Three distinct regions of cRaf kinase domain interact with membrane.

Raf kinases are downstream effectors of small GTPase Ras. Mutations in Ras and Raf are associated with a variety of cancers and genetic disorders. Of the three Raf isoforms, cRaf is most frequently involved in tumor initiation by Ras. Cytosolic Raf is auto-inhibited and becomes active upon recruitment to the plasma membrane. Since the catalytic domain of Raf is its kinase domain, we ask the following: does the kinase domain of Raf has potential to interact with membrane and if yes, what role does the membrane interaction play? We present a model of cRaf kinase domain in complex with a heterogeneous membrane bilayer using atomistic molecular dynamics simulation. We show that the kinase domain of cRaf has three distinct membrane-interacting regions: a polybasic motif (R.RKTR) from the regulatory αC-helix, an aromatic/hydrophobic cluster from the N-terminal acidic region (NtA) and positively charged/aromatic cluster from the activation segment (AS). We show that residues from these regions form an extended membrane-interacting surface that resembles the membrane-interacting residues from known membrane-binding domains. Activating phosphorylatable regions (NtA and AS), make direct contact with the membrane whereas R.RKTR forms specific multivalent salt bridges with PA. PA lipids dwell for longer times around the R.RKTR motif. Our results suggest that membrane interaction of monomeric cRaf kinase domain likely orchestrates the Raf activation process and modulates its function. We show that R.RKTR is a hotspot that interacts with membrane when cRaf is monomeric and becomes part of the interface upon Raf dimerization. We propose that in terms of utilizing a specific hotspot to form membrane interaction and dimer formation, both Raf and its upstream binding partner KRas, are similar.