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).

Keywords: Copper; Functional Differentiation; Metal specificity; Metallothionein; Tetrahymena thermophila.; Zinc.

Figure 1

(A) Multiple sequence alignment (Clustal Omega) of the five Tetrahymena thermophila MT isoforms. The Cys residues are in grey. The unique amino acid substitution between MTT2 and MTT4 is marked in bold. The Glu (Q) residues encoded by mutated codons are marked in bold italics. The initial GS residues (in italics) result from the recombinant synthesis rationale. (B) Comparison of the main sequence features of the five Tetrahymena thermophila MT isoforms.

Figure 2

Deconvoluted ESI-MS spectra of the demetalated Zn- and Cd-MTT complexes, recorded at acidic pH. The spectra correspond to the demetalated preparations in Zn- and/or Cd-enriched cultures of (A) MTT1, (B) MTT3, (C) MTT4, and (D) MTT5. For those isoforms that were resistant to demetalation, the ESI-MS was run at pH 2.4 and pH 1.8.

Figure 3

Deconvoluted ESI-MS spectra of the recombinant preparations of MTT1, MTT3 and MTT5. The metal-MTT complexes were synthesized in recombinant cultures supplemented with Zn, Cd, or Cu, and in the case of Cu-enriched media, the synthesis was carried out under regular and low aeration conditions. (---) denotes that no metal-MTT complexes could be purified from the corresponding cultures.

Figure 4

Circular dichroism spectra of the recombinant preparations of MTT1, MTT3 and MTT5. The metal-MTT complexes were synthesized in recombinant cultures supplemented with Zn, Cd, or Cu, and in the case of Cu-enriched media, the synthesis was carried out under regular (solid line) and low aeration (dashed line) conditions.

Figure 5

Characterization of in vitro prepared metal-MTT5 and metal-MTT4 complexes. (A) Circular dichroism -CD- spectra recorded after the addition of up to 6 Cd²⁺ eq to Zn-MTT5 at pH 7.0. (B) Deconvoluted ESI-MS spectrum recorded after the addition of 10 Cd²⁺ eq to Zn-MTT5. (C) CD spectra corresponding to the Cd-MTT5 preparation (solid line) and that recorded after the addition of 10 Cd²⁺ eq to Zn-MTT5. (D) CD spectra recorded after the addition of up to 14 Cu⁺ eq to Zn-MTT5 at pH 7.0. Comparison of (E) the deconvoluted ESI-MS and (F) the CD spectra of the recombinant Cu-MTT5 preparation (solid line) and those recorded at several stages of the titration of Zn-MTT5 with Cu⁺ (dashed line for the addition of 6 Cu⁺ eq). (G) CD spectra recorded after the addition of up to 8 Cu⁺ eq to Zn-MTT4 at pH 7.0. Comparison of (H) the deconvoluted ESI-MS and (I) the CD spectra of the recombinant Cu-MTT4 preparation (solid line) and those recorded after the addition of 6 Cu⁺ eq to Zn-MTT4 (dashed).

Figure 6

Deconvoluted ESI-MS spectra of the recombinant preparations of MTT2 and MTT4. The metal-MTT complexes were synthesized in recombinant cultures supplemented with Zn, Cd, or Cu, and in the case of Cu-enriched media, the synthesis was carried out under regular and low aeration conditions. ESI-MS was run at pH 7.0 and pH 2.4. (M=Zn or Cu). (---) denotes that no metal-MTT complexes could be purified from the corresponding cultures.

Figure 7

Circular dichroism spectra of the recombinant preparations of MTT2 and MTT4. The metal-MTT complexes were synthesized in recombinant cultures supplemented with Zn, Cd, or Cu, and in the case of Cu-enriched media, the synthesis was carried out under regular (solid line) and low aeration (dashed line) conditions. (---) denotes that no metal-MTT complexes could be purified from the corresponding cultures.