SUMMARY Ras proteins recruit and activate effectors, including Raf, that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, activation of Raf requires its dimerization. It has been suspected that dimeric Ras may promote dimerization and activation of Raf. Here we show that the GTP-bound catalytic domain of K-Ras4B, a highly oncogenic splice variant of the K-Ras isoform, forms stable homodimers. We observe two major dimer interfaces. The first, highly populated β-sheet dimer interface is at the Switch I and effector binding regions, overlapping Raf’s, PI3K’s, RalGDS’ and additional effectors’ binding surfaces. This interface has to be inhibitory to such effectors. The second, helical interface also overlaps some effectors’ binding sites. This interface may promote Raf‘s activation. Our data reveal how Ras self-association can regulate effector binding and activity, and suggest that disruption of the helical dimer interface by drugs may abate Raf’s signaling in cancer.

Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible.

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