RT Journal Article
T1 Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins
A1 Espart, Anna
A1 Marín, Maribel
A1 Gil Moreno, Selene
A1 Palacios, Óscar
A1 Amaro Torres, Francisco
A1 Martín González, Ana María
A1 Gutiérrez Fernández, Juan Carlos
A1 Capdevilla, Mercé
A1 Atrian, Sílvia
AB 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).
PB Ivyspring International Publisher
SN 1449-2288
YR 2015
FD 2015
LK https://hdl.handle.net/20.500.14352/35448
UL https://hdl.handle.net/20.500.14352/35448
LA eng
NO Ministerio de Economía y Competitividad (MINECO)
DS Docta Complutense
RD 27 abr 2024