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.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections

“…The molecular envelopes generated from the SAXS data unequivocally support a dimeric Ras/Raf-RBD structure for the contents of the first major peak eluted from the SEC column ( Fig. 1 and Supplemental Materials Fig S4), with excellent c 2 fits to either the crystallographic dimer (PDB ID 4G0N) 25 or NMR dimer (PDB ID 6W4E) 10 (with Raf-RBD added), both containing the α4-α5…”

“…Furthermore, full-length farnesylated and methylated KRas (KRas-FMe) does not form dimers by itself on supported membranes in vitro 5 . Paradoxically, indication that Ras functions through dimerization appeared in the early days of Ras research 6 and Ras dimers have been detected at low levels in supported membranes [7][8][9] , in nanodiscs 10 , in cells 11 , in solution by NMR 12 and mass spectrometry 13 . Importantly, dimerization has been shown to be essential for signaling through the Ras/Raf/MEK/ERK pathway in cells and in mice 14 .…”

“…Ras clustering on the membrane through these multiple interfaces may result in nanoclusters important for signaling, bringing together effector proteins and other components of the signaling machinery [18][19][20] . While nanoclusters may form through multiple Ras interfaces 18 , signaling through Ras/Raf occurs specifically through Ras dimers involving the α4-α5 interface 10,14 . Furthermore, the dimer of the Ras/Raf-RBD complex by itself is most likely not the full signaling unit, as scaffold proteins such as Galectins interact with Raf-RBD and are required for activation of the Ras/Raf/MEK/ERK pathway 21,22 .…”

Crystal structure reveals the full Ras:Raf interface and advances mechanistic understanding of Raf activation

“…The allosteric connections linking residues D113 at the galectin binding pocket of each Raf-RBD molecule in the dimer, which also includes residue D117 (46) ( Table S2, S3 & S4), leads to a model where Ras/Raf-RBD_CRD dimers couple with galectin dimers to form a higher order macromolecular platform involved in signal amplification and kinetic proofreading ( Interestingly, the location of the Raf-CRD in the context of the Ras/Raf dimer, combined with evidence supporting an approximately perpendicular orientation of helices 3, 4 and 5 with respect to the membrane (38,39), precludes insertion of the Raf-CRD into the membrane. In light of our Ras/Raf-RBD_CRD structure and the increasing evidence that the helix 4/helix 5 Ras dimer is an essential unit for signaling through Ras/Raf/MEK/ERK (34,(38)(39)(40)(41), we must question the presumed membrane-binding function of the Raf-CRD that has been characterized only in the context of monomeric Ras/Raf-RBD_CRD complexes (17,18,20). Instead we suggest that the primary function of previously proposed hydrophobic membrane-binding region, conserved across Raf isoforms, is to stabilize Raf autoinhibition in the absence of Ras as demonstrated in the inactive BRaf/MEK1/14-3-3 cryo-EM structure (27).…”

“…A second dimer model was constructed utilizing a closely related helix 4/helix 5 Ras dimerization interface, recently described for NMR-data driven nanodisc-bound KRas dimers in the absence of Raf (PDB ID 6W4E) ( Fig. S2A) (41). We performed three replicates of molecular dynamics simulations on both models.…”