Two Closed Conformations of CRAF Require the 14-3-3 Binding Motifs and Cysteine-Rich Domain to be Intact in Live Cells

[Display omitted] •Wild-type CRAF has two types of closed conformations.•Both closed conformations involve the binding of N- and C-termini and S259 to 14-3-3.•Two types of closed conformations have different responsiveness to EGF stimulation.•14-3-3 cannot interact with alanine but unphosphorylated serine with low affinity.•A 14-3-3 dimer is supposed to bridge pS259 and pS621 to form a closed structure. The protein rapidly accelerated fibrosarcoma (RAF) is a kinase downstream of the membrane protein RAS in the cellular signal transduction system. In the structure of RAF, the N- and C-terminus domains are connected with a flexible linker. The open/close dynamics and dimerization of RAF are thought to regulate its activity, although the details of these conformations are unknown, especially in live cells. In this work, we used alternating laser excitation to measure cytosolic CRAF in live HeLa cells and obtained single-molecule Förster resonance energy transfer (smFRET) distributions of the structural states. We compared the results for wild-type (WT)-CRAF before and after epidermal growth factor (EGF) stimulation, with mutations of the 14-3-3 binding sites and cysteine-rich domain, and an N-terminus truncation. The smFRET distributions of full-length CRAFs were analyzed by global fitting with three beta distributions. Our results suggested that a 14-3-3 dimer bound to two sites on a single CRAF molecule and induced the formation of the autoinhibitory closed conformation. There were two closed conformations, which the majority of WT-CRAF adopted. These two conformations showed different responsiveness to EGF stimulation.