Hsu Yuan-Hao, Burke John E, Stephens Daren L, Deems Raymond A, Li Sheng, Asmus Kyle M, Woods Virgil L, Dennis Edward A
Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, CA 92093-0601, USA.
J Biol Chem. 2008 Apr 11;283(15):9820-7. doi: 10.1074/jbc.M708143200. Epub 2008 Jan 21.
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.
GIVA磷脂酶A2（PLA2）包含两个结构域：一个钙结合结构域（C2）和一个催化结构域。这些结构域通过一个柔性连接链相连。GIVA PLA2的活性依赖于Ca2+，因为钙结合促进蛋白质与磷脂膜对接。此外，催化结构域有一个覆盖活性位点的盖子，推测其调节GIVA PLA2的活性。我们现在展示了利用肽酰胺氢/氘（H/D）交换结合液相色谱-质谱（DXMS）来探测C2结构域、催化结构域和完整的GIVA PLA2在溶液中的溶剂可及性和主链柔韧性，从而探索该酶在溶液中的动力学和构象变化的研究。我们还分析了Ca2+结合后完整的GIVA PLA2的H/D交换变化。DXMS结果显示有一个快速H/D交换的盖子和一个缓慢交换的中心核心。C2结构域显示出两个不同的区域：一个背离催化结构域的快速交换区域和一个存在于C2结构域与催化结构域之间“裂隙”区域的缓慢交换区域。C2结构域缓慢交换区域紧邻催化结构域。Ca2+结合对GIVA PLA2的影响局限于C2结构域，表明两个不同Ca2+离子的结合导致C2结构域顶端围绕阴离子空穴的区域收紧。这种构象变化可能是GIVA PLA2激活的初始步骤。
