Crystallographic identification of metal-binding sites in Escherichia coli inorganic pyrophosphatase.
Crystallographic identification of metal-binding sites in Escherichia coli inorganic pyrophosphatase.
复制标题
大肠杆菌无机焦磷酸酶中金属结合位点的晶体学鉴定。
DOI：
10.1021/bi952637e
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Goldman,A
中科院分区：
文献类型：
--
作者：
Kankare,J;Salminen,T;Lahti,R;Cooperman,BS;Baykov,AA;Goldman,A
我们报道了E-PPase(E-PPase)六聚体可溶性无机焦磷酸酶(E-PPase)的精细晶体结构，在2.2时和2.3时分别为18.3%和17.1%。这两种结构在R32细胞的不对称单位中都含有两个独立的单体。这两种结构的不同之处在于，后者每个单体含有1.5个镁离子。一种金属离子结合到平衡透析研究确定的“紧密的”金属结合部位，并与Asp65、Asp70和Asp102配位。另一个金属离子由两个单体在三聚体之间的二联体界面上一个未知的金属结合部位共享，通过水分子从两个单体配位到Asp26s和Asn24s。结合了金属的六角体比没有结合金属的六角体关联更紧密。结合我们的其他机制和结构数据，结果表明，在较高的金属浓度下，E-PPase可能至少与每个单体结合4.5金属： 在结合底物之前的活性部位有两个，与底物结合前有两个，在二联体界面上有0.5%。Glu20通过水分子与Asp70相互作用，并出现在相关的酵母PPase结构(Heikinheimo，手稿正在准备中)中，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来稳定六聚体形式。直接影响是通过亚基界面上更紧密的结合；间接影响发生是因为镁稳定了Glu20和Ile32之间的环的正确构象，该环参与了三聚体−三聚体的相互作用。因此，我们的结果为溶液研究提供了结构上的解释，这些研究表明E20D变体是部分六聚体，六聚体形式可以通过结合镁离子来稳定。
We report refined crystal structures of the hexameric soluble inorganic pyrophosphatase fromEscherichia coli(E-PPase) toR-factors of 18.3% and 17.1% at 2.2 and 2.3 Å, respectively. Both structures contain two independent monomers in the asymmetric unit of anR32 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 the 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 in trimer−trimer 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.