Molecular dynamics characterization of the C2 domain of protein kinase Cβ

Protein kinase C (PKC) isozymes comprise a family of related enzymes that play a central role in many intracellular eukaryotic signaling events. Isozyme specificity is mediated by association of each PKC isozyme with specific anchoring proteins, termed RACKs. The C2 domain of betaPKC contains at least part of the RACK-binding sites. Because the C2 domain contains also a RACK-like sequence (termed pseudo-RACK), it was proposed that this pseudo-RACK site mediates intramolecular interaction with one of the RACK-binding sites in the C2 domain itself, stabilizing the inactive conformation of betaPKC. betaPKC depends on calcium for its activation, and the C2 domain contains the calcium-binding sites. The x-ray structure of the C2 domain of betaPKC shows that three Ca2+ ions can be coordinated by two opposing loops at one end of the domain. Starting from this x-ray structure, we have performed molecular dynamics (MD) calculations on the C2 domain of betaPKC bound to three Ca2+ ions, to two Ca2+ ions, and in the Ca2+-free state, in order to analyze the effect of calcium on the RACK-binding sites and the pseudo-RACK sites, as well as on the loops that constitute the binding site for the Ca2+ ions. The results show that calcium stabilizes the,G-sandwich structure of the C2 domain and thus affects two of the three RACK-binding sites within the C2 domain. Also, the interactions between the third RACK-binding site and the pseudo-RACK site are not notably modified by the removal of Ca2+ ions. On that basis, we predict that the pseudo-RACK site within the C2 domain masks a RACK-binding site in another domain of betaPKC, possibly the V5 domain. Finally, the MD modeling shows that two Ca2+ ions are able to interact with two molecules of O-phospho-L-serine. These data suggest that Ca2+ ions may be directly involved in PKC binding to phosphatidylserine, an acidic lipid located exclusively on the cytoplasmic face of membranes, that is required for PKC activation.