Abstract 2763: Raf-1 cysteine-rich domain (CRD) promotes active KRas4B membrane orientation facilitating dimerization through the allosteric lobe interface
Authors: Jang Hyunbum , Nussinov Ruth 
Published: August 2019

Ras proteins are classical members of small GTPases, controlling signal transduction pathways and promoting cell proliferation. Ras consists of highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ significantly across Ras isoforms. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and activation is terminated by GTPase-activating proteins that induce the GTP hydrolysis. Ras has multiple partners, signals through several key pathways and fulfills critical functions in the cell life. Mutations in Ras are common in a variety of cancers; yet it is still undruggable. KRas4B is frequently mutated in cancer. Elucidation of Ras conformational ensembles at the signaling platforms in the plasma membrane including the ligand-bound conformations, membrane orientations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, and transition states are essential for deciphering Ras functions from its conformational landscapes. In the MAPK pathway, KRas4B preferentially recruits Raf-1 and activates it. Raf kinases contain Ras binding domain (RBD) and cysteine-rich domain (CRD) at conserved region 1 (CR1), and kinase domain at CR3. The high-affinity interaction of Raf RBD with Ras was solved in the absence of CRD. It is known that Raf CRD play a key role in the membrane anchoring mechanism. Recently, pivotal roles of CRD in the membrane interactions of KRas4B-Raf-1 RBD-CRD complex were reported. Here, we employ all-atom molecular dynamics (MD) simulations to investigate dimeric KRas4B-Raf-1 complex at the anionic lipid bilayer composed of DOPC and DOPS (4:1). Active KRas4B dimer modulates Raf-1 activation, promoting dimerization of Raf-1’s kinase domain. In the dimeric complex of KRas4B-Raf-1, Raf-1 CRD stably binds anionic lipid bilayers inserting its positively-charged loop into the amphipathic interface. The key basic residues at the loop are responsible for the membrane association, suggesting that CRD is an intrinsic membrane binding domain of Raf kinase. Our simulations suggest that Raf-1 CRD not only offers an additional membrane anchor for the KRas4B-Raf-1 complex, but also reduces the fluctuations of Ras-RBD, enhancing the high affinity interaction between Ras and Raf. Raf-1 CRD supports the active-state Ras orientation, promoting Ras dimerization through the allosteric lobe helical interface at the membrane. The enhanced stability of the complex at the membrane rendered by CRD promotes Raf dimerization in the MAPK signaling, which is the key Ras proliferative pathway. Here we suggest these significant roles of CRD at the membrane in the Raf activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.

Citation Format: Hyunbum Jang, Ruth Nussinov. Raf-1 cysteine-rich domain (CRD) promotes active KRas4B membrane orientation facilitating dimerization through the allosteric lobe interface [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2763.