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A Phospholipid Substrate Molecule Residing in the Membrane Surface Me‐diates Opening of the Lid Region in Group IVA Cytosolic Phospholipase A2
Author(s) -
Burke John Edmund,
Hsu YuanHao,
Woods Virgil L,
Dennis Edward A
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.873.1
Subject(s) - phospholipase a2 , chemistry , phospholipid , cytosol , biophysics , substrate (aquarium) , membrane , binding site , c2 domain , lipid bilayer , vesicle , phospholipase , intracellular , biochemistry , hydrogen–deuterium exchange , enzyme , chromatography , biology , mass spectrometry , ecology
The Group IVA phospholipase A2 (GIVA PLA 2 ) associates with natural membranes in response to an increase in intracellular Ca 2+ , or increases in certain lipid mediators. Employing deuterium exchange mass spectrometry (DXMS), we have identified the regions of the protein binding the lipid surface, and conformational changes upon a single substrate binding in the absence of a lipid surface. Lipid binding produced both increase and decreases in exchange. The regions with decreased exchange included the C2 domain, and a charged patch of residues on the catalytic domain. The regions with an increase in exchange are all located near the lid and hinge regions from 403‐457. Using the GIVA PLA 2 irreversible inhibitor methyl‐arachidonyl flourophosphonate (MAFP) we were able to isolate structural changes caused only by pseudo‐substrate binding. The changes seen in the catalytic domain are the same with substrate and inhibitor, which implies that most of the changes are due to a substrate mediated, not interface mediated, lid opening which exposes the active site to water. Finally experiments with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface. This represents a new methodology to study membrane associated proteins.