A structural determinant of the unique interfacial binding mode of bovine pancreatic phospholipase A(2)

The catalytic steps of the phospholipase A(2) (PLA(2))-catalyzed hydrolysis of phospholipids are preceded by interfacial binding. Among various pancre atic PLA(2)s, bovine pancreatic PLA(2) (bpPLA(2)) has a unique interfacial binding mode in which Lys-56 plays an important role in its binding to anio nic lipid surfaces. To identify the structural determinant of this unique i nterfacial binding mode of bpPLA(2), we systematically mutated bpPLA(2) and measured the effects of mutations on its interfacial binding and activity. First, different cationic clusters were generated in the amino-terminal al pha-helix by the N6R, G7K, and N6R/G7K mutations. These mutations enhanced the binding of bpPLA(2) to anionic liposomes up to 15-fold. For these mutan ts, however, the K56E mutation still caused a large drop in interfacial aff inity for and activity toward anionic liposomes, indicating that the genera tion of a cationic patch in the amino-terminal alpha-helix of bpPLA(2) did not change its interfacial binding mode. Second, residues 62-66 that form a part of the pancreatic loop were deleted. For this deletion mutant (Delta( 62-66)), which was as active as wild-type toward anionic liposomes, the K56 E and K116E mutations (Delta(62-66)/K56E and Delta(62-66)/K116E) did not ha ve significant effects on interfacial affinity. In contrast, the K10E mutat ion showed a much larger decrease in interfacial affinity (10-fold), indica ting the deletion of residues 62-66 caused a major change in the interfacia l binding mode. Finally, hydrophobic residues in positions 63 and 65 were r eplaced by bulkier ones (V63F and V63F/V65L) to pinpoint the structural det erminant of the interfacial binding mode of bpPLA(2). The effects of K10E a nd K56E mutations on the interfacial affinity and activity of these mutants showed that Val-63 and Val-65 of bpPLA(2) are the structural determinant o f its unique interfacial binding mode and that relatively conservative subs titutions at these positions result in large changes in the interfacial bin ding mode among mammalian pancreatic PLA(2)s. Taken together, this study re veals how minor structural differences among homologous PLA(2)s can lead to distinct interfacial binding behaviors.