Structure of recombinant Haemophilus influenzae e (P4) acid phosphatase reveals a new member of the haloacid dehalogenase superfamily
Structure of recombinant Haemophilus influenzae e (P4) acid phosphatase reveals a new member of the haloacid dehalogenase superfamily
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DOI：
10.1021/bi701016m
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发表时间：
2007-10-02
期刊：
影响因子：
2.9
通讯作者：
Tanner, John J.
中科院分区：
文献类型：
--
作者：
Felts, Richard L.;Ou, Zhonghui;Tanner, John J.
流感嗜血杆菌脂蛋白e（P4）属于磷酸水解酶的“DDDD”超家族，是C类非特异性酸性磷酸酶的原型。P4也是H的组分。流感疫苗我们报告的晶体结构的重组P4在配体的自由和钨酸盐抑制的形式，这是第一个结构的α/β类磷酸酶。P4具有由核心W结构域和较小的α结构域组成的双结构域架构。核心结构域的特点是一个五链的β-片层，两侧是螺旋，这让人想起卤酸脱卤酶超家族。α结构域似乎是独特的，并在底物结合和二聚化中发挥作用。活性位点是溶剂可及的，并且位于两个结构域之间的裂缝中。结构表明P4是一种金属酶，镁是晶体重组酶中最可能的金属离子。金属离子的配体是DDDD基序的第一个和第三个Asp残基的羧基、DDDD基序的第二个Asp的骨架羰基和两个水分子。钨酸盐结合酶的结构表明Asp 64是攻击底物P原子的亲核试剂。二聚化似乎是重要的催化，因为亚基间的接触稳定的活性位点。对疫苗研究工程化突变的结构背景的分析表明，最有希望的突变位于二聚体界面。这一观察结果表明了一种基于结构的疫苗设计策略，其中破坏二聚体界面以暴露埋藏在二聚体P4中的表位。
Lipoprotein e (P4) from Haemophilus influenzae belongs to the "DDDD" superfamily of phosphohydrolases and is the prototype of class C nonspecific acid phosphatases. P4 is also a component of a H. influenzae vaccine. We report the crystal structures of recombinant P4 in the ligand-free and tungstate-inhibited forms, which are the first structures of a class alpha/beta phosphatase. P4 has a two-domain architecture consisting of a core W domain and a smaller alpha domain. The core domain features a five-stranded beta-sheet flanked by helices on both sides that is reminiscent of the haloacid dehalogenase superfamily. The alpha domain appears to be unique and plays roles in substrate binding and dimerization. The active site is solvent accessible and located in a cleft between the two domains. The structure shows that P4 is a metalloenzyme and that magnesium is the most likely metal ion in the crystalline recombinant enzyme. The ligands of the metal ion are the carboxyl groups of the first and third Asp residues of the DDDD motif, the backbone carbonyl of the second Asp of the DDDD motif, and two water molecules. The structure of the tungstate-bound enzyme suggests that Asp64 is the nucleophile that attacks the substrate P atom. Dimerization appears to be important for catalysis because intersubunit contacts stabilize the active site. Analysis of the structural context of mutations engineered for vaccine studies shows that the most promising mutations are located in the dimer interface. This observation suggests a structure-based vaccine design strategy in which the dimer interface is disrupted in order to expose epitopes that are buried in dimeric P4.