechanistically, eIF2B acts as an allosteric regulator of SOS, selectively enhancing GDP–GTP exchange on mutant KRAS. This specificity arises from the translational activity of eIF2B, which upregulates glycosphingolipid (GSL) biosynthesis to remodel PM lipids and preferentially anchor mutant KRAS. Together, our results uncover an unexpected moonlighting function of eIF2B: acting both as a direct activator of SOS and as a regulator of GSL pathway that shapes the membrane landscape, both required for mutant KRAS activation. These insights redefine our understanding of eIF2B and mutant KRAS functions in cancer and have profound implications for KRAS-driven oncogenesis. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, PJT-168864 [1]: pending:yes

Mutations in KRAS drive 88% of pancreatic ductal adenocarcinomas (PDAC) and up to 40% of low-grade serous ovarian cancers (LGSOC), making KRAS a long-standing therapeutic target. We previously showed that DIRAS3 binds RAS, forming heterodimers that disrupt RAS clustering and downstream MAPK signaling. Building on this, we developed conformationally constrained DIRAS3-derived peptides using two cyclization strategies and characterized them by NMR and biolayer interferometry. These cyclic peptides attenuate the interaction between KRAS and the BRAF RAS-binding domain, penetrate cells efficiently, and inhibit KRAS nanoclustering on the inner leaflet of the plasma membrane. Functionally, they reduce cell viability and suppress PDAC and LGSOC growth in cell culture and xenograft models. Our findings demonstrate that DIRAS3-based cyclic peptides represent a distinct strategy to directly inhibit oncogenic KRAS signaling and provide a promising framework for therapeutic development in KRAS-driven cancers.

RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface.

The RAS isoforms (KRAS, HRAS and NRAS) have distinct cancer type-specific profiles. NRAS mutations are the second most prevalent RAS mutations in skin and hematological malignancies. Although RAS proteins were considered undruggable for decades, isoform and mutation-specific investigations have produced successful RAS inhibitors that are either specific to certain mutants, isoforms (pan-KRAS) or target all RAS proteins (pan-RAS). While extensive structural and biochemical investigations have focused mainly on K- and H-RAS mutations, NRAS mutations have received less attention, and the most prevalent NRAS mutations in human cancers, Q61K and Q61R, are rare in K- and H-RAS. This manuscript presents a crystal structure of the NRAS Q61K mutant in the GTP-bound form. Our structure reveals a previously unseen pocket near switch II induced by the binding of a ligand to the active form of the protein. This observation reveals a binding site that can potentially be exploited for development of inhibitors against mutant NRAS. Furthermore, the well-resolved catalytic site of this GTPase bound to native GTP provides insight into the stalled GTP hydrolysis observed for NRAS-Q61K.

DiRAS3, also called ARHI, is a RAS (sub)family small GTPase protein that shares 50-60% sequence identity with H-, K-, and N-RAS, with substitutions in key conserved G-box motifs and a unique 34 amino acid extension at its N-terminus. Unlike the RAS proto-oncogenes, DiRAS3 exhibits tumor suppressor properties. DiRAS3 function has been studied through genetics and cell biology, but there has been a lack of understanding of the biochemical and biophysical properties of the protein, likely due to its instability and poor solubility. To overcome this solubility issue, we engineered a DiRAS3 variant (C75S/C80S), which significantly improved soluble protein expression in E. coli. Recombinant DiRAS3 was purified by Ni-NTA and size exclusion chromatography (SEC). Concentration dependence of the SEC chromatogram indicated that DiRAS3 exists in monomer-dimer equilibrium. We then produced truncations of the N-terminal (ΔN) and both (ΔNC) extensions to the GTPase domain. Unlike full-length DiRAS3, the SEC profiles showed that ΔNC is monomeric while ΔN was monomeric with aggregation, suggesting that the N and/or C-terminal tail(s) contribute to dimerization and aggregation. The 1H-15N HSQC NMR spectrum of ΔNC construct displayed well-dispersed peaks similar to spectra of other GTPase domains, which enabled us to demonstrate that DiRAS3 has a GTPase domain that can bind GDP and GTP. Taken together, we conclude that, despite the substitutions in the G-box motifs, DiRAS3 can switch between nucleotide-bound states and that the N- and C-terminal extensions interact transiently with the GTPase domain in intra- and inter-molecular fashions, mediating weak multimerization of this unique small GTPase.

KRAS is a peripheral membrane protein that regulates multiple signaling pathways, and is mutated in ≈30 % of cancers. Transient self-association of KRAS is essential for activation of the downstream effector RAF and oncogenicity. The presence of anionic phosphatidylserine (PS) lipids in the membrane was shown to promote KRAS self-assembly, however, the structural mechanisms remain elusive. Here, we employed nanodisc bilayers of defined lipid compositions, and probed the impact of PS concentration on KRAS self-association. Paramagnetic NMR experiments demonstrated the existence of two transient dimer conformations involving alternate electrostatic contacts between R135 and either D153 or E168 on the "α4/5-α4/5" interface, and revealed that lipid composition and salt modulate their dynamic equilibrium. These dimer interfaces were validated by charge-reversal mutants. This plasticity demonstrates how the dynamic KRAS dimerization interface responds to the environment, and likely extends to the assembly of other signaling complexes on the membrane.

