Bofill R, Capdevila M, Cols N, Atrian S, Gonzàlez-Duarte P
Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
J Biol Inorg Chem. 2001 Apr;6(4):405-17. doi: 10.1007/s007750100216.
We postulate that zinc(II) is a keystone in the structure of physiological mouse copper metallothionein 1 (Cu-MT 1). Only when Zn(II) is coordinated does the structure of the in vivo- and in vitro-conformed Cu-MT species consist of two additive domains. Therefore, the functionally active forms of the mammalian Cu-MT may rely upon a two-domain structure. The in vitro behaviour of the whole protein is deduced from the Cu titration of the apo and Zn-containing forms and compared with that of the independent fragments using CD, UV-vis, ESI-MS and ICP-AES. We propose the formation of the following Cu, Zn-MT species during Zn/Cu replacement in Zn7-MT: (Zn4)alpha(Cu4Zn1)beta-MT, (Cu3Zn2)alpha(Cu4Zn1)beta-MT and (Cu4Zn1)alpha(Cu6)beta-MT. The cooperative formation of (Cu3Zn2)alpha(Cu4Zn1)beta-MT from (Zn4)alpha(Cu4Zn1)beta-MT indicates that the preference of Cu(I) for binding to the beta domain is only partial and not absolute, as otherwise accepted. Homometallic Cu-MT species have been obtained either from the apoform of MT or from Zn7-MT after total replacement of zinc. In these species, copper distribution cannot be inferred from the sum of the independent alpha and beta fragments. The in vivo synthesis of the entire MT in Cu-supplemented media has afforded Cu7Zn3-MT [(Cu3Zn2)alpha(Cu4Zn1)beta-MT], while that of alpha MT has rendered a mixture of Cu4Zn1-alpha MT (40%), Cu5Zn1-alpha MT (20%) and Cu7-alpha MT (40%). In the case of beta MT, a mixture of Cu6-beta MT (25%) and Cu7-beta MT (75%) was recovered [1]. These species correspond to some of those conformed in vitro and confirm that Zn(II) is essential for the in vivo folding of Cu-MT in a Cu-rich environment. A final significant issue is that common procedures used to obtain mammalian Cu6-beta MT from native sources may not be adequate.
我们推测锌（II）是生理状态下小鼠铜金属硫蛋白1（Cu-MT 1）结构的关键所在。只有当锌（II）配位时，体内和体外形成的铜金属硫蛋白物种的结构才由两个加性结构域组成。因此，哺乳动物铜金属硫蛋白的功能活性形式可能依赖于双结构域结构。通过对脱辅基形式和含锌形式进行铜滴定来推断整个蛋白质的体外行为，并使用圆二色光谱（CD）、紫外可见光谱（UV-vis）、电喷雾质谱（ESI-MS）和电感耦合等离子体发射光谱（ICP-AES）将其与独立片段的行为进行比较。我们提出在锌7-金属硫蛋白（Zn7-MT）的锌/铜置换过程中会形成以下铜、锌-金属硫蛋白物种：(Zn4)α(Cu4Zn1)β-MT、(Cu3Zn2)α(Cu4Zn1)β-MT和(Cu4Zn1)α(Cu6)β-MT。从(Zn4)α(Cu4Zn1)β-MT协同形成(Cu3Zn2)α(Cu4Zn1)β-MT表明，铜（I）与β结构域结合的偏好只是部分的而非绝对的，并非如通常所认为的那样。通过金属硫蛋白的脱辅基形式或锌完全被置换后的锌7-金属硫蛋白已获得同金属铜-金属硫蛋白物种。在这些物种中，无法从独立的α和β片段的总和推断铜的分布。在补充铜的培养基中体内合成整个金属硫蛋白得到了铜7锌3-金属硫蛋白[(Cu3Zn2)α(Cu4Zn1)β-MT]，而α金属硫蛋白的合成得到了铜4锌1-α金属硫蛋白（40%）、铜5锌1-α金属硫蛋白（20%）和铜7-α金属硫蛋白（40%）的混合物。就β金属硫蛋白而言，回收了铜6-β金属硫蛋白（25%）和铜7-β金属硫蛋白（75%）的混合物[1]。这些物种与一些体外形成的物种相对应，并证实锌（II）对于在富铜环境中铜-金属硫蛋白的体内折叠至关重要。最后一个重要问题是，从天然来源获得哺乳动物铜6-β金属硫蛋白的常用方法可能并不适用。
