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International Journal Of Bilogical Sciences

.: Home > International Journal Of Bilogical Sciences > 2015 > Volume 11 Number 4 > Anna Espart1, Maribel Marín2, Selene Gil-Moreno2, Òscar Palacios2, Francisco Amaro3, Ana Martín-González3, Juan C. Gutiérrez3, Mercè Capdevila2 and Sílvia Atrian1

Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins

Anna Espart1, Maribel Marín2, Selene Gil-Moreno2, Òscar Palacios2, Francisco Amaro3, Ana Martín-González3, Juan C. Gutiérrez3, Mercè Capdevila2 and Sílvia Atrian1
1. Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, 08028-Barcelona, Spain; 2. Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Cerdanyola del Vallès (Barcelona), Spain; 3. Departamento de Microbiología-III, Facultad de Biología, Universidad Complutense, 28040-Madrid, Spain.  Corresponding author: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 643, 08028-Barcelona, Spain, Phone: +34 934021501, FAX: +34 934034420, E-mail: satrian@ub.edu
Abstract :

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). 

Keywords :
Metallothionein, Functional Differentiation, Metal specificity, Zinc, Copper, Tetrahymena thermophila

Date Deposited : 22 Feb 2016 10:14

Last Modified : 22 Feb 2016 10:14

Official URL: http://www.ijbs.com/v11i4

Volume 11, Number 4, - 2015 , ISSN 1449-2288

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