Abstract 2270: Membrane organization of KRas4B with regulatory domain of Raf-1

Abstract

A major pathway in RAS-driven cell proliferation is the MAPK (Raf/MEK/ERK) pathway. In cancer, membrane-anchored, proximally located, oncogenic Ras isoforms promote Raf dimerization and fully activate MAPK signaling. Among Ras isoforms, KRas4B is frequently mutated in cancer. In the MAPK pathway, active KRas4B dimer or nanocluster can lead to dimerization of Raf kinase domain enhancing the affinity of the KRas4B-Raf interaction. Recently, we modeled the binary KRas4B-Raf-1 complex and its quaternary assembly with the regulatory domain of Raf-1 containing the Ras binding domain (RBD) and cysteine-rich domain (CRD), and suggested that Raf-1 CRD bolsters the high-affinity interaction of Raf-1 with KRas4B by reducing the KRas4B-RBD fluctuations. For the quaternary assembly, recent in silico studies suggested that KRas4B dimer is aligned through the α3 and α4 helical interface, not the α4 and α5 helical interface due to hindrance to the membrane contact of Raf-1 CRD. The Raf-1 regulatory domain contains the N-terminal region (residues 1-55), RBD, and CRD. However, previous modeling efforts were conducted for the KRas4B-Raf-1 complex in the absence of the N-terminal region. Here, computational studies were performed for the KRas4B-Raf-1 complex with full sequence of Raf-l regulatory domain interacting with monomeric and dimeric KRas4B-GTP at the membrane. Increasing experimental evidences suggested that the N-terminal region of Raf plays a significant role in the Ras interaction and the membrane anchorage of Raf. The membrane interaction of isolated N-terminal segment was investigated as the first step toward the complex model construction. Since oncogenic KRas4B mutants form dimers/nanoclusters at the membrane microdomains that provide the signaling platform for Raf, KRas4B in different mutation states interacting with the membrane microdomains with different lipid composition was also studied. The presence of the N-terminal segment of Raf-1 regulatory domain at the membrane can enhance the stability of KRas4B-Raf-1 and thereby relieving Raf9s autoinhibition toward a kinase domain-accessible state, providing a complete mechanistic picture of the KRas4B-Raf-1 interaction. Our recent work uncovers the mechanism of Raf-1 activation by KRas4B at atomic resolution and highlight how this may help in elucidating vital mechanistic questions in Ras biology. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Hyunbum Jang, Mingzhen Zhang, Ruth Nussinov. Membrane organization of KRas4B with regulatory domain of Raf-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2270.