A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.

Ral Guanine Nucleotide Dissociation Stimulator Like 1 (RGL1) is a RAS effector protein that activates Ral GTPase by stimulating nucleotide exchange. Most structures of RAS-effector complexes are for the HRAS isoform; relatively few KRAS-effector structures have been solved, even though KRAS mutations are more frequent in human cancers. We determined crystal structures of KRAS/RGL1-RAS-association (RA) domain complexes and characterized the interaction in solution using nuclear magnetic resonance spectroscopy, size-exclusion chromatography combined with multi-angle light scattering and biolayer interferometry. We report structures of wild-type KRAS and the oncogenic G12V mutant in complex with the RA domain of RGL1 at < 2 angstrom resolution. KRAS(WT)/RGL1-RA crystallized as a 1:1 heterodimer, whilst KRAS(G12V)/RGL1-RA crystallized as a heterotetrameric structure in which RGL1-RA dimerized via domain-swapping the C-terminal beta-strand. Solution data indicated that KRAS(WT) and KRAS(G12V )in complex with RGL1-RA both exist predominantly as 1:1 dimers, while tetramerization occurs through very slow association. Through detailed structural analyses, the distance and angle between RAS alpha 1 helix and RBD/RA alpha 1 helix were found to differ significantly among RAS and RBD/RA complexes. The KRAS/RGL1-RA structures possess some of the largest alpha 1(RAS)/alpha 1(Effector) distances (21.7-22.2 angstrom), whereas the corresponding distances in previously reported RAS/RAF complexes are significantly shorter (15.2-17.7 angstrom). Contact map analysis identified unique structural signatures involving contacts between the beta 1-beta 2 loop of RA and the alpha 1 helix of RAS, clearly distinguishing the KRAS/RGL1-RA (and other RAS/RA complexes) from RAS/RBD complexes. These results demonstrate that RAS effectors employ an assortment of finely-tuned docking surfaces to achieve optimal interactions with RAS. (C) 2022 Elsevier Ltd. All rights reserved.

Despite well-established tumorigenic roles of KRAS mutants, targeting their smooth surfaces was a challenge, which was overcome through the development of G12C-specific covalent inhibitors. A new study shows that optimizing non-covalent interactions with a cryptic pocket produces remarkable potency for another hotspot mutation.

Recent reports suggest that specific KRAS mutations are associated with either resistance or sensitivity of cancer cells to SHP2 inhibitors (SHP2i). However, a lack of understanding of the underlying mechanism(s) is hindering the clinical advancement of SHP2i therapy for cancer. Here we report that cancer cells that harbor KRAS Q61H mutation (found in about 5% of pancreatic ductal adenocarcinoma tumors) or HEK293 cells that over-express KRAS Q61H are resistant to SHP2i. Our biochemical studies supported by all atom molecular dynamic simulations show that the Q61H mutation impairs intrinsic GTP hydrolysis and impedes stimulation of the GTPase cycle by both SOS1 and RASA1, but has negligible impact on binding to BRAF-RBD. Similar to wild-type KRAS, the Q61H mutant can be phosphorylated by Src at Tyr-32 and Tyr-64, and both site scan be dephosphorylated by SHP2, although SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. In vitro, phosphorylation of KRAS Q61H increased intrinsic nucleotide exchange without affecting its insensitivity to SOS1 and RASA1. Phosphorylation of wild-type and SHP2i-sensitive mutants (e.g., G12V) confers resistance to SOS1/RASA1 activities and impairs binding to BRAF-RBD, thus the constitutive resistance to upstream regulation and uncompromised ability of phosphorylated KRAS Q61H to activate MAPK signaling are distinct properties of this mutant. Decoupling of KRAS Q61H from upstream signaling and impaired intrinsic nucleotide hydrolysis lead to a highly GTP-loaded pool that is insensitive to the suppressive effects of Src phosphorylation. While SHP2 plays multiple roles in stimulating RAS signaling, including promoting the GEF function of SOS1, reducing p120 GAP-mediated inactivation of KRAS, and reversing Src phosphorylation of KRAS, we revealed that none of these are required by KRAS Q61H. These insights provide a mechanistic understanding of oncogenic KRAS mutants that can guide clinical trials of SHP2 inhibitors for patients with pancreatic and other cancers bearing KRAS Q61H. Citation Format: Teklab Gebregiworgis, Yoshihito Kano, Michael Ohh, Christopher B. Marshall, Mitsuhiko Ikura. Distinct biochemical properties of KRAS Q61H mutant render cancer cells resistant to SHP2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 670.

