Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
J Mol Biol. 2012 Jan 13;415(2):288-306. doi: 10.1016/j.jmb.2011.10.050. Epub 2011 Nov 12.
Nucleoside triphosphate diphosphohydrolases (NTPDases) are a physiologically important class of membrane-bound ectonucleotidases responsible for the regulation of extracellular levels of nucleotides. CD39 or NTPDase1 is the dominant NTPDase of the vasculature. By hydrolyzing proinflammatory ATP and platelet-activating ADP to AMP, it blocks platelet aggregation and supports blood flow. Thus, great interest exists in understanding the structure and dynamics of this prototype member of the eukaryotic NTPDase family. Here, we report the crystal structure of a variant of soluble NTPDase1 lacking a putative membrane interaction loop identified between the two lobes of the catalytic domain. ATPase and ADPase activities of this variant are determined via a newly established kinetic isothermal titration calorimetry assay and compared to that of the soluble NTPDase1 variant characterized previously. Complex structures with decavanadate and heptamolybdate show that both polyoxometallates bind electrostatically to a loop that is involved in binding of the nucleobase. In addition, a comparison of the domain orientations of the four independent proteins in the crystal asymmetric unit provides the first direct experimental evidence for a domain motion of NTPDases. An interdomain rotation angle of up to 7.4° affects the active site cleft between the two lobes of the protein. Comparison with a previously solved bacterial NTPDase structure indicates that the domains may undergo relative rotational movements of more than 20°. Our data support the idea that the influence of transmembrane helix dynamics on activity is achieved by coupling to a domain motion.
核苷酸三磷酸二磷酸水解酶（NTPDases）是一类重要的膜结合胞外核苷酸酶，负责调节细胞外核苷酸的水平。CD39 或 NTPDase1 是血管中主要的 NTPDase。通过水解促炎的 ATP 和血小板激活的 ADP 为 AMP，它可阻止血小板聚集并维持血流。因此，人们对理解这种真核 NTPDase 家族原型成员的结构和动力学非常感兴趣。在这里，我们报告了一种可溶性 NTPDase1 变体的晶体结构，该变体缺失了在催化结构域的两个叶之间发现的假定膜相互作用环。通过新建立的动力学等温滴定量热法测定了该变体的 ATPase 和 ADPase 活性，并与先前表征的可溶性 NTPDase1 变体进行了比较。与 decavanadate 和 heptamolybdate 的复合物结构表明，两种多金属氧酸盐都通过静电相互作用与一个参与核碱基结合的环结合。此外，对晶体不对称单位中四个独立蛋白质的结构域取向的比较提供了 NTPDases 结构域运动的第一个直接实验证据。高达 7.4°的结构域旋转角度会影响蛋白质两个叶之间的活性位点裂缝。与以前解决的细菌 NTPDase 结构的比较表明，结构域可能发生超过 20°的相对旋转运动。我们的数据支持这样一种观点，即跨膜螺旋动力学对活性的影响是通过与结构域运动的偶联来实现的。
