Localizing the Membrane Binding Region of Group VIA Ca2+-independent Phospholipase A2 Using Peptide Amide Hydrogen/Deuterium Exchange Mass Spectrometry
Localizing the Membrane Binding Region of Group VIA Ca2+-independent Phospholipase A2 Using Peptide Amide Hydrogen/Deuterium Exchange Mass Spectrometry
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DOI：
10.1074/jbc.m109.021857
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发表时间：
2009-08-28
影响因子：
4.8
通讯作者：
Dennis, Edward A.
中科院分区：
文献类型：
--
作者：
Hsu, Yuan-Hao;Burke, John E.;Dennis, Edward A.
基团VIA-2 Ca2+独立磷脂酶A(2) （GVIA-2 iPLA(2)）由7个连续的n端锚蛋白重复序列、一个连接体区域和一个c端磷脂酶催化结构域组成。没有这种酶的结构信息，也没有关于膜结合表面的信息。我们用GVIA-2 iPLA(2)在磷脂底物和共价抑制剂甲基花生四烯酰基氟膦酸盐的存在下进行了氘交换实验，并定位了蛋白质中随着脂质结合而变化的区域。仅存在甲基花生四烯酰基氟膦酸盐时未见变化。脂质结合后变化最大的区域是708-730区域，在许多时间点上氘化水平下降了约70%。由于磷脂结合，在蛋白质的锚蛋白重复结构域中没有看到交换减少。为了定位酶上交换变化的区域，我们基于同源结构构建了计算同源模型。通过将氘交换结果与预测结构进行比较，验证了该模型的正确性。我们的模型与存在脂质底物的氘交换结果相结合，使我们能够提出GVIA-2 iPLA的第一个结构模型(2)以及界面脂质结合区。
The Group VIA-2 Ca2+-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.