Structural and functional insights into alpha-kinases and their regulation

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Protein (de)phosphorylation (i.e. the addition/removal of phosphate groups) is a crucial and fundamental process in living organisms, underlying the regulatory control of virtually all intracellular signal transduction pathways. A small subclass of atypical kinases, the alpha-kinases, was originally discovered as they recognized an alpha-helical structure rather than a particular sequence, yet very little is known with respect to their structure and function. The object of this proposal is to gain an in-depth understanding into the mechanism and regulation of the atypical kinases. Two alpha-kinase structures have been determined: TRMP7 (an Mg2+-sensing channel kinase) and MHCK (a threonine-specific kinase which acts on myosin II). Our determination of the MHCK kinase domain structure revealed a phosphorylated aspartyl residue, suggesting a novel 2-step reaction mechanism via a phosphoaspartyl intermediate in contrast to the direct phosphorylation of most kinases. An additional Mg2+ ion, in a conserved Gly-rich N/D loop (named for a catalytically critical Asn/Asp residue) may act as Mg2+-sensing conformational switch which, if confirmed, would be a novel, regulatory mechanism. While similar mechanisms presumably underlie TRPM7 function, what is unknown is TRMP7's substrate. Substrate identification, using peptide arrays and other approaches, will allow further analyses regarding the additional Mg2+-dependence of its activity. Another alpha-kinase of unknown structure, eEF2K, is a "master controller" of protein synthesis, and is unique in its regulation by and dependence upon calmodulin for activation. Development of specific inhibitors and the subsequent determination of enzyme-inhibitor complexes will help decipher the mode of action of these atypical kinases while optimizing further inhibitor design. Trainees will utilize a combination of biochemical, biophysical and structural techniques to uncover the structural basis and molecular mechanisms governing substrate specificity, catalytic activity and regulation. Our collaborative efforts gave us a "niche" in the "overcrowded" mammalian kinase field where we can make novel contributions, while our MHCK work gives us a "competitive edge".