Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy.
Biochem Biophys Res Commun. 2013 Jan 11;430(2):769-73. doi: 10.1016/j.bbrc.2012.11.073. Epub 2012 Dec 1.
ZnuA is the soluble component of the high-affinity ZnuABC zinc transporter belonging to the ATP-binding cassette-type periplasmic Zn-binding proteins. The zinc transporter ZnuABC is composed by three proteins: ZnuB, the membrane permease, ZnuC, the ATPase component and ZnuA, the soluble periplasmic metal-binding protein which captures Zn and delivers it to ZnuB. The ZnuA protein contains a charged flexible loop, rich in histidines and acidic residues, showing significant species-specific differences. Various studies have established that this loop contributes to the formation of a secondary zinc binding site, which has been proposed to be important in the acquisition of periplasmic Zn for its delivery to ZnuB or for regulation of zinc uptake. Due to its high mobility the structure of the histidine-rich loop has never been solved by X-ray diffraction studies. In this paper, through a combined use of molecular modeling, mutagenesis and fluorescence spectroscopy, we confirm the presence of two zinc binding sites characterized by different affinities for the metal ion and show that the flexibility of the loop is modulated by the binding of the zinc ions to the protein. The data obtained by fluorescence spectroscopy have then be used to validate a 3D model including the unsolved histidine-rich loop.
ZnuA 是高亲和力 ZnuABC 锌转运体的可溶性成分，属于 ATP 结合盒型周质 Zn 结合蛋白。锌转运体 ZnuABC 由三种蛋白组成：ZnuB，膜渗透酶；ZnuC，ATP 酶成分；ZnuA，可溶性周质金属结合蛋白，它捕获 Zn 并将其递送至 ZnuB。ZnuA 蛋白含有带电荷的柔性环，富含组氨酸和酸性残基，具有显著的物种特异性差异。多项研究已经确立，该环有助于形成二级锌结合位点，这对于从周质中获取锌以递送至 ZnuB 或调节锌摄取可能很重要。由于其高迁移率，通过 X 射线衍射研究从未解决过富含组氨酸环的结构。在本文中，我们通过分子建模、突变和荧光光谱学的综合使用，证实了存在两个锌结合位点，其特征为对金属离子的不同亲和力，并表明环的柔韧性受锌离子与蛋白质结合的调节。然后，我们使用荧光光谱学获得的数据来验证包括未解决的富含组氨酸环的 3D 模型。
