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首页> 外文期刊>Journal of biological inorganic chemistry: JBIC: a publication of the Society of Biological Inorganic Chemistry >Nickel binding properties of Helicobacter pylori UreF, an accessory protein in the nickel-based activation of urease
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Nickel binding properties of Helicobacter pylori UreF, an accessory protein in the nickel-based activation of urease

机译：幽门螺杆菌UreF的镍结合特性，它是尿素酶基于镍的激活中的辅助蛋白

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摘要

Helicobacter pylori UreF (HpUreF) is involved in the insertion of Ni~(2+) in the urease active site. The recombinant protein in solution is a dimer characterized by an extensive a-helical structure and a wellfolded tertiary structure. HpUreF binds two Ni~(2+) ions per dimer, with a micromolar dissociation constant, as shown by calorimetry. X-ray absorption spectroscopy indicated that the Ni~(2+) ions reside in a five-coordinate pyramidal geometry comprising exclusively N/O-donor ligands derived from the protein, including one or two histidine imidazole and carboxylate ligands. Binding of Ni~(2+) does not affect the solution properties of the protein. Mutation to alanine of His229 and/or Cys231, a pair of residues located on the protein surface that interact with H. pylori UreD, altered the affinity of the protein for Ni~(2+). This result, complemented by the findings from X-ray absorption spectroscopy, indicates that the Ni~(2+) binding site involves His229, and that Cys231 has an indirect structural role in metal binding. An in vivo assay of urease activation demonstrated that H229A HpUreF, C231A HpUreF, and H229/C231 HpUreF are significantly less competent in this process, suggesting a role for a Ni~(2+) complex with UreF in urease maturation. This hypothesis was supported by calculations revealing the presence of a tunnel that joins the Cys-Pro-His metal binding site on UreG and an opening on the UreD surface, passing through UreF close to His229 and Cys231, in the structure of the H. pylori UreDFG complex. This tunnel could be used to transfer nickel into the urease active site during apoenzyme-to-holoenzyme activation.
机译：幽门螺杆菌UreF（HpUreF）参与在尿素酶活性位点插入Ni〜（2+）。溶液中的重组蛋白是二聚体，其特征在于广泛的α-螺旋结构和折叠良好的三级结构。 HpUreF与每个二聚体结合两个Ni〜（2+）离子，具有微摩尔解离常数，如量热法所示。 X射线吸收光谱法表明，Ni〜（2+）离子位于五坐标的锥体几何结构中，仅包含衍生自蛋白质的N / O供体配体，包括一个或两个组氨酸咪唑和羧酸盐配体。 Ni〜（2+）的结合不会影响蛋白质的溶液性质。 His229和/或Cys231（位于蛋白表面的一对残基与幽门螺杆菌UreD相互作用）的突变为丙氨酸，改变了蛋白对Ni〜（2+）的亲和力。该结果与X射线吸收光谱法的发现相补充，表明Ni〜（2+）结合位点涉及His229，而Cys231在金属结合中具有间接的结构作用。尿素酶激活的体内分析表明，H229A HpUreF，C231A HpUreF和H229 / C231 HpUreF在此过程中的能力明显较低，这表明与UreF形成的Ni〜（2+）复合物在尿素酶成熟中的作用。该假设得到了计算的支持，该计算揭示了在幽门螺杆菌的结构中存在一条隧道，该隧道连接UreG上的Cys-Pro-His金属结合位点和UreD表面的一个开口，穿过靠近His229和Cys231的UreF。 UreDFG复合体。该通道可用于在脱辅酶至全酶活化过程中将镍转移至脲酶活性位点。

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