Department of Chemistry, The University of Western Ontario, London, Canada N6A 5B7.
Biochemistry. 2013 Apr 9;52(14):2461-71. doi: 10.1021/bi400021b. Epub 2013 Mar 29.
Mammalian metallothioneins bind up to seven Zn(2+) ions in two distinct domains: an N-terminal β-domain that binds three Zn(2+) ions and a C-terminal α-domain that binds four Zn(2+) ions. Domain specificity has been invoked in the metalation mechanism with cluster formation and bridging of the 20 Cys residues taking place prior to saturation with seven Zn(2+) ions. We report a novel experiment that examines Zn(2+) metalation by exploiting the expected decrease in K(F) at the onset of clustering using electrospray ionization mass spectrometry (ESI-MS). During the titration with Zn(2+), the ESI-MS data show that several metalated species coexist until the fully saturated proteins are formed. The relative Zn binding affinities of the seven total sites in the α- and β-fragments were determined through direct competition for added Zn(2+). The K(F) values for each Zn(2+) are expected to decrease as a function of the remaining available sites and the onset of clustering. Analysis shows that Zn(2+) binds to β-rhMT with a greater affinity than α-rhMT. The incremental distribution of Zn(2+) between the competing fragments and apo-βα-rhMT (essentially three and four sites competing with seven sites) identifies the exact point at which clustering begins in the full protein. Analysis of the speciation data shows that Zn(5)-MT forms before clustering begins. This means that all 20 Cys residues of apo-βα-rhMT are bound terminally to Zn(2+) as Zn(Cys)(4) units before clustering begins; there is no domain preference in this first metalation stage. Preferential binding of Zn(2+) to β- and α-rhMT at the point where βα-rhMT must form clusters is caused by a significant decrease in the affinity of βα-rhMT for further Zn(2+). The single-domain Zn(5)-rhMT, in which there are no exposed cysteine sulfurs, is a key component of the metalation pathway because the lower affinities of the two clustered Zn(2+) ions allow donation to apoenzymes.
哺乳动物金属硫蛋白在两个不同的结构域中结合多达七个锌（2+）离子：一个 N 端的β-结构域结合三个锌（2+）离子，一个 C 端的α-结构域结合四个锌（2+）离子。在金属化机制中，域特异性被认为涉及簇形成和 20 个半胱氨酸残基的桥接，这些反应在七个锌（2+）离子饱和之前发生。我们报告了一项新的实验，利用电喷雾电离质谱（ESI-MS）检测簇形成时预期的 K（F）下降，来研究锌（2+）的金属化。在锌（2+）滴定过程中，ESI-MS 数据表明，几种金属化物种共存，直到形成完全饱和的蛋白质。通过直接竞争添加的锌（2+），确定了α-和β-片段中七个总位点的相对锌结合亲和力。每个锌（2+）的 K（F）值预计会随着剩余可用位点的减少和簇形成的开始而降低。分析表明，锌（2+）与β-rhMT 的结合亲和力大于α-rhMT。竞争片段和 apo-αβ-rhMT（实际上是三个和四个位点与七个位点竞争）之间锌（2+）的增量分布确定了在完整蛋白质中簇形成开始的确切位置。物种形成数据的分析表明，Zn（5）-MT 先于簇形成开始形成。这意味着 apo-αβ-rhMT 的所有 20 个半胱氨酸残基在簇形成开始之前都作为 [Zn（Cys）（4）]（2-）单元末端结合到锌（2+）上；在这个第一个金属化阶段没有结构域偏好。当βα-rhMT 必须形成簇时，β-和α-rhMT 对锌（2+）的优先结合是由于 βα-rhMT 对进一步锌（2+）的亲和力显著降低所致。没有暴露的半胱氨酸硫的单结构域 Zn（5）-rhMT 是金属化途径的关键组成部分，因为两个簇状锌（2+）离子的较低亲和力允许捐赠给无酶蛋白。
