Two Distinct Structures of Membrane-Associated Homodimers of GTP- and GDP-Bound KRAS4B Revealed by Paramagnetic Relaxation Enhancement

Abstract

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.

Keywords: KRAS; NMR spectroscopy; dimerization; membrane proteins.

Figure 1.

Experimental design of nanodisc-based system for paramagnetic relaxation enhancement (PRE) studies of membrane-dependent KRAS dimerization. ¹³C- and ¹⁵N-labeled KRAS is irreversibly attached to the lipid bilayer of a nanodisc by linking Cys185 (the site of farnesylation in native KRAS) to a DOPE head group through maleimide conjugation (MC-KRAS). These nanodiscs are then titrated with isotopically unlabeled, farnesylated and fully processed KRAS (FP-KRAS). PRE spin labels including (i) TEMPO nitroxide tags attached to specific Cys residues of FP-KRAS, (ii) Gd³⁺/Cu²⁺ ions chelated by a DTPA-modified lipid head group, and (iii) Gd-DTPA-BMA in the bulk solvent are used to identify dimerization and membrane interfaces.

Figure 2.

Intermolecular PRE effects between KRAS-GTPγS molecules. (A) Intensity ratios of peaks in the presence of paramagnetic versus diamagnetic (reduced) spin labels (I_para/I_dia) for ILV ¹³C-methyls and Lys ¹⁵N-amides of MC-KRAS-GTPγS in the presence of FP-KRAS-GTPγS (‘homo-dimerization’, black lines) and FP-KRAS-GDP (‘cross-dimerization’, green lines) bearing TEMPO labels at one of Cys118, Cys169, or Cys39. Probes are categorized according to the extent of PRE (I_para/I_dia threshold values < 0.8, moderate, yellow and < 0.6, strong, red). (B) Mapping PRE-affected probes onto the crystal structure of KRAS-GTPγS (PDB ID: 4DSO). Probes that exhibit moderate and strong PREs are colored as in panel (A), and PRE-unaffected probes are gray. Dotted lines represent PRE effects that arise from TEMPO conjugated to Sγ atoms of Cys118 (red) and Cys169 (blue) in the opposing (arbitrarily positioned) FP-KRAS-GTPγS protomer.

Figure 3.

PRE effects induced by membrane-associated and free soluble spin labels on nanodisc-bound MC-KRAS-GTPγS alone and in the presence of FP-KRAS-GTPγS. The I_para/I_dia values of ILV ¹³C-methyls and Lys ¹⁵N-amides of MC-KRAS-GTPγS were measured with nanodiscs containing 5% Gd³⁺- or Cu²⁺-chelated PE-DTPA lipids (as indicated), or with 2 mM Gd-DTPA-BMA in solution, with Lu³⁺ serving as the diamagnetic control. NMR probes that exhibit increased or decreased PRE upon addition of FP-KRAS-GTPγS are color-coded red or blue, respectively, according to changes in ¹H transverse PRE rates of MC-KRAS-GTPγS (see Supporting Figure S7 and S9).

Figure 4.

Structures of membrane-bound KRAS homodimers in the GTPγS-bound (A) and GDP-bound (B) states. Regions that exhibit increased or decreased PRE from membrane or solvent spin labels upon dimerization are indicated, and the switch I and II sites that undergo conformational changes upon GDP/GTP cycling are colored red and yellow, respectively. The right panels highlight the differences in the α4-α5 dimer interfaces between (A) KRAS-GTPγS and (B) KRAS-GDP homodimers, in which the axes of the interfacial α4 and α5 helices are oriented in parallel and perpendicular fashions, respectively.

Figure 5.

Intermolecular interactions at the α4-α5 dimer interface of the KRAS homodimer in the GTPγS-bound (A) and GDP-bound (B) states, with key side chains shown. (C) Validation of an electrostatic interaction between R135 and E168 within the dimer interface of KRAS-GTPγS using charge-reversal mutations. The mutants R135E, E168R, and the double R135E/E168R mutant were prepared, and intermolecular PRE effects for key probes (V45γ and I142δ of KRAS-GTPγS as well as I133δ and I142δ of KRAS-GDP), induced by Cys118 TEMPO-labeled FP-KRAS (same mutant and nucleotide-bound state), were monitored as indicators of dimerization.

Figure 6.

Binding of the monobody NS1 to the α4-α5 interface of KRAS disrupts dimerization. Reductions of intermolecular PRE effects between FP-KRAS molecules in the GTPγS-bound (A) and GDP-bound (B) states induced by titration of NS1. The I_para/I_dia values for several ILVT probes in the α4-α5 region are plotted against the KRAS:NS1 molar ratios (1:0, 1:0.3, and 1:1). “UB” and “B” represent split peaks that correspond to the NS1-unbound and bound forms of FP-KRAS, respectively. (C) Mapping of the probes that exhibit reduced PRE effects upon NS1 addition onto the structure of the KRAS-NS1 complex. These probes, in the GTPγS- and GDP-bound states, are colored in red and blue, respectively, and the superscript “*” represents the probes common to both nucleotide states. ILVT probes that do not exhibit substantial PRE changes upon NS1 addition are colored in dark gray (see Figure S18). The structure of the KRAS (residues 1-172) in complex with NS1 was modeled using the crystal structure of the HRAS-NS1 complex (PDB ID: 5E95).