MAPPING THE INTERFACIAL BINDING SURFACE OF HUMAN SECRETORY GROUP IIA PHOSPHOLIPASE A(2)

Human secretory group IIa phospholipase A(2) (hIIa-PLA(2)) contains a large number of prominent cationic patches on its molecular surface an d has exceptionally high affinity for anionic surfaces, including anio nic membranes. To identify the cationic amino acid residues that suppo rt binding of hIIa-PLA(2) to anionic membranes, we have performed exte nsive site-directed mutagenesis of this protein and measured vesicle b inding and interfacial kinetic properties of the mutants using polymer ized liposomes and nonpolymerized anionic vesicles. Unlike other secre tory PLA(2)s, which have a few cationic residues that support binding of enzyme to anionic membranes, interfacial binding of hIIa-PLA(2) is driven in part by electrostatic interactions involving a number of cat ionic residues forming patches on the putative interfacial binding sur face. Among these residues, the amino-terminal patch composed of Arg-7 , Lys-10, and Lys-16 makes the most significant contribution to interf acial adsorption, and this is supplemented by contributions from other patches, most notably Lys-74/Lys-87/Arg-92 and Lys-124/Arg-127. For t hese mutants, complete vesicle binding occurs in the presence of high vesicle concentrations, and under these conditions the mutants display specific activities comparable to that of wild-type enzyme. These stu dies indicate that electrostatic interactions between surface lysine a nd arginine residues and the interface contribute to interfacial bindi ng of hIIa-PLA(2) to anionic vesicles and that cationic residues close st to the opening of the active-site slot make the most important inte ractions with the membrane, However, because the wild type binds extre mely tightly to anionic vesicles, it was not possible to exactly deter mine what fraction of the total interfacial binding energy is due to e lectrostatics.