Calcium Binding Rigidifies the C2 Domain and the Intradomain Interaction of GIVA Phospholipase A sub(2) as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry

The GIVA phospholipase A sub(2) (PLA sub(2)) contains two domains: a calcium-binding domain (C2) and a catalytic domain. These domains are linked via a flexible tether. GIVA PLA sub(2) activity is Ca super(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 sub(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 chromatographymass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the C2 domain, the catalytic domain, and the intact GIVA PLA sub(2). We also analyzed the changes in H/D exchange of the intact GIVA PLA sub(2) upon Ca super(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 super(2+) binding on GIVA PLA sub(2) are localized in the C2 domain and suggest that binding of two distinct Ca super(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 sub(2) activation.