Crystal structure of phospholipase A2 complex with the hydrolysis products of platelet activating factor: equilibrium binding of fatty acid and lysophospholipid-ether at the active site may be mutually exclusive.
We have solved the 1.55 A crystal structure of the anion-assisted dimer of porcine pancreatic group IB phospholipase A2 (PLA2), complexed with the products of hydrolysis of the substrate platelet activating factor. The dimer contains five coplanar phosphate anions bound at the contact surface between the two PLA2 subunits. This structure parallels a previously reported anion-assisted dimer that mimics the tetrahedral intermediate of PLA2 bound to a substrate interface [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. The dimer structure has a molecule of the product acetate bound in subunit A and the other product 1-octadecyl-sn-glycero-3-phosphocholine (LPC-ether) to subunit B. Therefore, this structure is of the two individual product binary complexes and not of a ternary complex with both products in one active site of PLA2. Protein crystals with bound products were only obtained by cocrystallization starting from the initial substrate. In contrast, an alternate crystal form was obtained when PLA2 was cocrystallized with LPC-ether and succinate, and this crystal form did not contain bound products. The product bound structure has acetate positioned in the catalytic site of subunit A such that one of its oxygen atoms is located 3.5 A from the catalytic calcium. Likewise, a longer than typical Ca-to-Gly(32) carbonyl distance of 3.4 A results in a final Ca coordination that is four-coordinate and has distorted geometry. The other oxygen of acetate makes hydrogen bonds with N(delta)(1)-His(48), O(delta)(1)-Asp(49), and the catalytic assisting water (w7). In contrast, the glycerophosphocholine headgroup of LPC-ether in subunit B makes no contacts with calcium or with the catalytic residues His(48) or Asp(49). The tail of the LPC-ether is located near the active site pocket with the last nine carbons of the sn-1- acyl chain refined in two alternate conformations. The remaining atoms of the LPC-ether product have been modeled into the solvent channel but have their occupancies set to zero in the refined model due to disorder. Together, the crystallographic and equilibrium binding results with the two products show that the simultaneous binding of both the products in a single active site is not favored.
Pan
Ying H
YH
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, and Department of Molecular Evolution, Uppsala University Evolutionary Biology Center, Uppsala, Sweden.
Yu
Bao-Zhu
BZ
Berg
Otto G
OG
Jain
Mahendra K
MK
Bahnson
Brian J
BJ
D000818:Animals; D000838:Anions; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D008024:Ligands; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism*; D054467:Phospholipases A2; D010742:Phospholipid Ethers / Q000737:chemistry*; D010972:Platelet Activating Factor / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine
D000838:Anions; D008024:Ligands; D010742:Phospholipid Ethers; D010972:Platelet Activating Factor; D021122:Protein Subunits; C026659:edelfosine; D006639:Histidine; D010741:Phospholipases A; D054467:Phospholipases A2