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. 2015 May 26;112(21):6625-30.
doi: 10.1073/pnas.1419895112. Epub 2015 May 4.

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

Mohammad T Mazhab-Jafari  1 , Christopher B Marshall  1 , Matthew J Smith  1 , Geneviève M C Gasmi-Seabrook  1 , Peter B Stathopulos  1 , Fuyuhiko Inagaki  2 , Lewis E Kay  3 , Benjamin G Neel  1 , Mitsuhiko Ikura  4
Affiliations

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

Mohammad T Mazhab-Jafari et al. Proc Natl Acad Sci U S A. .

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.

Keywords: KRAS; Noonan syndrome; lipid bilayer nanodisc; nuclear magnetic resonance; oncogenic mutation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
K-RAS4B signaling at the plasma membrane and the nanodisc lipid bilayer model for NMR studies. (A) Schematic illustration of K-RAS4B signaling on plasma membrane. (Inset) Schematic of a K-RAS4B:nanodisc complex. (B) Distribution of eleven isoleucine Cδ throughout the K-RAS4B GTPase-domain. (Right) 1H-13C HMQC spectra of nanodisc-conjugated K-RAS4B in the GDP- (Upper) and GMPPNP- (Lower) bound forms.
Fig. 2.
Fig. 2.
Activated K-RAS4B adopts an occluded orientation on anionic membranes. (A) PRE-induced broadening of K-RAS4B H(13C) resonances by Gd3+-conjugated lipid incorporated into nanodiscs. PRE effects detected for each isoleucine residue of K-RAS4B presented as ratios of H(13C) resonance intensities of K-RAS4B conjugated to nanodiscs in the presence (I*) to those in the absence (Io) of Gd3+. Residues are grouped according to their location with respect to the α and β membrane interfaces defined below. Error bars based on spectral noise. (B) Map of the PRE effect on K-RAS4B Ile-Cδ. Residues broadened more than 20% are highlighted in red and the remainder in cyan. (C) PRE-driven models of K-RAS4B-nanodisc complexes (see Materials and Methods for details). (D) Surface electrostatics of K-RAS4B-GDP (Left; PDB ID code 4LPK) and K-RAS4B-GMPPNP (Right; modeled from PDB ID code 3GFT). K-RAS4B is oriented to view the occluded interface from the membrane. Blue, positive; red, negative. Surface exposed positive (bold) and negative charged residues are indicated. Increased exposure of R102 and reorientation of E63 contribute to a more positively charged occluded interface in the GMPPNP-bound form. (E) Schematic illustration of K-RAS4B reorientation on GTPase cycling. The α- and β-interfaces are shown with a helix and an arrow, respectively.
Fig. 3.
Fig. 3.
Occluded K-RAS4B orientation is incompatible with RBD binding. (A) Effect of interaction with ARAF-RBD on the PRE profile of nanodisc-tethered K-RAS4B. (Upper) PRE profile of ARAF-RBD Ile-Cδ resonances in the presence of Gd3+-containing (I*) vs. Gd3+-free (Io) nanodisc-tethered K-RAS4B-GMPPNP. (Lower) PRE profile of nanodisc-tethered K-RAS4B-GMPPNP (red) vs. nanodisc-tethered K-RAS4B-GMPPNP in complex with ARAF-RBD (magenta). Residues are grouped according to their location with respect to the α and β membrane interfaces. (B) PRE-based model of nanodisc-tethered K-RAS4B in complex with ARAF-RBD. The K-RAS4B:ARAF-RBD complex was modeled on the basis of homology to H-RAS:C-RAFRBD (PDB ID code 4G0N) (37), and this complex was docked to the bilayer surface based on PRE-derived constraints using HADDOCK. The lowest HADDOCK-scored structure within the major cluster is shown. (C) Schematic illustration of membrane orientations of K-RAS4B complexed with RBDs of ARAF vs. RALGDS.
Fig. 4.
Fig. 4.
K-RAS4B oncogenic and Noonan syndrome mutations relieve the occluded orientation and alter RBD binding kinetics on lipid bilayer. (A) PRE profiles of WT K-RAS4B vs. K5N, D153V, and G12D mutants. 1H-13C HMQC spectra are shown in Fig. S1. Error bars based on spectral noise. Residues are grouped according to their location with respect to the α and β membrane interfaces. (B) Location of mutations and Ile36/Ile139 probes on exposed and occluded K-RAS4B models. (C and D) Relative association rates (kON), dissociation rates (kOFF), and dissociation constants (Kd) for (C) free K-RAS4B and (D) nanodisc-tethered K-RAS4B binding to ARAF-RBD. Binding kinetics of K-RAS4B (analyte) to immobilized ARAF-RBD (ligand) was measured by biolayer interferometry (Fig. S7). Each kON and kOFF rate was determined using three concentrations of K-RAS4B. Error bars represent SD. Kd values were determined from kON and kOFF, and SD was propagated accordingly. (E) Schematic illustration of K-RAS4B reorientation induced by mutations. The mutation locations are shown by cyan spheres and changes in net charge are depicted by ±1. The wavy arrow represents the increased dynamics of the β-interface associated with the state 1 population of the G12D mutant. The curved green arrow represents the reorientation.
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