Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins
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, in-cluding 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tet-rahym...
| Autores: | , , , , , , , , |
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| Tipo de recurso: | artículo |
| Fecha de publicación: | 2015 |
| País: | España |
| Institución: | Universidad Complutense de Madrid (UCM) |
| Repositorio: | Docta Complutense |
| Idioma: | inglés |
| OAI Identifier: | oai:docta.ucm.es:20.500.14352/35448 |
| Acceso en línea: | https://hdl.handle.net/20.500.14352/35448 |
| Access Level: | acceso abierto |
| Palabra clave: | 579 593.1 Metallothionein Functional Differentiation Metal specificity Zinc Copper Tetrahymena thermophila. Invertebrados Microbiología (Biología) 2401.17 Invertebrados 2414 Microbiología |
| Sumario: | 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, in-cluding 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tet-rahymena 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 in-ternal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and op-timization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination). |
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