SOLUTION STRUCTURE AND MEMBRANE INTERACTIONS OF THE C2 DOMAIN OF CYTOSOLIC PHOSPHOLIPASE A(2)

The amino-terminal, 138 amino acid C2 domain of cytosolic phospholipas e A(2) (cPLA(2)-C2) mediates an initial step in the production of lipi d mediators of inflammation: the Ca2+-dependent translocation of the e nzyme to intracellular membranes with subsequent liberation of arachid onic acid. The high resolution solution structure of this Ca2+-depende nt, lipid-binding domain (CaLB) has been determined using heteronuclea r three-dimensional NMR spectroscopy. Secondary structure analysis, de rived from several sets of spectroscopic data, shows that the domain i s composed of eight antiparallel P-strands with six interconnecting lo ops that fits the ''type II'' topology for C2 domains. Using a total o f 2370 distance and torsional restraints, the structure was found to b e a P-sandwich in the ''Greek key'' motif. The solution structure of c PLA(2)-C2 domain is very similar to the X-ray crystal structure of the C2 domain of phospholiyase-C-delta and phylogenetic analysis clarifie s the structural role of highly conserved residues. Calorimetric studi es further demonstrate that cPLA(2)-C2 binds two Ca2+ with observed K( d)s of approximately 2 mu M in an entropically assisted process. Moreo ver, regions on cPLA(2)-C2 interacting with membranes were identified by N-15-HSQC-spectroscopy of cPLA(2)-C2 in the presence of low molecul ar weight lipid micelles. An extended binding site was identified that binds the phosphocholine headgroup in a Ca2+-dependent manner and als o interacts with proximal regions of the membrane surface. Based upon these results, a structural model is presented for the mechanism of as sociation of cPLA(2) with its membrane substrate. (C) 1998 Academic Pr ess.