Response regulators undergo regulated phosphorylation and dephosphorylation at conserved aspartic acid residues in bacterial signal transduction systems. OmpR is a winged helix-turnhelix DNA-binding protein that functions as a global regulator in bacteria and is also important in pathogenesis. A detailed mechanistic picture of how OmpR binds to DNA and activates transcription is lacking. We used NMR spectroscopy to solve the solution structure of the C-terminal domain of OmpR (OmpR C ) and to analyze the chemical shift changes that occur upon DNA binding. There is little overlap in the interaction surface with residues of PhoB that were reportedly involved in protein/protein interactions in its head-to-tail dimer. Multiple factors account for the lack of overlap. One is that the spacing between the OmpR half-sites is shorter than observed with PhoB, requiring the arrangement of the two OmpR molecules to be different from that of the PhoB dimer on DNA. A second is the demonstration herein that OmpR can bind to its high affinity site as a monomer. As a result, OmpR C appears to be capable of adopting alternative orientations depending on the precise base composition of the binding site, which also contributes to the lack of overlap. In the presence of DNA, chemical shift changes occur in OmpR in the recognition ␣-helix 3, the loop between -strand 4 and ␣-helix 1, and the loop between -strands 5 and 6. DNA contact residues are Val 203 (T), Arg 207 (G), and Arg 209 (phosphate backbone). Our results suggest that OmpR binds to DNA as a monomer and then forms a symmetric or asymmetric dimer, depending on the binding site. We propose that during activation OmpR binds to DNA and undergoes a conformational change that promotes phosphorylation of the N-terminal receiver domain, the receiver domains dimerize, and then the second monomer binds to DNA. The flexible linker of OmpR enables the second monomer to bind in multiple orientations (head-to-tail and head-to-head), depending on the specific DNA contacts.
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-...
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