Cryo-EM structure of a RAS/RAF recruitment complex

Abstract

RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer. Analysis of this KRAS/BRAF/MEK1/14-3-3 complex reveals KRAS bound to the RAS-binding domain of BRAF, captured in two orientations. Core autoinhibitory interactions in the complex are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by RAS and suggest stabilization of this pre-activation intermediate as an alternative therapeutic strategy to blocking binding of KRAS.

Fig. 1. Preparation of KRAS/BRAF complexes for structural analysis.

a Schematic of domain structures of BRAF, KRAS, and MEK1. Key phosphorylation sites are indicated above the schematics. Binding sites for the 14-3-3 domain in BRAF are indicated in red. BRS BRAF-specific domain, RBD RAS-binding domain, CRD cysteine-rich domain, HVR RAS hypervariable region. b Size-exclusion chromatography of the KRAS/BRAF/MEK1/14-3-3 complex. Elution profile of the complex on Superdex S200 is shown on the left, and a Coomassie-stained SDS-PAGE gel of the indicated fractions is shown on the right. The experiment was performed more than three times with similar results. c Size-exclusion chromatography of a BS3-cross-linked KRAS/BRAF/MEK1/14-3-3 sample. The complex analyzed in (b) was subjected to cross-linking with BS3 and re-examined with size-exclusion chromatography. Elution profile of the complex on Superose 6 is shown on the left, and a Coomassie-stained SDS-PAGE gel of the indicated fractions is shown on the right. Aliquots of the input sample before and after approximately sevenfold concentration, but prior to cross-linking, are also shown on the gel. The experiment was performed three times with similar results.

Fig. 2. Cryo-EM structures of a KRAS/BRAF/MEK1/14-3-3 complex.

a Structure of the KRAS/BRAF/MEK1/14-3-3 complex in the “RAS up” conformation. A ribbon diagram of the structure, colored as in Figure 1a, is shown together with the transparent cryo-EM density map. b Structure of the KRAS/BRAF/MEK1/14-3-3 complex in the “RAS front” conformation, determined with the BS3 cross-linked sample. A ribbon diagram of the structure, colored as in (a) above, is shown together with the transparent cryo-EM density map.

Fig. 3. Activation studies of the autoinhibited BRAF/MEK1/14-3-3 complex.

a–c The time course of phosphorylation of ERK2 by the purified BRAF/MEK1/14-3-3 complex, with or without the addition of the indicated KRAS construct and/or liposomes, is measured by western blotting with an anti-pERK1/2 antibody. Integrated band intensities, normalized to the intensity of the autoinhibited complex alone at the 80 min time point, are shown under the blots. a Activity of the autoinhibited complex alone, or with the addition of either GMP-PNP loaded KRAS^GTPase or KRAS4b-FME at a 1:1 molar ratio. b Activity of the autoinhibited complex alone or with the addition of phosphatidylserine (PS) liposomes, KRAS4b-FME, or both liposomes and KRAS4b-FME, as indicated. Liposomes were added to a final concentration of 0.1 mg/ml, and GMP-PNP loaded KRAS4b-FME was added in a 2:1 molar ratio to the autoinhibited BRAF/MEK1/14-3-3 complex (5 nM). c Comparison of the relative activity of the BRAF/MEK1/14-3-3 complex in the autoinhibited vs. active, dimeric state, with or without the addition of KRAS4b-FME. The samples for the autoinhibited complex with and without KRAS4b-FME are aliquots of the same reaction as the corresponding samples in (b), re-run on the same gel and blot with the active dimer reactions to allow comparison of their relative activities. Note that the exposure of the blot in (c) (5 s) is much shorter than that in (b) (2 min). Original uncropped blots are provided in the Source Data File. Experiments in (a–c) were performed three times each with similar results.

Fig. 4. Assembling a model for RAS-driven activation of RAF.

In the quiescent state, BRAF is maintained in an autoinhibited complex with MEK and a 14-3-3 dimer in the cytosol. As a first step in activation (1), this complex is recruited to the membrane by GTP-bound RAS to form a recruitment complex, as visualized in this study. The interaction with farnesylated RAS in a membrane context induces release of autoinhibitory interactions in the BRAF/MEK1/14-3-3 complex, resulting in formation of an “open” monomer complex (2). This second step in activation may result from extraction of the CRD domain from its relatively buried site in the autoinhibited complex, due to preferential binding to RAS and the plasma membrane. Opening exposes pS365 for dephosphorylation by the SHOC2 phosphatase complex, and also allows the 14-3-3 dimer to rearrange to bind the C-terminal pS729 site of two BRAF molecules, driving and stabilizing the active BRAF dimer (3). Because the pS365 site is buried and not accessible for dephosphorylation in the recruitment complex, and because S365 plays no known role in the formation of the active dimer, we hypothesize that the SHOC2 phosphatase complex acts on the open monomer state and contributes to activation by preventing its “reclosure”. Once pS365 is dephosphorylated, BRAF cannot reassume the closed, autoinhibited state observed in the recruitment complex, and the resulting accumulation of open monomers favors rearrangement into active dimers. Protein Data Bank accession codes for structures supporting distinct states and components of this model include: 6NYB and 7MFD (autoinhibited complex); 8DGS and 8DGT (recruitment complex, this study); 6PP9 and 6U2G (BRAF/MEK kinase domain portion of open monomer); 6XHB and 7JHP (RAS complexes with RBD/CRD fragments of RAF, relevant to the open monomer and active dimer states); 6Q0J, 6Q0K, 6UAN, 6XAG, 7MFF (14-3-3-bound BRAF dimers, with or without MEK); and 7SD0, 7UPI, 7TXH, and 7TVF (ternary SHOC2, MRAS, PP1C phosphatase complex).