Allosteric Effects of the Oncogenic RasQ61L Mutant on Raf-RBD

Fetics, Susan; Guterres, Hugo; Kearney, B.; Buhrman, G.; Ma, Buyong; Nussinov, Ruth; Mattos, Carla

doi:10.1016/j.str.2014.12.017

Cited by 221 publications

(299 citation statements)

References 30 publications

(78 reference statements)

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“…Switch II contains the catalytic residue Q61 and is normally highly disordered (11), resulting in a very slow hydrolysis rate in the absence of GAPs (12). We have shown an association between allosteric modulation and intrinsic hydrolysis in the presence of RAF that would be expected to take place independent of GAPs (13)(14)(15)(16). In our current model, the GTP hydrolysis rate is expected to be increased in the RAS/RAF complex by binding of Ca 2þ at a remote allosteric site, resulting in a conformational shift from a catalytically impaired T state (disordered active site) to the competent R state in which the catalytic residue Q61 is ordered in the active site (Fig.…”

Section: Introductionmentioning

confidence: 86%

“…Thus, targeting the site between switch II and helix 3 might inadvertently result in prolonged signaling through RAS-RAF in some situations. Because RAF does not bind switch II (15), a compound at this site would be unlikely to have a significant effect on RAF binding while abrogating hydrolysis of GTP, working against the goal of inhibiting RAS signaling. Although no natural binding partners have been identified to interact with RAS at site 3, importin-b occupies it in RAN GTPase to halt hydrolysis during transport of cargo into the nucleus (65).…”

Section: Binding Pockets On the Allosteric Lobe Of Ras Differ Betweenmentioning

confidence: 99%

“…The G12D and Q61 mutants directly impair the chemistry of GTP hydrolysis by either placing a negative charge right over the nucleotide or removing an important catalytic residue. Furthermore, Q61L is also unique in that it has a strong tendency to stabilize the T state in the anticatalytic conformation when bound to RAF, abolishing even the slow hydrolysis rate observed in the uncomplexed protein (14,15,73). In the absence of binding partners, both switch I and switch II are disordered in solution (11,61), and they become stabilized by interactions with other proteins (15,30,76,77).…”

Section: Effects Of Oncogenic Mutations On Intramolecular Communicatimentioning

confidence: 99%

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Direct Attack on RAS: Intramolecular Communication and Mutation-Specific Effects

Marcus

Mattos

2015

Clinical Cancer Research

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

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Section: Introductionmentioning

confidence: 86%

Section: Binding Pockets On the Allosteric Lobe Of Ras Differ Betweenmentioning

confidence: 99%

Section: Effects Of Oncogenic Mutations On Intramolecular Communicatimentioning

confidence: 99%

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Direct Attack on RAS: Intramolecular Communication and Mutation-Specific Effects

Marcus

Mattos

2015

Clinical Cancer Research

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“…For simulation of K-RAS4B-GDP, isoleucines 24, 46, 55, 100, 139, 142, and 163 were defined as active and residues 23,25,45,47,54,56,99,101,138,140,141,143,162, and 164 were defined as passive. For simulation of K-RAS4B-GMPPNP, isoleucines 24, 36, 46, 55, 100, 139, 142, and 163 were identified as active residues and 23, 25,35,37,45,47,54,56,99,101,138,140,141,143,162, and 164 as passive residues. Surface electrostatics of K-RAS4B were generated by the Poisson-Boltzmann Solver (34)(35)(36).…”

Section: Methodsmentioning

confidence: 99%

“…Surface electrostatics of K-RAS4B were generated by the Poisson-Boltzmann Solver (34)(35)(36). The K-RAS4B:ARAF-RBD complex was modeled by assembling the K-RAS4B model described above with the NMR structure of ARAF-RBD [Protein Data Bank (PDB) ID code 1WXM], guided by the H-RAS:C-RAFRBD crystal structure (PDB ID code 4G0N) (37). In simulations of the complex, isoleucines 24, 36, 46, 55, 100, 139, 142, and 163 of K-RAS4B and isoleucines 54 and 76 of ARAF-RBD were identified as active residues, and the flanking residues were selected as passive.…”

Section: Methodsmentioning

confidence: 99%

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

Mazhab-Jafari

Marshall

Smith

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

203

292

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K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery. KRAS | nuclear magnetic resonance | lipid bilayer nanodisc | oncogenic mutation | Noonan syndrome T he K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) protein product of the KRAS gene undergoes posttranslational farnesylation and C-terminal processing, which, in conjunction with a poly-basic hypervariable region (HVR), targets K-RAS4B to anionic lipid rafts on the intracellular side of the plasma membrane (Fig. 1A) (1). This localization is essential for K-RAS4B function and enhances signaling fidelity (2). Although the significance of membrane tethering of K-RAS4B is well appreciated, a high-resolution map of how K-RAS4B interacts with the membrane is lacking. Because membrane-anchored RAS presents a major challenge to crystallization, current structural insights into the behavior of membrane-anchored RAS have come from a variety of lower-resolution techniques including in vivo FRET-based studies (3), fluorescence and infrared spectroscopic studies (4-6), and in silico models (3). These pioneering studies suggested that the K-RAS4B GTPase domain adopts certain p...

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Allosterism and Drug Discovery

Bell

2021

Burger's Medicinal Chemistry and Drug Discovery

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Interest in allostery and drug development has significantly expanded with increasingly broad ranges of targets and diseases. Experimental and computational approaches have led to advances in understanding of the mechanisms and roles of protein dynamics and conformational states, and come together in the concept of conformational selection. The models of Monod, Wyman, and Changeux and Koshland, Nemethy, and Filmer provide conceptual explanations of homotropic cooperativity and heterotropic allostery, while conformational selection provides realistic detail that can be exploited in drug design. Distinction between allostery and cooperativity and development of approaches to identify cryptic sites on proteins significantly expands the range of targets for allosteric drug design. High‐throughput screening and SAR optimization remain a staple of drug design, however, increased understanding of the thermodynamics of protein–ligand interactions and conformational changes, and the mechanisms of information flow between existing allosteric sites and across subunit interfaces or between allosteric and active sites, rational design of ligands to interact with a cryptic “allosteric” site on any protein becomes possible. Recent “omics” approaches to identify druggable targets both genome wide and in vivo further enhance the range of targets for drug design, both covalent and noncovalent, exploiting allosteric and cooperative properties of proteins.

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Allosteric Effects of the Oncogenic RasQ61L Mutant on Raf-RBD

Cited by 221 publications

References 30 publications

Direct Attack on RAS: Intramolecular Communication and Mutation-Specific Effects

Direct Attack on RAS: Intramolecular Communication and Mutation-Specific Effects

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

Allosterism and Drug Discovery

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