title: Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.
abstract: The Group VIA-2 Ca(2+)-independent phospholipase A(2) (GVIA-2 iPLA(2)) is composed of seven consecutive N-terminal ankyrin repeats, a linker region, and a C-terminal phospholipase catalytic domain. No structural information exists for this enzyme, and no information is known about the membrane binding surface. We carried out deuterium exchange experiments with the GVIA-2 iPLA(2) in the presence of both phospholipid substrate and the covalent inhibitor methyl arachidonoyl fluorophosphonate and located regions in the protein that change upon lipid binding. No changes were seen in the presence of only methyl arachidonoyl fluorophosphonate. The region with the greatest change upon lipid binding was region 708-730, which showed a >70% decrease in deuteration levels at numerous time points. No decreases in exchange due to phospholipid binding were seen in the ankyrin repeat domain of the protein. To locate regions with changes in exchange on the enzyme, we constructed a computational homology model based on homologous structures. This model was validated by comparing the deuterium exchange results with the predicted structure. Our model combined with the deuterium exchange results in the presence of lipid substrate have allowed us to propose the first structural model of GVIA-2 iPLA(2) as well as the interfacial lipid binding region.
authors: Department of Chemistry, University of California, San Diego, La Jolla, California 92093-0601, USA.
authors: Yuan-Hao
authors: YH
authors: Hsu
authors: John E
authors: JE
authors: Burke
authors: Sheng
authors: S
authors: Li
authors: Virgil L
authors: VL
authors: Woods
authors: Edward A
authors: EA
authors: Dennis
mesh_terms: D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D054522:Group VI Phospholipases A2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D013058:Mass Spectrometry; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D000577:Amides; D010743:Phospholipids; D006859:Hydrogen; D054522:Group VI Phospholipases A2; D002118:Calcium