期刊名称:Proceedings of the National Academy of Sciences
印刷版ISSN:0027-8424
电子版ISSN:1091-6490
出版年度:2020
卷号:117
期号:22
页码:12101-12108
DOI:10.1073/pnas.1914076117
出版社:The National Academy of Sciences of the United States of America
摘要:Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD–CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD–CRD complex. We found that the nucleotide-dependent KRAS–RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD–CRD, and the KRAS:RBD–CRD complex. RBD–CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD β7–8), as well as an alternative interface comprising β6 and the C terminus of CRD and β2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD β7–8 and KRAS α4–α5 while state B involved the C terminus of CRD, β3–5 of RBD, and part of KRAS α5. Notably, α4–α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS–CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD–CRD, and the membrane.