Understanding the 7-Cys module amplification ofC. neoformansmetallothioneins: how high capacity Cu-binding polypeptides are built to neutralize host nutritional immunity

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Cryptococcus neoformansmetallothioneins (MTs), CnMT1 and CnMT2, have been identified as essential infectivity and virulence factors of this pathogen. Both MTs are unusually long Cu-thioneins, exhibiting protein architecture and metal-binding abilities compatible with the hypothesis of resulting from three and five tandem repetitions of 7-Cys motives, respectively, each of them folding into Cu5-clusters. Through the study of the Zn(II)- and Cu(I)-binding capabilities of several CnMT1 truncated mutants, we show that a 7-Cys segment of CnMT1 folds into Cu5-species, of additive capacity when joined in tandem. All the obtained Cu-complexes share practically similar architectural features, if judging by their almost equivalent CD fingerprints, and they also share their capacity to restore copper tolerance in MT-devoid yeast cells. Besides the analysis of the modular composition of these long fungal MTs, we evaluate the features of the Cys-rich stretch spacer and flanking sequences that allow the construction of stable metal clusters by adjacent union of binding modules. Overall, our data support a mechanism by which some microbial MTs may have evolved to enlarge their original metal co-ordination capacity under the specific selective pressure of counteracting the Cu-based immunity mechanisms evolved by the infected hosts.

C. neoformans metallothioneins have a modular structure built by the repetition of three (CnMT1) or five (CnMT2) 7-Cys units, separated by spacers devoid of Cys, plus flanking Cys doublets.

Each 7-Cys box optimally coordinates 5 Cu(I) ions, and the presence of spacers and flanking regions is necessary to endow cluster stability, both as separate or concatenated entities.

The Cu5 species constitute an unreported Cu-MT cluster, which may represent a primeval microbial MT structure.

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