Additional binding sites for anionic phospholipids and calcium ions in the crystal structures of complexes of the C2 domain of protein kinase Cα

The C2 domain of protein kinase Calpha (PKCalpha) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCalpha isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-L-serine. Intrinsic Ca binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCalpha-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-L-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCa crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-L-serine (DAPS) or 1,2-dicaproyl-sn-phos-phatidic acid (DCPA) have now been determined and refined at 1.9 Angstrom and at 2.0 Angstrom, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-L-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCa activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed. (C) 2002 Elsevier Science Ltd. All rights reserved.