KRAS forms transient dimers and higher-order multimers (nanoclusters) on the plasma membrane, which drive MAPK signaling and cell proliferation. KRAS is a frequently mutated oncogene, and while it is well known that the most prevalent mutation, G12D, impairs GTP hydrolysis, thereby increasing KRAS activation, G12D has also been shown to enhance nanoclustering. Elucidating structures of dynamic KRAS assemblies on a membrane has been challenging, thus we have refined our NMR approach that uses nanodiscs to study KRAS associated with membranes. We incorporated paramagnetic relaxation enhancement (PRE) titrations and interface mutagenesis, which revealed that, in addition to the symmetric 'α-α' dimerization interface shared with wild-type KRAS, the G12D mutant also self-associates through an asymmetric 'α-β' interface. The 'α-β' association is dependent on the presence of phosphatidylserine lipids, consistent with previous reports that this lipid promotes KRAS self-assembly on the plasma membrane in cells. Experiments using engineered mutants to spoil each interface, together with PRE probes attached to the membrane or free in solvent, suggest that dimerization through the primary 'α-α' interface releases β interfaces from the membrane promoting formation of the secondary 'α-β' interaction, potentially initiating nanoclustering. In addition, the small molecule BI-2852 binds at a β-β interface, stabilizing a new dimer configuration that outcompetes native dimerization and blocks the effector-binding site. Our data indicate that KRAS self-association involves a delicately balanced conformational equilibrium between transient states, which is sensitive to disease-associated mutation and small molecule inhibitors. The methods developed here are applicable to biologically important transient interactions involving other membrane-associated proteins.

Lung Cancer Driven by BRAF Mutation Is Targetable by EGFR Kinase Inhibitors Ku-Geng Huo, PhD, Hirotsugu Notsuda, MD, PhD, Zhenhao Fang, PhD, Ningdi Feng Liu, BScH, Teklab Gebregiworgis, PhD, Quan Li, PhD, Nhu-An Pham, PhD, Ming Li, MD, Ni Liu, MSc, Frances A. Shepherd, MD, Christopher B. Marshall, PhD, Mitsuhiko Ikura, PhD, Nadeem Moghal, PhD,* Ming-Sound Tsao, MD, FRCPC* Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada Department of Thoracic Surgery Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Rab11 GTPase proteins are required for cytokinesis, ciliogenesis, and lumenogenesis. Rab11a is critical for apical delivery of podocalyxin (PODXL) during lumen formation in epithelial cells. SH3BP5 and SH3BP5L are guanine nucleotide exchange factors (GEFs) for Rab11. We show that SH3BP5 and SH3BP5L are required for activation of Rab11a and cyst lumen formation. Using proximity-dependent biotin identification (BioID) interaction proteomics, we have identified SH3BP5 and its paralogue SH3BP5L as new substrates of the poly-ADP-ribose polymerase Tankyrase and the E3 ligase RNF146. We provide data demonstrating that epithelial polarity via cyst lumen formation is governed by Tankyrase, which inhibits Rab11a activation through the suppression of SH3BP5 and SH3BP5L. RNF146 reduces Tankyrase protein abundance and restores Rab11a activation and lumen formation. Thus, Rab11a activation is controlled by a signaling pathway composed of the sequential inhibition of SH3BP5 paralogues by Tankyrase, which is itself suppressed by RNF146.

Cancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant.

Calmodulin (CaM) is a Ca2+-sensor that regulates a wide variety of target proteins, many of which interact through short basic helical motifs bearing two hydrophobic 'anchor' residues. CaM comprises two globular lobes, each containing a pair of EF-hand Ca2+-binding motifs that form a Ca2+-induced hydrophobic pocket that binds an anchor residue. A central flexible linker allows CaM to accommodate diverse targets. Several reported CaM interactors lack these anchors but contain Lys/Arg-rich polybasic sequences adjacent to a lipidated N- or C-terminus. Ca2+-CaM binds the myristoylated N-terminus of CAP23/NAP22 with intimate interactions between the lipid and a surface comprised of the hydrophobic pockets of both lobes, while the basic residues make electrostatic interactions with the negatively charged surface of CaM. Ca2+-CaM binds farnesylcysteine, derived from the farnesylated polybasic C-terminus of KRAS4b, with the lipid inserted into the C-terminal lobe hydrophobic pocket. CaM sequestration of the KRAS4b farnesyl moiety disrupts KRAS4b membrane association and downstream signaling. Phosphorylation of basic regions of N-/C-terminal lipidated CaM targets can reduce affinity for both CaM and the membrane. Since both N-terminal myristoylated and C-terminal prenylated proteins use a Singly Lipidated Polybasic Terminus (SLIPT) for CaM binding, we propose these polybasic lipopeptide elements comprise a non-canonical CaM-binding motif.

KRAS homo-dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR spectroscopy, and determined distinct structures of membrane-anchored KRAS dimers in the active GTP- and inactive GDP-loaded states. Both dimerize through an α4-α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS-GTP, stabilized by electrostatic interactions between R135 and E168, favors an orientation on the membrane that promotes accessibility of the effector-binding site. Remarkably, "cross"-dimerization between GTP- and GDP-bound KRAS molecules is unfavorable. These models provide a platform to elucidate the structural basis of RAF activation by RAS and to develop inhibitors that can disrupt the KRAS dimerization. The methodology is applicable to many other farnesylated small GTPases.