Abstract 2918: Comprehensive survey of KRas4B membrane interaction and orientation
Authors: Jang Hyunbum , Zhang Mingzhen , Gaponenko Vadim , Nussinov Ruth 
Published: June 2022

Three RAS genes encode four Ras proteins, HRas, NRas, and KRas splice variants, KRas4A and KRas4B. In human cancers, KRAS oncogene is highly expressed (85%) as compared to other RAS oncogenes, NRAS (11%) and HRAS (4%). Oncogenic Ras leads to aberrant cell proliferation signaling, primarily through the MAPK (Raf/MEK/ERK) pathway. At the plasma membrane, nanoclustered Ras proteins promote Raf activation, mediating the downstream signaling pathway. Among the Ras isoforms, KRas4B in the membrane has been studied extensively due to its higher level of mutations in cancer. Although a number of experimental studies have focused on KRas4B subcellular localization and their nanocluster formation at the signaling platform, the mechanistic details of the membrane anchorage and the membrane orientation of KRas4B at atomic resolution are still unknown. Here, comprehensive computational studies were performed for KRas4B at the anionic lipid bilayers, composed of the phospholipids, PS and PA, and the phosphoinositides, PIP2, PI4P, and PI5P. Wild-type and two oncogenic mutants, G12D and G12V KRas4B in the GTP- and GDP-bound states were considered. The lysine rich HVR was post-translationally modified with the farnesyl and methyl groups. Previous studies showed that wild-type KRas4B-GDP interacts weakly and nonspecifically with anionic phospholipids, while oncogenic mutations alter selective anionic lipid binding toward anionic lipids, PA, PI3P, PI4P, and PI5P. Our studies indicate that the membrane interaction of KRas4B is highly sensitive depending on the types of nucleotides, mutational states, and anionic lipids enriched the lipid bilayer. While the GTP-bound spontaneously inserts its prenyl group into the hydrophobic core of the bilayer, the occurrence of spontaneous insertion is reduced in the GDP-bound form. The distance of GTP-bound catalytic domain from the bilayer surface is longer than the GDP-bound form. The HVR is sequestered by the GDP-bound catalytic domain, which causes the catalytic domain to be occluded and increases the membrane interaction by the catalytic domain. In contrast, the GTP-bound catalytic domain releases its HVR and liberates from the membrane interaction. The occlusion of catalytic domain is less prevalent in the GTP-bound state. KRas4BG12D/G12V-GTP exhibits the active-state orientation, exposing its effector binding site for recruiting the effectors. While PA and PIP2 support the active-state orientation, the PIP4 enriched bilayer prevents the spontaneous prenyl insertion resulting in the occlusion of catalytic domain. Our results underscore the importance of detailed structural mechanisms of KRas4B membrane interaction and orientation, elucidating vital mechanistic role of KRas4B, a key membrane-anchored protein in cancer. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261201500003I.

Citation Format: Hyunbum Jang, Mingzhen Zhang, Vadim Gaponenko, Ruth Nussinov. Comprehensive survey of KRas4B membrane interaction and orientation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2918.