title: Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.
abstract: The GIVA phospholipase A(2) (PLA(2)) contains two domains: a calcium-binding domain (C2) and a catalytic domain. These domains are linked via a flexible tether. GIVA PLA(2) activity is Ca(2+)-dependent in that calcium binding promotes protein docking to the phospholipid membrane. In addition, the catalytic domain has a lid that covers the active site, presumably regulating GIVA PLA(2) activity. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium (H/D) exchange coupled with liquid chromatography-mass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the C2 domain, the catalytic domain, and the intact GIVA PLA(2). We also analyzed the changes in H/D exchange of the intact GIVA PLA(2) upon Ca(2+) binding. The DXMS results showed a fast H/D-exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the "cleft" region between the C2 and catalytic domains. The slow exchanging region of the C2 domain is in tight proximity to the catalytic domain. The effects of Ca(2+) binding on GIVA PLA(2) are localized in the C2 domain and suggest that binding of two distinct Ca(2+) ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step in GIVA PLA(2) activation.
authors.affiliation: Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, CA 92093-0601, USA.
authors.forename: Yuan-Hao
authors.initials: YH
authors.lastname: Hsu
authors.forename: John E
authors.initials: JE
authors.lastname: Burke
authors.forename: Daren L
authors.initials: DL
authors.lastname: Stephens
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Deems
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: Kyle M
authors.initials: KM
authors.lastname: Asmus
authors.forename: Virgil L
authors.initials: VL
authors.lastname: Woods
authors.forename: Edward A
authors.initials: EA
authors.lastname: Dennis
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D002853:Chromatography, Liquid; D003903:Deuterium / Q000737:chemistry; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D013058:Mass Spectrometry; D054467:Phospholipases A2 / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology
chemical_list: D006859:Hydrogen; D003903:Deuterium; D054467:Phospholipases A2; D002118:Calcium