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首页> 美国卫生研究院文献>Biochemical Journal >A putative glutathione-binding site in CdZn-metallothionein identified by equilibrium binding and molecular-modelling studies.
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A putative glutathione-binding site in CdZn-metallothionein identified by equilibrium binding and molecular-modelling studies.

机译：通过平衡结合和分子模型研究鉴定了CdZn-金属硫蛋白中的一个假定的谷胱甘肽结合位点。

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摘要

Glutathione (GSH) has been found to form a complex with both vertebrate and invertebrate copper-metallothionein (CuMT) [Freedman, Ciriolo and Peisach (1989) J. Biol. Chem. 264, 5598-5605; Brouwer and Brouwer-Hoexum (1991) Arch. Biochem. Biophys. 290, 207-213]. In this paper we report on the interaction of GSH with CdZnMT-I and CdZnMT-II from rabbit liver and with CdMT-I from Blue crab hepatopancreas. Ultrafiltration experiments showed that all three MTs combined with GSH. The measured binding data for the three MTs could be described by a single binding isotherm. The GSH/MT stoichiometry was 1.4 +/- 0.3 and Kdiss. = 14 +/- 6 microM. Partially Zn-depleted MT does not significantly bind GSH, indicating that the GSH-binding site is located on MT's Zn-containing N-terminal domain. The putative GSH-binding site on rabbit liver MT was investigated using molecular-graphics analysis. A cleft on the MT's N-terminal domain, which has the labile Zn-2 at its base, could easily accommodate GSH. Cysteine-ligand exchange between the terminal (non-bridging) Cys-26, bound to Zn-2, and the cysteine in GSH is stereochemically possible. Based on these considerations a model of MT-GSH was built in which GSH's cysteine replaces Cys-26 as a terminal Zn-2 ligand. This complex was energy-minimized by molecular-mechanics calculations, taking into account computed partial electrostatic charges on all atoms, including Cd and Zn. These calculations showed that the MT-GSH complex was thermodynamically more stable than MT, due to favourable non-bonded, electrostatic and van der Waals interactions. Six hydrogen bonds can form between GSH and MT. The average pairwise root-mean-square deviations (RMSD) of the metals in energy-minimized MT and MT-GSH, compared with the metals in the crystal structure, were 0.0087 +/- 0.0028 nm (0.087 +/- 0.028 A) and 0.0168 +/- 0.0087 nm (0.168 +/- 0.087 A) respectively. The RMSD values for the polypeptide-backbone alpha carbons were 0.0136 +/- 0.0060 nm (0.136 +/- 0.060 A) and 0.0491 +/- 0.0380 nm (0.491 +/- 0.380 A) respectively. No other docking sites for GSH were found. The energy-minimized structure of an MT-2-mercaptoethanol complex was somewhat less stable than the native MT domain, attesting to the specificity of the MT-GSH interaction. The possible physiological significance of the MT-GSH interaction is discussed.
机译：已经发现谷胱甘肽（GSH）与脊椎动物和无脊椎动物铜-金属硫蛋白（CuMT）形成复合物[Freedman，Ciriolo和Peisach（1989）J.Biol.Chem.Soc.Sci。，Vol.5，pp.3（1）]。化学264，5598-5605； Brouwer和Brouwer-Hoexum（1991）拱。生化。生物物理学。 290，207-213]。在本文中，我们报道了GSH与来自兔肝的CdZnMT-I和CdZnMT-II以及与蓝蟹肝胰腺的CdMT-I的相互作用。超滤实验表明，所有三种MT均与GSH结合。可以通过单个结合等温线描述三个MT的测量结合数据。 GSH / MT化学计量比为1.4 +/- 0.3，Kdiss。 = 14 +/- 6 microM。部分缺锌的MT不能显着结合GSH，表明GSH结合位点位于MT的含锌N末端结构域上。使用分子图谱分析研究了假定的GSH在兔肝MT上的结合位点。 MT的N末端区域的裂口（其基部具有不稳定的Zn-2）可以轻易地容纳GSH。立体化学上，与Zn-2结合的末端（非桥接）Cys-26与半胱氨酸之间的半胱氨酸-配体交换是可能的。基于这些考虑，建立了MT-GSH模型，其中GSH的半胱氨酸替代Cys-26作为末端Zn-2配体。考虑到在包括Cd和Zn在内的所有原子上计算出的部分静电荷，通过分子力学计算使该配合物的能量最小化。这些计算表明，由于有利的非键，静电和范德华相互作用，MT-GSH配合物在热力学上比MT更稳定。 GSH和MT之间可以形成六个氢键。与晶体结构中的金属相比，能量最小化MT和MT-GSH中金属的平均成对均方根偏差（RMSD）为0.0087 +/- 0.0028 nm（0.087 +/- 0.028 A）和0.0168 +/- 0.0087 nm（0.168 +/- 0.087 A）。多肽骨架α碳的RMSD值分别为0.0136 +/- 0.0060nm（0.136 +/- 0.060A）和0.0491 +/- 0.0380nm（0.491 +/- 0.380A）。未找到GSH的其他停靠站点。 MT-2-巯基乙醇复合物的能量最小化结构比天然MT结构域稳定性稍差，证明了MT-GSH相互作用的特异性。讨论了MT-GSH相互作用的可能的生理意义。

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