Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site.
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.
Department of Medical Biophysics, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada M5G 2M9;
Mohammad T
MT
Mazhab-Jafari
Department of Medical Biophysics, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada M5G 2M9;
Christopher B
CB
Marshall
Department of Medical Biophysics, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada M5G 2M9;
Matthew J
MJ
Smith
Department of Medical Biophysics, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada M5G 2M9;
Geneviève M C
GM
Gasmi-Seabrook
Department of Medical Biophysics, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada M5G 2M9;
Peter B
PB
Stathopulos
Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan;
Fuyuhiko
F
Inagaki
Departments of Molecular Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; and Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8.
Lewis E
LE
Kay
Department of Medical Biophysics, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada M5G 2M9;
Benjamin G
BG
Neel
Department of Medical Biophysics, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University of Toronto, Toronto, ON, Canada M5G 2M9; mikura@uhnresearch.ca.
Mitsuhiko
M
Ikura
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D011905:Genes, ras; D006801:Humans; D008051:Lipid Bilayers; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D055672:Static Electricity; D020726:ral Guanine Nucleotide Exchange Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C117307:KRAS protein, human; D008051:Lipid Bilayers; D020726:ral Guanine Nucleotide Exchange Factor; D016283:Proto-Oncogene Proteins p21(ras)
KRAS; Noonan syndrome; lipid bilayer nanodisc; nuclear magnetic resonance; oncogenic mutation