title: Full characterization of the Cu-, Zn-, and Cd-binding properties of CnMT1 and CnMT2, two metallothioneins of the pathogenic fungus Cryptococcus neoformans acting as virulence factors.
abstract: We report here the full characterization of the metal binding abilities of CnMT1 and CnMT2, two Cryptococcus neoformans proteins recently identified as metallothioneins (MTs), which have been shown to play a crucial role in the virulence and pathogenicity of this human-infecting fungus. In this work, we first performed a thorough in silico study of the CnMT1 and CnMT2 genes, cDNAs and corresponding encoded products. Subsequently, the Zn(II)-, Cd(II)- and Cu(I) binding abilities of both proteins were fully determined through the analysis of the metal-to-protein stoichiometries and the structural features (determined by ESI-MS, CD, ICP-AES and UV-vis spectroscopies) of the corresponding recombinant Zn-, Cd- and Cu-MT preparations synthesized in metal-enriched media. Finally, the analysis of the Zn/Cd and Zn/Cu replacement processes of the respective Zn-MT complexes when allowed to react with Cd(II) or Cu(I) aqueous solutions was performed. Comprehensive consideration of all gathered results allows us to consider both isoforms as genuine copper-thioneins, and led to the identification of unprecedented Cu5-core clusters in MTs. CnMT1 and CnMT2 polypeptides appear to be evolutionarily related to the small fungal MTs, probably by ancient tandem-duplication events responding to a highly selective pressure to chelate copper, and far from the properties of Zn- and Cd-thioneins. Finally, we propose a modular structure of the Cu-CnMT1 and Cu-CnMT2 complexes on the basis of Cu5 clusters, concordantly with the modular structure of the sequence of CnMT1 and CnMT2, constituted by three and five Cys-rich units, respectively.
authors: Dept. de Química, Fac. de Ciències, Universitat Autònoma de Barcelona, 08193-Cerdanyola del Vallès, Barcelona, Spain.
authors: Òscar
authors: Ò
authors: Palacios
authors: Anna
authors: A
authors: Espart
authors: Jordi
authors: J
authors: Espín
authors: Chen
authors: C
authors: Ding
authors: Dennis J
authors: DJ
authors: Thiele
authors: Sílvia
authors: S
authors: Atrian
authors: Mercè
authors: M
authors: Capdevila
mesh_terms: D000595:Amino Acid Sequence; D002104:Cadmium / Q000378:metabolism*; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D003455:Cryptococcus neoformans / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D005800:Genes, Fungal; D006801:Humans; D008668:Metallothionein / Q000737:chemistry / Q000378:metabolism*; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D037521:Virulence Factors / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D010455:Peptides; D011994:Recombinant Proteins; D037521:Virulence Factors; D002104:Cadmium; D003300:Copper; D008668:Metallothionein; D015032:Zinc