Pinter Tyler B J, Stillman Martin J
Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7.
Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7
Biochem J. 2015 Nov 1;471(3):347-56. doi: 10.1042/BJ20150676. Epub 2015 Aug 20.
Mammalian metallothioneins (MTs) bind up to seven Zn(2+) using a large number of cysteine residues relative to their small size and can act as zinc-chaperones. In metal-saturated Zn7-MTs, the seven zinc ions are co-ordinated tetrahedrally into two distinct clusters separated by a linker; the N-terminal β-domain [(Zn3Cys9)(3-)] and C-terminal α-domain [(Zn4Cys11)(3-)]. We report on the competitive zinc metalation of apo-carbonic anhydrase [CA; metal-free CA (apo-CA)] in the presence of apo-metallothionein 1A domain fragments to identify domain specific determinants of zinc binding and zinc donation in the intact two-domain Znn-βαMT1A (human metallothionein 1A isoform; n=0-7). The apo-CA is shown to compete effectively only with Zn2-3-βMT and Zn4-αMT. Detailed modelling of the ESI mass spectral data have revealed the zinc-binding affinities of each of the zinc-binding sites in the two isolated fragments. The three calculated equilibrium zinc affinities [log(KF)] of the isolated β-domain were: 12.2, 11.7 and 11.4 and the four isolated α-domain affinities were: 13.5, 13.2, 12.7 and 12.6. These data provide guidance in identification of the location of the strongest-bound and weakest-bound zinc in the intact two-domain Zn7βαMT. The β-domain has the weakest zinc-binding site and this is where zinc ions are donated from in the Zn7-βαMT. The α-domain with the highest affinity binds the first zinc, which we propose leads to an unscrambling of the cysteine ligands from the apo-peptide bundle. We propose that stabilization of the intact Zn6-MT and Zn7-MT, relative to that of the sum of the separated fragments, is due to the availability of additional cysteine ligand orientations (through interdomain interactions) to support the clustered structures.
哺乳动物金属硫蛋白（MTs）相对其较小的尺寸而言，利用大量半胱氨酸残基结合多达7个Zn(2+)，并可作为锌伴侣。在金属饱和的Zn7-MTs中，7个锌离子以四面体方式配位形成两个由连接子隔开的不同簇；N端β结构域[（Zn3Cys9）(3-)]和C端α结构域[（Zn4Cys11）(3-)]。我们报道了在无金属硫蛋白1A结构域片段存在的情况下，无锌碳酸酐酶[CA；无金属CA（无锌CA）]的竞争性锌金属化过程，以确定完整的双结构域Znn-βαMT1A（人金属硫蛋白1A异构体；n = 0 - 7）中锌结合和锌捐赠的结构域特异性决定因素。结果表明，无锌CA仅与Zn2-3-βMT和Zn4-αMT有效竞争。对电喷雾质谱数据的详细建模揭示了两个分离片段中每个锌结合位点的锌结合亲和力。分离的β结构域的三个计算平衡锌亲和力[log(KF)]分别为：12.2、11.7和11.4，分离的α结构域的四个亲和力分别为：13.5、13.2、12.7和12.6。这些数据为确定完整双结构域Zn7βαMT中结合最强和最弱的锌的位置提供了指导。β结构域具有最弱的锌结合位点，这也是Zn7-βαMT中锌离子捐赠的位置。具有最高亲和力的α结构域结合第一个锌，我们认为这导致了无锌肽束中半胱氨酸配体的解缠。我们提出，相对于分离片段的总和，完整的Zn6-MT和Zn7-MT的稳定性是由于存在额外的半胱氨酸配体取向（通过结构域间相互作用）来支持簇状结构。
