Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane

Abstract

Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD–CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD–CRD complex. We found that the nucleotide-dependent KRAS–RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD–CRD, and the KRAS:RBD–CRD complex. RBD–CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD β7–8), as well as an alternative interface comprising β6 and the C terminus of CRD and β2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD β7–8 and KRAS α4–α5 while state B involved the C terminus of CRD, β3–5 of RBD, and part of KRAS α5. Notably, α4–α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS–CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD–CRD, and the membrane.

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

“…The position of the CRD observed in our crystal structure is consistent with recently published NMR chemical shift perturbation (CSP) data (Fig. 1C & S1A), and with Ras N26G and V45E mutations shown to disrupt the Ras/Raf-CRD interaction (12,17). Given its close contact with loop 3 on Ras, the Raf-CRD is allosterically connected to helix 5 through salt bridges that form between Ras D47 and E49 on loop 3 and R161 and R164 on helix 5 (Fig.…”

“…S1B), where the Ras/Raf-CRD interaction was modelled utilizing chemical shift perturbation data that are also consistent with our model ( Fig. S1A) and paramagnetic relaxation enhancement (PRE) data with the unfortunate placement of the probe at a cysteine engineered in place of Q43 (17), an important residue at the Ras/Raf-CRD interface (Fig. 1C).…”

“…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).…”

“…The Raf-CRD requires coordination of two zinc ions and has been shown to interact with various phospholipids and supported lipid bilayers in vitro (3,(13)(14)(15)(16). Its membrane-binding role has further been studied by molecular dynamics (MD) simulations and recently supported by NMR guided characterization of nanodisc-bound KRas/CRaf-RBD_CRD complexes (17)(18)(19)(20)(21)(22).…”

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

“…Following recruitment of RAF to the membrane, the CRD forms contacts with phospholipids, disrupting the autoinhibited state. Based on mutagenesis and NMR experiments, CRD residues involved in membrane interaction have been proposed to be located in the two hydrophobic loops surrounded by basic residues [6][7][8] . In addition to the known RBD-RAS interactions, CRD has been reported as a second RAS-binding domain of RAF 9-13 that can bind to RAS independently of RBD with weaker (micromolar) affinity 14 .…”