K-Ras4B is a ubiquitous p21 GTPase that controls cell survival and proliferation. KRas4B is among the frequently mutated oncogenes in human tumors. Ras proteins consist of highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVR) that differ significantly across Ras isoforms. The roles of these structural differences are poorly understood. Unexpectedly, we found that the HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of GDP-bound, but not GTP-bound protein. NMR studies of the 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 tight interaction of HVR with 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 �-strand conformation, extending the �-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. 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.

High affinity interaction of K-Ras4B hypervariable region with Ras active site

248th National Meeting (2014)

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