Cells rely on sophisticated molecular communications for their essential functions. The importance of the membrane surface environment in cell signaling is now widely recognized as it actively regulates the protein behavior beyond being a passive physical boundary. At the heart of this regulation are the emergent properties from multivalent interaction between modular protein domains and membranes, which is a common theme broadly observed in many signaling pathways. However, much remains to be understood about how these processes alter the structure and function of proteins to produce the desired signaling outcome. In this talk, I will discuss how protein kinase Raf integrates distinct signaling inputs to initiate its activation process, focusing on membrane binding. Raf is a major eukaryotic signaling protein that initiates the three-tiered kinase cascade Raf-MEK-ERK in response to small GTPase Ras activation. Given a relatively low expression level of Raf, Raf activation likely forms a kinetic bottleneck (rate-limiting step) of the mitogen-activated protein kinase (MAPK) pathways controlling cell survival and organism development, with direct relevance to cancer. To study the interaction between Raf and membranes, we characterized membrane binding and molecular diffusion on supported lipid membranes at the single-molecule level. Our results show that the tandem Ras binding domain (RBD) and cysteine-rich domain (CRD) in the regulatory region integrate protein and lipid interactions through kinetically distinct binding modes, showing a synergistic binding effect. This mechanism may allow Raf to achieve rapid turn on/off behavior while maintaining a high affinity to membranes. The discussion will be extended to illuminating dynamics of Raf activation mechanisms in conjunction with static structures recently revealed via Cryo-EM, x-ray crystallography, and NMR.

Mechanisms for membrane binding of Raf: Integration of protein and lipid interactions via modular protein domains

ACS Spring 2022

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