Kleinboelting Silke, van den Heuvel Joop, Kambach Christian, Weyand Michael, Leipelt Martina, Steegborn Clemens
Department of Biochemistry, University of Bayreuth, 95445 Bayreuth, Germany.
Helmholtz-Zentrum für Infektionsforschung, 38124 Braunschweig, Germany.
Acta Crystallogr F Struct Biol Commun. 2014 Apr;70(Pt 4):467-9. doi: 10.1107/S2053230X14004014. Epub 2014 Mar 25.
The second messenger cAMP is synthesized in mammals by ten differently regulated adenylyl cyclases (AC1-10). These ACs are grouped into nucleotidyl cyclase class III based on homologies in their catalytic domains. The catalytic domain of AC10 is unique, however, in being activated through direct interaction with calcium and bicarbonate. Here, the production, crystallization and X-ray diffraction analysis of the catalytic domain of human AC10 are described as a basis for structural studies of regulator binding sites and mechanisms. The recombinant protein had high specific AC activity, and crystals of AC10 in space group P63 diffracted to ∼2.0 Å resolution on a synchrotron beamline. A complete diffraction data set revealed unit-cell parameters a = b = 99.65, c = 98.04 Å, indicating one AC10 catalytic domain per asymmetric unit, and confirmed that the obtained crystals are suitable for structure solution and mechanistic studies.
在哺乳动物中，第二信使环磷酸腺苷（cAMP）由十种调控方式不同的腺苷酸环化酶（AC1 - 10）合成。基于其催化结构域的同源性，这些腺苷酸环化酶被归类为III类核苷酸环化酶。然而，AC10的催化结构域独特之处在于它通过与钙和碳酸氢根直接相互作用而被激活。在此，描述了人AC10催化结构域的产生、结晶及X射线衍射分析，作为研究调节剂结合位点和机制的结构基础。重组蛋白具有高特异性AC活性，空间群为P63的AC10晶体在同步加速器光束线上衍射分辨率达到约2.0 Å。完整的衍射数据集显示晶胞参数a = b = 99.65，c = 98.04 Å，表明每个不对称单元中有一个AC10催化结构域，并证实所获得的晶体适用于结构解析和机制研究。
