2MSD

NMR data-driven model of GTPase KRas-GNP tethered to a lipid-bilayer nanodisc

Summary for 2MSD

• Entry DOI: 10.2210/pdb2msd/pdb
• Related: 1AV1 2M4A 2M4B 2MSC 2MSE 3GFT 4DSN
• NMR Information: BMRB: 25115
• Descriptor: Apolipoprotein A-I, GTPase KRas, 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE, ... (6 entities in total)
• Functional Keywords: k-ras, nanodisc, pre, docking, lipid binding protein
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 3
• Total formula weight: 131259.77

Authors

Mazhab-Jafari, M.,Stathopoulos, P.,Marshall, C.,Ikura, M. (deposition date: 2014-07-29, release date: 2015-06-03, Last modification date: 2024-05-01)

Primary citation

Mazhab-Jafari, M.T.,Marshall, C.B.,Smith, M.J.,Gasmi-Seabrook, G.M.,Stathopulos, P.B.,Inagaki, F.,Kay, L.E.,Neel, B.G.,Ikura, M.
Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site.
Proc.Natl.Acad.Sci.USA, 112:6625-6630, 2015

PubMed Abstract: 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.

PubMed: 25941399
DOI: 10.1073/pnas.1419895112

Experimental method

SOLUTION NMR