Effector Binding Sequentially Alters KRAS Dimerization on the Membrane: New Insights Into RAS‐Mediated RAF Activation

• Published in: Advanced science Vol. 11; no. 38; pp. e2401530 - n/a
• Main Authors: Lee, Soo‐Yeon, Eun, Hyun‐Jong, Lee, Ki‐Young
• Format: Journal Article
• Language: English
• Published: Germany John Wiley & Sons, Inc 01.10.2024; John Wiley and Sons Inc; Wiley
• Subjects: Binding sites; Cell Membrane - metabolism; Dimerization; Humans; Interfaces; Kinases; KRAS dimerization; Lipids; Membranes; nanodisc; paramagnetic relaxation enhancement; Plasma; Protein Binding; Protein Multimerization; Proteins; Proto-Oncogene Proteins c-raf - chemistry; Proto-Oncogene Proteins c-raf - genetics; Proto-Oncogene Proteins c-raf - metabolism; Proto-Oncogene Proteins p21(ras) - chemistry; Proto-Oncogene Proteins p21(ras) - genetics; Proto-Oncogene Proteins p21(ras) - metabolism; RBD‐CRD; sequential binding; Signal Transduction - genetics; RBD‐CRD; paramagnetic relaxation enhancement; sequential binding; KRAS dimerization; nanodisc
• ISSN: 2198-3844; 2198-3844
• DOI: 10.1002/advs.202401530

RAS proteins are peripheral membrane GTPases that activate multiple downstream effectors for cell proliferation and differentiation. The formation of a signaling RAS–RAF complex at the plasma membrane is implicated in a quarter of all human cancers; however, the underlying mechanism remains unclear. In this work, nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses to determine the structure of a hetero‐tetrameric complex comprising KRAS and the RAS‐binding domain (RBD) and cysteine‐rich domain (CRD) of activated RAF1 are employed. The binding of the RBD or RBD–CRD differentially alters the dimerization modes of KRAS on both anionic and neutral membranes, validated by interface‐specific mutagenesis. Notably, the RBD binding allosterically generated two distinct KRAS dimer interfaces in equilibrium, favored by KRAS free and in complex with the RBD–CRD, respectively. Additional interactions of the CRD with both KRAS protomers are mutually cooperative to stabilize a new dimer configuration of KRAS bound to the RBD–CRD. The RAF binding sequentially alters KRAS dimerization, providing new insights into RAF activation, including a configurational transition of the KRAS dimer to provide an interaction site for the CRD and release the autoinhibited RAF complex. These methods are applicable to many other signaling protein complexes on the membrane. Nanodisc platforms and paramagnetic relaxation enhancement analyses revealed that the binding of the RAS‐binding domain (RBD) generates two distinct KRAS dimer interfaces in equilibrium, favored by KRAS free and in complex with the RBD–cysteine‐rich domain (CRD). Additional interactions of the CRD with both KRAS protomers are mutually cooperative to stabilize a new dimer conformation of KRAS.