No abstract

CLC secondary active transporters exchange Cl- for H+. Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using 19F NMR, we show that as [H+] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.DOI: http://dx.doi.org/10.7554/eLife.11189.001

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 belongs to the family of p21 Ras GTPases, which play an important role in cell proliferation, survival and motility. The p21 Ras proteins such as K-Ras4B, K-Ras4A, H-Ras, and N-Ras, share 85% sequence homology and activate very similar signaling pathways. Only the C-terminal hypervariable regions differ significantly. A growing body of literature demonstrates that each Ras isoform possesses unique functions in normal physiological processes as well as in pathogenesis. One of the central questions in the field of Ras biology is how these very similar proteins achieve such remarkable specificity in protein-protein interactions that regulate signal transduction pathways. Here we explore specific binding of K-Ras4B to calmodulin. Using NMR techniques and isothermal titration calorimetry we demonstrate that the hypervariable region of K-Ras contributes in a major way to the interaction with calmodulin while the catalytic domain of K-Ras4B provides a way to control the interaction by nucleotide binding. The hypervariable region of K-Ras4B binds specifically to the C-terminal domain of Ca 2+ -loaded calmodulin with micromolar affinity, while the GTP-γ-S loaded catalytic domain of K-Ras4B may interact with the N-terminal domain of calmodulin. KeywordsK-Ras4B; calmodulin; hypervariable region; catalytic domain Members of the Ras family of proto-oncogenes are mutated in up to a third of human malignancies(1,2). These are small p21 GTPases that cycle between the GDP-bound inactive and the GTP-bound active states to transmit intracellular signals. How Ras proteins contribute to cancer development is not fully understood, in spite of much study. They have many signaling partners and regulate a variety of cellular processes including proliferation, transformation, differentiation, metastasis, and apoptosis. There are four isoforms in the Ras family, and of these K-Ras, almost exclusively, is mutated in common epithelial cancers including those of the pancreas, colon and lung. Two forms of K-Ras are generated by alternate mRNA splicing, namely K-Ras4A and K-Ras4B. K-Ras4B is more abundant in most tissues, and has been demonstrated to cause tumor formation in studies with genetically engineered mice(3-5). Noonan syndrome, a developmental disorder, is caused by a mutation specifically in K-Ras4B(6).*To whom correspondence should be addressed: Vadim Gaponenko, Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607, vadimg@uic Thus, K-Ras4B has a particularly important role in human cancer as well as human development. It differs from the other highly homologous Ras isoforms in the C-terminal region where the alternate 4B exon provides a polylysine region in addition to posttranslational farnesylation. The other Ras isoforms lack the polylysine tail and are modified with a palmitoyl group in addition to the farnesyl moiety. Some of the unique properties of K-Ras4B have been revealed in studies of comparative physiology. These include induced Raf-...