Crystallization of ectonucleotide phosphodiesterase/pyrophosphatase-3 and orientation of the SMB domains in the full-length ectodomain
Crystallization of ectonucleotide phosphodiesterase/pyrophosphatase-3 and orientation of the SMB domains in the full-length ectodomain
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DOI：
10.1107/s2053230x18011111
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发表时间：
2018-11-01
影响因子：
0.9
通讯作者：
Straeter, Norbert
中科院分区：
文献类型：
--
作者：
Doehler, Christoph;Zebisch, Matthias;Straeter, Norbert
外核苷酸磷酸二酯酶/焦磷酸酶-3（NPP 3，ENPP 3）是一种位于细胞外空间的ATP水解糖蛋白。在HEK 293 S GntI（-）细胞中表达大鼠NPP 3的全长胞外域，使用两个色谱步骤纯化并结晶。其在2.77埃分辨率下的结构显示活性位点锌离子缺失，并且大部分活性位点和周围残基是柔性的。SMB样结构域在不对称单元的所有四个分子中具有相同的取向。SMB 2结构域的取向与NPP 2相同，但SMB 1结构域不与PDE结构域相互作用，而是进一步远离PDE结构域延伸。删除SMB域导致衍射至2.4埃分辨率的晶体，并且适合于基底结合研究。
Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3, ENPP3) is an ATP-hydrolyzing glycoprotein that is located in the extracellular space. The full-length ectodomain of rat NPP3 was expressed in HEK293S GntI(-) cells, purified using two chromatographic steps and crystallized. Its structure at 2.77 angstrom resolution reveals that the active-site zinc ions are missing and a large part of the active site and the surrounding residues are flexible. The SMB-like domains have the same orientation in all four molecules in the asymmetric unit. The SMB2 domain is oriented as in NPP2, but the SMB1 domain does not interact with the PDE domain but extends further away from the PDE domain. Deletion of the SMB domains resulted in crystals that diffracted to 2.4 angstrom resolution and are suitable for substrate-binding studies.