Mapping the Interfacial Binding Surface of Human Secretory Group IIa Phospholipase A₂

Abstract

Human secretory group IIa phospholipase A2 (hIIa-PLA2) contains a large number of prominent cationic patches on its molecular surface and has exceptionally high affinity for anionic surfaces, including anionic membranes. To identify the cationic amino acid residues that support binding of hIIa-PLA2 to anionic membranes, we have performed extensive site-directed mutagenesis of this protein and measured vesicle binding and interfacial kinetic properties of the mutants using polymerized liposomes and nonpolymerized anionic vesicles. Unlike other secretory PLA2s, which have a few cationic residues that support binding of enzyme to anionic membranes, interfacial binding of hIIa-PLA2 is driven in part by electrostatic interactions involving a number of cationic residues forming patches on the putative interfacial binding surface. Among these residues, the amino-terminal patch composed of Arg-7, Lys-10, and Lys-16 makes the most significant contribution to interfacial adsorption, and this is supplemented by contributions from other patches, most notably Lys-74/Lys-87/Arg-92 and Lys-124/Arg-127. For these 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 studies indicate that electrostatic interactions between surface lysine and arginine residues and the interface contribute to interfacial binding of hIIa-PLA2 to anionic vesicles and that cationic residues closest to the opening of the active-site slot make the most important interactions with the membrane. However, because the wild type binds extremely tightly to anionic vesicles, it was not possible to exactly determine what fraction of the total interfacial binding energy is due to electrostatics.

Role of Charge Properties of Bacterial Envelope in Bactericidal Action of Human Group IIA Phospholipase A2against Staphylococcus aureus

“…Subsequent interactions at the bacterial membrane, however, likely differ. Killing by CAP is generally believed to require membrane insertion of the peptide (28 -31) whereas PL degradation and killing by PLA 2 can be mediated by protein bound to the extracytoplasmic surface of the membrane (32)(33)(34). Membrane insertion of CAP may require very extensive electrostatic interactions between the peptide and negatively charged membrane lipids to trigger an amphiphilic transition of the peptide and membrane insertion (for review see Ref.…”

Mechanism of Human Group V Phospholipase A2(PLA2)-induced Leukotriene Biosynthesis in Human Neutrophils

“…Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) and used without further purification. Recombinant hIIaPLA 2 was prepared as described (10).…”

“…Spectral bandwidth was set at 5 nm for both excitation and emission. Values of k cat */K m * were determined from reaction progress curves as described previously (10). The PLA 2 -catalyzed hydrolysis of diC 6 PE monomers was performed with 0.5 mM phospholipid, 0.16 M NaCl, and 10 mM CaCl 2 .…”

Human Group V Phospholipase A2 Induces Group IVA Phospholipase A2-independent Cysteinyl Leukotriene Synthesis in Human Eosinophils

“…Expression and Purification of sPLA 2 -Recombinant human group IIa PLA 2 (hIIaPLA 2 ) was prepared as described (19). Recombinant hVPLA 2 and mutants were expressed in Escherichia coli, refolded, and purified as described previously (20,21).…”