Hints for metal-preference protein sequence determinants: different metal binding features of the five tetrahymena thermophila metallothioneins.
Hints for metal-preference protein sequence determinants: different metal binding features of the five tetrahymena thermophila metallothioneins.
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DOI：
10.7150/ijbs.11060
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发表时间：
2015
影响因子：
9.2
通讯作者：
Atrian S
中科院分区：
文献类型：
--
作者：
Espart A;Marín M;Gil-Moreno S;Palacios Ò;Amaro F;Martín-González A;Gutiérrez JC;Capdevila M;Atrian S
金属硫蛋白（MT）的金属结合偏好将其分为两个极端的子集，Zn/Cd-和Cu-硫蛋白。纤毛虫是迄今为止报道的最大的MT基因/蛋白家族，除了MTT 2和MTT 4外，还有5个同源基因，它们的序列相似性相对较低。根据基因表达诱导和系统发育分析，在嗜热四膜虫中发现3个MT（MTT 1、MTT 3和MTT 5）为Cd硫蛋白，2个MT（MTT 2和MTT 4）为Cu硫蛋白。在这项研究中，五个MTT蛋白的金属结合能力的特点，以获得有关其同源和非同源金属络合物的折叠和稳定性的信息，并表征T。在蛋白水平上对嗜热菌MT系统进行了初步研究。因此，五个MTTs重组合成为Zn 2 +-，Cd 2 +-或Cu+-配合物，通过电喷雾质谱（ESI-MS），圆二色性（CD），紫外-可见分光光度法进行分析。在镉硫蛋白，MTT 1和MTT 5是最佳的Cd 2+的协调，产生独特的Cd 17-和Cd 8-复合物，分别。当与Zn ~（2+）结合时，它们呈现Zn-物种的混合物。只有MTT 5能够配位Cu+，虽然产生杂合的Zn-、Cu-物种或高度不稳定的Cu-同型物种。MTT 3对Cd 2+和Cu+都表现出较差的结合能力，尽管不是最佳的，但当配位Zn 2+时，它产生了最好的结果。两种Cu-硫蛋白MTT 2和MTT 4同种型在补充Cu的宿主中合成后形成同源Cu-络合物（主要Cu 20-MTT）。首先，它们不能折叠成稳定的Cd-复合物，而Zn-MTT物质仅回收MTT 4（主要为Zn 10-MTT 4）。因此，五种T.嗜热菌MT与其先前分类为Cd-和Cu-硫蛋白相关性良好，并且在全球范围内，它们可以根据等级从Zn/Cd-到Cu-硫蛋白分类：MTT 1> MTT 5> MTT 3> MTT 4> MTT 2。MTT金属结合偏好的演变和专业化的主要机制可能是内部串联重复，存在的双峰和三重态Cys模式的Zn/Cd-硫蛋白，和优化的位点特异性氨基酸决定簇（赖氨酸的Zn/Cd-和Asn的Cu-协调）。
The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, including 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tetrahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn2+-, Cd2+- or Cu+-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd2+ coordination, yielding unique Cd17- and Cd8- complexes, respectively. When binding Zn2+, they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu+, although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd2+ and for Cu+, and although not optimally, it yielded the best result when coordinating Zn2+. The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu20-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn10-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been internal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and optimization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination).