Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 117, Issue 22, Pages 12101-12108Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1914076117
Keywords
KRAS; protein membrane interactions; RAF kinase; cysteine-rich domain (CRD); RAS-binding domain (RBD)
Categories
Funding
- Cancer Research Society (Canada) [14014]
- Canadian Cancer Society Research Institute [703209]
- Princess Margaret Cancer Foundation
- Canadian Institutes of Health Research Foundation [410008598]
- Connaught Fund
- Canada Foundation for Innovation
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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 beta 7-8), as well as an alternative interface comprising beta 6 and the C terminus of CRD and beta 2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD beta 7-8 and KRAS alpha 4-alpha 5 while state B involved the C terminus of CRD, beta 3-5 of RBD, and part of KRAS alpha 5. Notably, alpha 4-alpha 5, 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.
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