title: Interaction of group IA phospholipase A2 with metal ions and phospholipid vesicles probed with deuterium exchange mass spectrometry.
abstract: Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A 2 (GIA PLA 2) was carried out in the presence of metal ions Ca (2+) and Ba (2+) and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein-lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large alpha-helices of residues 43-53 and 89-101, which suggests that these alpha-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca (2+) or Ba (2+) ions decreased the deuterium exchange rates for five regions of the protein (residues 24-27, 29-40, 43-53, 103-110, and 111-114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24-27 and 103-110 which showed greater changes for Ca (2+). The crystal structure of the N. naja naja GIA PLA 2 contains a single Ca (2+) bound in the catalytic site, but the crystal structures of related PLA 2s contain a second Ca (2+) binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA 2 does in fact bind two Ca (2+) ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid vesicles with 100 microM Ca (2+) present at 0 degrees C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3-8, 18-21, and 56-64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate.
authors.affiliation: Department of Chemistry and Biochemistry, School of Medicine, University of California at San Diego, La Jolla, California 92093-0601, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Burke
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Karbarz
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Deems
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: Virgil L
authors.initials: VL
authors.lastname: Woods
authors.forename: Edward A
authors.initials: EA
authors.lastname: Dennis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004546:Elapid Venoms / Q000201:enzymology*; D054499:Group IA Phospholipases A2 / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D053719:Tandem Mass Spectrometry / Q000379:methods; D053835:Unilamellar Liposomes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D004546:Elapid Venoms; D010446:Peptide Fragments; D010743:Phospholipids; D053835:Unilamellar Liposomes; D001464:Barium; D054499:Group IA Phospholipases A2; D002118:Calcium