Abstract
The Helix pomatia metallothionein (MT) system, namely, its two highly specific forms, HpCdMT and HpCuMT, has offered once again an optimum model to study metal–protein specificity. The present work investigates the most unexplored aspect of the coordination behavior of MT polypeptides with respect to either cognate or noncognate metal ions, as opposed to the standard studies of cognate metal ion coordination. To this end, we analyzed the in vivo synthesis of the corresponding complexes with their noncognate metals, and we performed a detailed spectroscopic and spectrometric study of the Zn2+/Cd2+ and Zn2+/Cu+ in vitro replacement reactions on the initial Zn-HpMT species. An HpCuMTAla site-directed mutant, exhibiting differential Cu+-binding abilities in vivo, was also included in this study. We demonstrate that when an MT binds its cognate metal, it yields well-folded complexes of limited stoichiometry, representative of minimal-energy conformations. In contrast, the incorporation of noncognate metal ions is better attributed to an unspecific reaction of cysteinic thiolate groups with metal ions, which is dependent on their concentration in the surrounding milieu, where no minimal-energy structure is reached, and otherwise, the MT peptide acts as a multidentate ligand that will bind metal ions until its capacity has been saturated. Additionally, we suggest that previous binding of an MT polypeptide with its noncognate metal ion (e.g., binding of Zn2+ to the HpCuMT isoform) may preclude the correct folding of the complex with its cognate metal ion.
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Acknowledgments
This work was supported by the Spanish Ministerio de Economía y Competitividad, (grants BIO2012-39682-C02-01 and BIO2012-39682-C02-02 to S.A. and M.C., respectively; these grants were partially supported by FEDER funding) and by the Austrian Science Foundation (grant P 23635-B20) to R.D. The authors from Barcelona are members of the 2009SGR-1457 Grup de Recerca de la Generalitat de Catalunya. The Spanish and Austrian groups were also financed by Acciones Integradas grants HU2006-0027 (Spain) and ES 02/2007 (Austria). S.P.R. received a predoctoral fellowship from the Departament de Química, Universitat Autònoma de Barcelona. We thank the Centres Científics i Tecnològics de la Universitat de Barcelona (ICP-AES, DNA sequencing) and the Servei d’Anàlisi Química de la Universitat Autònoma de Barcelona (CD spectroscopy, UV–vis spectroscopy, ESI-MS) for allocating instrument time.
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Palacios, Ò., Pérez-Rafael, S., Pagani, A. et al. Cognate and noncognate metal ion coordination in metal-specific metallothioneins: the Helix pomatia system as a model. J Biol Inorg Chem 19, 923–935 (2014). https://doi.org/10.1007/s00775-014-1127-4
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DOI: https://doi.org/10.1007/s00775-014-1127-4