Work in this laboratory is aimed at elucidating the mechanism of stimulus- response coupling mediated by Ca2+ and calmodulin. The calmodulin- stimulated protein phosphatase, calcineurin, is used as a model system. Our ultimate goal is to elucidate the structure of the calcineurin/calmodulin complex in order to understand how calmodulin activates this important enzyme. Our efforts, this year, were focused on the role of the Ca2+-binding regulatory subunit of calcineurin, calcineurin B, in the regulation of the protein phosphatase activity. Ca2+ binding to calcineurin B was shown to be absolutely required for enzymatic activity as well as for the calmodulin stimulation of the enzyme. Myristoylated and unmyristoylated calcineurin B have been expressed in high yield in E. coli. The Ca2+ binding properties of the recombinant proteins are similar to those of native calcineurin B but substitution of the myristoylated calcineurin B of the native enzyme by unmyristoylated calcineurin B yields inactive enzyme. The three- dimensional structure of recombinant calcineurin B has been determined in collaboration with Ad Bax and Jacob Anglister (NIDDK). Recombinant calcineurin A-beta has also been expressed in high yield in E. coli. It is being recombined with myristoylated calcineurin B to reconstitute enzymatically active enzyme and to obtain homogenous preparations of calcineurin suitable for crystal structure determination.
