STRUCTURE OF THE PROTEIN-KINASE C-BETA PHOSPHOLIPID-BINDING C2 DOMAINCOMPLEXED WITH CA2+

Background: Conventional isoforms (alpha, beta and gamma) of protein k inase C (PKC) are synergistically activated by phosphatidylserine and Ca2+; both bind to C2 domains located within the PKC amino-terminal re gulatory regions. C2 domains contain a bipartite or tripartite Ca2+-bi nding site formed by opposing loops at one end of the protein. Neither the structural basis for cooperativity between phosphatidylserine and Ca2+, nor the binding site for phosphatidylserine are known. Results: The structure of the C2 domain from PKC beta complexed with Ca2+ and o-phospho-L-serine has been determined to 2.7 Angstrom resolution usin g X-ray crystallography. The eight-stranded, Greek key beta-sandwich f old of PKC beta-C2 is similar to that of the synaptotagmin I type I C2 domain. Three Ca2+ ions, one at a novel site, were located, each shar ing common aspartate ligands. One of these ligands is donated by a dya d-related C2 molecule. A phosphoserine molecule binds to a lysine-rich cluster in C2. Conclusions: Shared ligation among the three Ca2+ ions suggests that they bind cooperatively to PKC beta-C2. Cooperativity m ay be compromised by the accumulation of positive charge in the bindin g site as successive ions are bound. Model building shows that the C1 domain could provide carboxylate and carbonyl ligands for two of the t hree Ca2+ sites. Ca2+-mediated interactions between the two domains co uld contribute to enzyme activation as well as to the creation of a po sitively charged phosphatidylserine-binding site.