Understanding the 7-Cys module amplification of C. neoformans metallothioneins: how high capacity Cu-binding...

Cryptococcus neoformans metallothioneins (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.