Abstract 3224: High affinity interaction of K-Ras4B hypervariable region with Ras active site
Authors: Chavan Tanmay S. , Jang Hyunbum , Khavrutskii Lyuba , Freed Benjamin C. , Johannessen Liv , Tarasov Sergey G. , Abraham Sherwin J. , Gaponenko Vadim , Nussinov Ruth , Tarasova Nadya 
Published: May 2015

Abstract
K-Ras4B is a ubiquitous p21 GTPase that controls cell survival and proliferation. Oncogenic mutations in K-Ras4B impair GTP hydrolysis and lead to constitutive activation of signaling. Ras proteins consist of highly homologous catalytic domains (residues 1-166) and C-terminal stretches of 18-20 amino acids called hypervariable regions (HVR) that differ significantly for the four Ras isoforms, H-Ras, N-Ras, K-Ras4A and K-Ras4B. The roles of these structural differences are poorly understood. Despite the central role of K-Ras4B in oncogenesis and widespread efforts to develop Ras-directed anti-cancer therapeutics, no selective, specific inhibitor of K-Ras4B is available for clinical use. This is primarily because its catalytic domain lacks pockets for high affinity small molecule binders. Unexpectedly, Microscale Thermophoresis studies have shown that analogs of HVR, thought to minimally impact the catalytic domain, directly interact with GDP-bound, but not GTP-bound recombinant K-Ras4B (1-166). The KD of HVR mimetics interaction with truncated GDP-loaded K-Ras4B is in the nanomolar range, while replacement of GDP with γ-S-GTP lowered the affinity almost two orders of magnitude. HVR analogs significantly slowed down GDP to GTP exchange. NMR studies of full-length K-Ras4B have revealed that HVR forms extensive interactions with the active site of GDP, but not GTP-bound K-Ras4B. GTP binding induces translocation of HVR and interaction of its side chains with cellular membrane. Molecular dynamics simulations confirmed the tight interaction of HVR with the catalytic domain in the GDP-bound state, but not GTP-bound K-Ras4B, suggesting that in the GDP-bound state, an HVR domain could adopt a β-strand conformation, extending the β-sheet in the active site of the catalytic domain. Interaction surface identified by NMR and computational studies involves primarily residues in the switch I and the effector binding regions. In addition, large structural differences have been observed between catalytic domains of the full-length GDP- and GTP-bound states, in contrast to the truncated protein, for which crystal structures of the GDP- and GTP-bound catalytic domains are almost identical. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles not only in membrane anchoring, but also in regulation of K-Ras4B signaling. High affinity binding of short peptide analogs of HVR to K-ras4B active site suggests that targeting this surface with inhibitory synthetic molecules is feasible. Structure-based optimization of compounds that bind at the interface between the HVR and the catalytic domain and thus interfere with GDP to GTP exchange is a promising approach for the development of potent inhibitors for so far non-druggable Ras oncoproteins.
Citation Format: Tanmay S. Chavan, Hyunbum Jang, Lyuba Khavrutskii, Benjamin C. Freed, Liv Johannessen, Sergey G. Tarasov, Sherwin J. Abraham, Vadim Gaponenko, Ruth Nussinov, Nadya Tarasova. High affinity interaction of K-Ras4B hypervariable region with Ras active site. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3224. doi:10.1158/1538-7445.AM2014-3224