title: Hints for metal-preference protein sequence determinants: different metal binding features of the five tetrahymena thermophila metallothioneins.
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 Zn(2+)-, Cd(2+)- 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 Cd(2+) coordination, yielding unique Cd17- and Cd8- complexes, respectively. When binding Zn(2+), 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 Cd(2+) and for Cu(+), and although not optimally, it yielded the best result when coordinating Zn(2+). 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).
authors: 1. Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, 08028-Barcelona, Spain;
authors: Anna
authors: A
authors: Espart
authors: 2. Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Cerdanyola del Vallès (Barcelona), Spain;
authors: Maribel
authors: M
authors: Marín
authors: 2. Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Cerdanyola del Vallès (Barcelona), Spain;
authors: Selene
authors: S
authors: Gil-Moreno
authors: 2. Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Cerdanyola del Vallès (Barcelona), Spain;
authors: Òscar
authors: Ò
authors: Palacios
authors: 3. Departamento de Microbiología-III, Facultad de Biología, Universidad Complutense, 28040-Madrid, Spain.
authors: Francisco
authors: F
authors: Amaro
authors: 3. Departamento de Microbiología-III, Facultad de Biología, Universidad Complutense, 28040-Madrid, Spain.
authors: Ana
authors: A
authors: Martín-González
authors: 3. Departamento de Microbiología-III, Facultad de Biología, Universidad Complutense, 28040-Madrid, Spain.
authors: Juan C
authors: JC
authors: Gutiérrez
authors: 2. Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Cerdanyola del Vallès (Barcelona), Spain;
authors: Mercè
authors: M
authors: Capdevila
authors: 1. Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, 08028-Barcelona, Spain;
authors: Sílvia
authors: S
authors: Atrian
mesh_terms: D003300:Copper / Q000378:metabolism; D008668:Metallothionein / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D016808:Tetrahymena thermophila / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
chemical_list: C041582:Cd-Zn-metallothionein; D003300:Copper; D008668:Metallothionein; D015032:Zinc
keywords: Copper; Functional Differentiation; Metal specificity; Metallothionein; Tetrahymena thermophila.; Zinc