Solution structure and membrane interactions of the C2 domain of cytosolic phospholipase A2

The amino-terminal, 138 amino acid C2 domain of cytosolic phospholipase A(2) (cPLA(2)-C2) mediates an initial step in the production of lipid mediators of inflammation: the Ca2+-dependent translocation of the enzyme to intracellular membranes with subsequent liberation of arachidonic acid. The high resolution solution structure of this Ca2+-dependent, lipid-binding domain (CaLB) has been determined using heteronuclear three-dimensional NMR spectroscopy. Secondary structure analysis, derived from several sets of spectroscopic data, shows that the domain is composed of eight antiparallel P-strands with six interconnecting loops that fits the "type II" topology for C2 domains. Using a total of 2370 distance and torsional restraints, the structure was found to be a P-sandwich in the "Greek key" motif. The solution structure of cPLA(2)-C2 domain is very similar to the X-ray crystal structure of the C2 domain of phospholiyase-C-delta and phylogenetic analysis clarifies the structural role of highly conserved residues. Calorimetric studies further demonstrate that cPLA(2)-C2 binds two Ca2+ with observed K(d)s of approximately 2 mu M in an entropically assisted process. Moreover, regions on cPLA(2)-C2 interacting with membranes were identified by N-15-HSQC-spectroscopy of cPLA(2)-C2 in the presence of low molecular weight lipid micelles. An extended binding site was identified that binds the phosphocholine headgroup in a Ca2+-dependent manner and also interacts with proximal regions of the membrane surface. Based upon these results, a structural model is presented for the mechanism of association of cPLA(2) with its membrane substrate. (C) 1998 Academic Press.