Extended Data Figure 10: Mutational and functional characterization of molecular interactions involving the NTRs and kinase domains of BRAF and KSR1.

a, Peak intensity change and CSP versus residue number for the [¹H–¹⁵N]HSQC spectra of the [¹⁵N]mCC-SAM L45D mutant (black) and the [¹⁵N]mCC-SAM L45D mutant in the presence of hBRS (green) (left), the [¹H,¹⁵N]HSQC spectra of the [¹⁵N]mCC-SAM C60D (black) versus [¹⁵N]mCC-SAM C60D in the presence of hBRS (green) (middle), and the [¹H–¹⁵N]HSQC spectra of [¹⁵N]mCC-SAM (black) and [¹⁵N]mCC-SAM in the presence of the hBRS M53D mutant (green) (right) at the indicated molar ratio. Cut-off values indicated by the red line were calculated using the corrected s.d. method⁴⁰. Residue coordinates of the CC-SAM correspond to mouse KSR1; the corresponding human CC-SAM mutations are L47D and C62D. b, The BRS and CC-SAM domains specifically interact as determined by BRET. The CC-SAM L47D and C62D mutations reduce the BRS:CC-SAM BRET binding signal. Similarly, the BRS M53D mutation disrupts the BRS:CC-SAM BRET binding signal. c, Disruption of the K88–E72 salt interaction disrupts BRS:CC-SAM BRET interaction. Restoring the salt bridge with reverse polarity as in the K88E–E72K mutant pair rescues complex formation. d, MEK1 expression induces the formation of full-length BRAF–KSR1 heterodimers, but does not induce the formation of heterodimers between the isolated NTRs of BRAF and KSR1 as detected by co-IP. e, BRET analysis demonstrating that MEK expression promotes KSR1 kinase domain homodimerization. Helix αG mutations in either KSR1 (W831R) or MEK1 (F311S) abolish MEK-induced homodimerization of the KSR1 kinase domain. BRET log₂-transformed fold-changes were reported as a function of log₁₀-transformed mCherry relative fluorescence units. f, Co-IP analysis of Pyo-KSR1 with Flag–KSR1 reveals that MEK1 promotes full-length KSR1 homodimerization. Experiments in b–f were repeated at least three times. For gel source data, see Supplementary Fig. 1.