2026-09-30T16:50:47Zhttps://docta.ucm.es/rest/oai/request

oai:docta.ucm.es:20.500.14352/354482023-08-27T16:19:59Zcom_20.500.14352_14col_20.500.14352_15

Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins Espart, Anna Marín, Maribel Gil Moreno, Selene Palacios, Óscar Amaro Torres, Francisco Martín González, Ana María Gutiérrez Fernández, Juan Carlos Capdevilla, Mercé Atrian, Sílvia 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). 2023-06-19T15:09:24Z 2023-06-19T15:09:24Z 2023-06-19T15:09:24Z 2015 journal article https://hdl.handle.net/20.500.14352/35448 1449-2288 10.7150/ijbs.11060 eng BIO2012-39682-C02-01 BIO2012-39682-C02-02 CGL2008-00317/BOS BES-2010-036553 https://creativecommons.org/licenses/by/3.0/es/ open access Atribución 3.0 España Ivyspring International Publisher