A structural determinant of the unique interfacial binding mode of bovine pancreatic phospholipase A2.
The catalytic steps of the phospholipase A2 (PLA2)-catalyzed hydrolysis of phospholipids are preceded by interfacial binding. Among various pancreatic PLA2s, bovine pancreatic PLA2 (bpPLA2) has a unique interfacial binding mode in which Lys-56 plays an important role in its binding to anionic lipid surfaces. To identify the structural determinant of this unique interfacial binding mode of bpPLA2, we systematically mutated bpPLA2 and measured the effects of mutations on its interfacial binding and activity. First, different cationic clusters were generated in the amino-terminal alpha-helix by the N6R, G7K, and N6R/G7K mutations. These mutations enhanced the binding of bpPLA2 to anionic liposomes up to 15-fold. For these mutants, however, the K56E mutation still caused a large drop in interfacial affinity for and activity toward anionic liposomes, indicating that the generation of a cationic patch in the amino-terminal alpha-helix of bpPLA2 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 (Delta62-66), which was as active as wild-type toward anionic liposomes, the K56E and K116E mutations (Delta62-66/K56E and Delta62-66/K116E) did not have significant effects on interfacial affinity. In contrast, the K10E mutation showed a much larger decrease in interfacial affinity (10-fold), indicating the deletion of residues 62-66 caused a major change in the interfacial binding mode. Finally, hydrophobic residues in positions 63 and 65 were replaced by bulkier ones (V63F and V63F/V65L) to pinpoint the structural determinant of the interfacial binding mode of bpPLA2. The effects of K10E and K56E mutations on the interfacial affinity and activity of these mutants showed that Val-63 and Val-65 of bpPLA2 are the structural determinant of its unique interfacial binding mode and that relatively conservative substitutions at these positions result in large changes in the interfacial binding mode among mammalian pancreatic PLA2s. Taken together, this study reveals how minor structural differences among homologous PLA2s can lead to distinct interfacial binding behaviors.
Lee
B I
BI
Department of Chemistry, University of Illinois at Chicago 60607-7061, USA.
Dua
R
R
Cho
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D008081:Liposomes; D010446:Peptide Fragments; D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2