6PTW

NMR data-driven model of KRas-GMPPNP:RBD-CRD complex tethered to a nanodisc (state B)

Summary for 6PTW

• Entry DOI: 10.2210/pdb6ptw/pdb
• NMR Information: BMRB: 30640
• Descriptor: Apolipoprotein A-I, GTPase KRas, RAF proto-oncogene serine/threonine-protein kinase, ... (8 entities in total)
• Functional Keywords: protein-protein-bilayer complex, small gtpase, ras-raf-nanodisc complex, signaling protein
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 4
• Total formula weight: 146205.05

Authors

Fang, Z.,Lee, K.,Gasmi-Seabrook, G.,Ikura, M.,Marshall, C.B. (deposition date: 2019-07-16, release date: 2020-05-13, Last modification date: 2024-05-15)

Primary citation

Fang, Z.,Lee, K.Y.,Huo, K.G.,Gasmi-Seabrook, G.,Zheng, L.,Moghal, N.,Tsao, M.S.,Ikura, M.,Marshall, C.B.
Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane.
Proc.Natl.Acad.Sci.USA, 117:12101-12108, 2020

PubMed Abstract: 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.

PubMed: 32414921
DOI: 10.1073/pnas.1914076117

Experimental method

SOLUTION NMR