Merceron Romain, Awama Ayman M, Montserret Roland, Marcillat Olivier, Gouet Patrice
From the Institut de Biologie et Chimie des Protéines, BMSSI-IBCP, UMR 5086 CNRS Université Lyon 1, 7, Passage du Vercors, 69367 Lyon Cedex 07, France and.
the Institut de Chimie et Biochimie Moléculaire et Supramoléculaire, UMR 5246 CNRS Université Lyon 1, 69622 Villeurbanne, France.
J Biol Chem. 2015 May 15;290(20):12951-63. doi: 10.1074/jbc.M114.628909. Epub 2015 Apr 2.
The taurocyamine kinase from the blood fluke Schistosoma mansoni (SmTK) belongs to the phosphagen kinase (PK) family and catalyzes the reversible Mg(2+)-dependent transfer of a phosphoryl group between ATP and taurocyamine. SmTK is derived from gene duplication, as are all known trematode TKs. Our crystallographic study of SmTK reveals the first atomic structure of both a TK and a PK with a bilobal structure. The two unliganded lobes present a canonical open conformation and interact via their respective C- and N-terminal domains at a helix-mediated interface. This spatial arrangement differs from that observed in true dimeric PKs, in which both N-terminal domains make contact. Our structures of SmTK complexed with taurocyamine or l-arginine compounds explain the mechanism by which an arginine residue of the phosphagen specificity loop is crucial for substrate specificity. An SmTK crystal was soaked with the dead end transition state analog (TSA) components taurocyamine-NO3 (2-)-MgADP. One SmTK monomer was observed with two bound TSAs and an asymmetric conformation, with the first lobe semiclosed and the second closed. However, isothermal titration calorimetry and enzyme kinetics experiments showed that the two lobes function independently. A small angle x-ray scattering model of SmTK-TSA in solution with two closed active sites was generated.
曼氏血吸虫（SmTK）的牛磺酸胺激酶属于磷酸原激酶（PK）家族，催化ATP与牛磺酸胺之间可逆的、依赖Mg(2+)的磷酰基转移。SmTK与所有已知的吸虫TK一样，源自基因复制。我们对SmTK的晶体学研究揭示了具有双叶结构的TK和PK的首个原子结构。两个未结合配体的叶呈现出典型的开放构象，并通过它们各自的C端和N端结构域在螺旋介导的界面处相互作用。这种空间排列不同于在真正的二聚体PK中观察到的情况，在真正的二聚体PK中，两个N端结构域相互接触。我们的SmTK与牛磺酸胺或L-精氨酸化合物复合的结构解释了磷酸原特异性环的精氨酸残基对底物特异性至关重要的机制。用终产物过渡态类似物（TSA）成分牛磺酸胺-NO3(2-)-MgADP浸泡SmTK晶体。观察到一个SmTK单体与两个结合的TSA和不对称构象，第一个叶半封闭，第二个叶封闭。然而，等温滴定量热法和酶动力学实验表明，两个叶独立发挥作用。生成了具有两个封闭活性位点的溶液中SmTK-TSA的小角X射线散射模型。
