Crystal structure and substrate binding mode of ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3)
Crystal structure and substrate binding mode of ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3)
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DOI：
10.1038/s41598-018-28814-y
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发表时间：
2018-07-18
影响因子：
4.6
通讯作者：
Straeter, Norbert
中科院分区：
文献类型：
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作者：
Doehler, Christoph;Zebisch, Matthias;Straeter, Norbert
外核苷酸磷酸二酯酶/焦磷酸酶-3（NPP 3）是调节细胞外核苷酸水平的膜结合糖蛋白。已知NPP 3通过水解ATP促进嗜碱性粒细胞的免疫应答，并调节Neuro 2a细胞中的糖基转移酶活性。在这里，我们报告的晶体结构的核酸酶和磷酸二酯酶结构域的大鼠NPP 3在复杂的不同的基板，产品和基板类似物提供洞察细节的催化机制。具有磷酸根离子、产物AMP和底物类似物AMPNPP的复合物结构提供了底物的协调的一致的图像，其中一个锌离子激活苏氨酸亲核试剂，而另一个锌离子结合磷酸根基团。与二核苷酸底物Ap 4A和UDPGlcNAc的共晶体结构揭示了这些底物的较大离去基团的结合口袋。晶体结构以及突变和动力学分析表明，较大的离去基团仅与酶发生微弱的相互作用，使得底物亲和力由第一个核苷基团的相互作用主导。对于该部分，核碱基堆叠在Y290和F207之间，并且与蛋白质的极性相互作用仅通过水分子形成，因此解释了有限的核碱基选择性。
Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3) is a membrane-bound glycoprotein that regulates extracellular levels of nucleotides. NPP3 is known to contribute to the immune response on basophils by hydrolyzing ATP and to regulate the glycosyltransferase activity in Neuro2a cells. Here, we report on crystal structures of the nuclease and phosphodiesterase domains of rat NPP3 in complex with different substrates, products and substrate analogs giving insight into details of the catalytic mechanism. Complex structures with a phosphate ion, the product AMP and the substrate analog AMPNPP provide a consistent picture of the coordination of the substrate in which one zinc ion activates the threonine nucleophile whereas the other zinc ion binds the phosphate group. Co-crystal structures with the dinucleotide substrates Ap4A and UDPGlcNAc reveal a binding pocket for the larger leaving groups of these substrates. The crystal structures as well as mutational and kinetic analysis demonstrate that the larger leaving groups interact only weakly with the enzyme such that the substrate affinity is dominated by the interactions of the first nucleoside group. For this moiety, the nucleobase is stacked between Y290 and F207 and polar interactions with the protein are only formed via water molecules thus explaining the limited nucleobase selectivity.