Kankare J, Salminen T, Lahti R, Cooperman B S, Baykov A A, Goldman A
Centre for Biotechnology, SF-20521 Turku, Finland.
Biochemistry. 1996 Apr 16;35(15):4670-7. doi: 10.1021/bi952637e.
We report refined crystal structures of the hexameric soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) to R-factors of 18.3% and 17.1% at 2.2 and 2.3 angstroms, respectively. Both structures contain two independent monomers in the asymmetric unit of an R32 cell. The difference between the structures is that the latter contains 1.5 Mg2+ ions per monomer. One metal ion binds to the "tight" metal-binding site identified by equilibrium dialysis studies, and is coordinated to Asp65, Asp70, and Asp102. The other metal ion, shared between two monomers at a hitherto unidentified metal-binding site in the dyad interface between trimers, is coordinated through water molecules to Asp26s and Asn24s from two monomers. The hexamers with metal bound to them are more tightly associated than the ones without metal bound to them. Combined with our other mechanistic and structural data, the results suggest that, at high metal concentrations, E-PPase may bind at least 4.5 metals per monomer: two in the active site before binding substrate, two with substrate, and 0.5 in the dyad interface. Glu20 interacts via a water molecule with Asp70 and appears in the related yeast PPase structure (Heikinheimo, manuscript in preparation) to be involved in binding the second metal ion. Magnesium ion therefore stabilizes the hexamer form through both direct and indirect effects. The direct effect is by tighter association at the subunit interface; the indirect effect occurs because magnesium stabilizes the correct conformation of the loop between Glu20 and Ile32, a loop involved a trimer-trimmer interactions. Our results thus provide a structural explanation for the solution studies that show that the E20D variant is partially hexameric and that the hexamer form can be stabilized by binding magnesium ion.
我们报道了来自大肠杆菌的六聚体可溶性无机焦磷酸酶（E-PPase）的精细晶体结构，在2.2埃和2.3埃分辨率下，R因子分别为18.3%和17.1%。这两种结构在R32晶胞的不对称单元中均包含两个独立的单体。两种结构的差异在于，后者每个单体含有1.5个Mg2+离子。一个金属离子结合到通过平衡透析研究确定的“紧密”金属结合位点，并与Asp65、Asp70和Asp102配位。另一个金属离子在三聚体之间二元界面处一个迄今未明确的金属结合位点上由两个单体共享，通过水分子与来自两个单体的Asp26和Asn24配位。结合了金属的六聚体比未结合金属的六聚体结合得更紧密。结合我们其他的机制和结构数据，结果表明，在高金属浓度下，E-PPase每个单体可能结合至少4.5个金属：在结合底物之前活性位点中有两个，与底物结合时有两个，在二元界面中有0.5个。Glu20通过一个水分子与Asp70相互作用，并且在相关的酵母PPase结构中（Heikinheimo，正在准备的手稿）似乎参与结合第二个金属离子。因此，镁离子通过直接和间接作用稳定六聚体形式。直接作用是通过在亚基界面处更紧密的结合；间接作用的发生是因为镁稳定了Glu20和Ile32之间环的正确构象，该环参与三聚体-三聚体相互作用。我们的结果因此为溶液研究提供了一个结构解释，该研究表明E20D变体部分为六聚体，并且六聚体形式可以通过结合镁离子而稳定。
