Zebisch Matthias, Sträter Norbert
Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
Proc Natl Acad Sci U S A. 2008 May 13;105(19):6882-7. doi: 10.1073/pnas.0802535105. Epub 2008 May 5.
Cell surface-located nucleoside triphosphate diphosphohydrolases (NTPDase1, -2, -3, and -8) are oligomeric integral membrane proteins responsible for signal conversion and inactivation in extracellular nucleotide-mediated "purinergic" signaling. They catalyze the sequential hydrolysis of the signaling molecule ATP via ADP to AMP. Here we present the structure of the extracellular domain of Rattus norvegicus NTPDase2 in an active state at resolutions between 1.7 A and 2.1 A in four different forms: (i) apo form, (ii) ternary complex with the nonhydrolyzable ATP analog AMPPNP and cofactor Ca(2+), (iii) quaternary complex with Ca(2+) and bound products AMP and phosphate, and (iv) binary product complex with AMP only. Analysis of the ATP (analog) binding mode explains the importance of several residues for activity and allows suggestion of a catalytic mechanism. The carboxylate group of E165 serves as a catalytic base and activates a water molecule, which is well positioned for nucleophilic attack on the terminal phosphate. Based on analysis of the two product complex structures in which AMP adopts different conformations, a substrate binding mode for ADP hydrolysis is proposed. This allows for an understanding of how the same hydrolytic site can be engaged in ATP and ADP but not AMP hydrolysis.
细胞表面定位的核苷三磷酸二磷酸水解酶（NTPDase1、-2、-3和-8）是寡聚整合膜蛋白，负责细胞外核苷酸介导的“嘌呤能”信号传导中的信号转换和失活。它们催化信号分子ATP依次通过ADP水解为AMP。在这里，我们展示了褐家鼠NTPDase2细胞外结构域在四种不同形式下处于活性状态的结构，分辨率在1.7 Å至2.1 Å之间：（i）无配体形式，（ii）与不可水解的ATP类似物AMPPNP和辅因子Ca(2+)的三元复合物，（iii）与Ca(2+)以及结合产物AMP和磷酸的四元复合物，以及（iv）仅与AMP的二元产物复合物。对ATP（类似物）结合模式的分析解释了几个残基对活性的重要性，并提出了一种催化机制。E165的羧基作为催化碱并激活一个水分子，该水分子处于对末端磷酸进行亲核攻击的有利位置。基于对AMP采用不同构象的两种产物复合物结构的分析，提出了ADP水解的底物结合模式。这有助于理解同一个水解位点如何参与ATP和ADP的水解，但不参与AMP的水解。
