Cognate and noncognate metal ion coordination in metal-specific metallothioneins: the Helix pomatia system as a model

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 Zn(2+)/Cd(2+) and Zn(2+)/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 Zn(2+) to the HpCuMT isoform) may preclude the correct folding of the complex with its cognate metal ion.

Cantareus aspersus metallothionein metal binding abilities: The unspecific CaCd/CuMT isoform provides hints about the metal preference determinants in metallothioneins

“…CaCdMT and CaCuMT synthesis under Zn-supplementation yielded the expected results, fully comparable to those previously reported for the H. pomatia orthologous isoforms. Hence, CaCdMT afforded unique, well-folded Zn 6 -CaCdMT complexes, with features totally alike to those of Zn 6 -HpCdMT [11,15], while CaCuMT exhibited its unsuitability for Zn 2+ coordination by yielding two complexes, Zn 5 -and Zn 6 -CaCuMT, with rather poor folding. Interestingly, the CaCd/CuMT isoform showed even worse Zn-binding aptitudes, since three species of significant abundance were recovered here (Zn 4 -, Zn 5 -and Zn 6 -CaCd/CuMT) showing a CD spectrum revealing a complete lack of chirality.…”

“…The third isoform was natively isolated as a mixed Cd and Cu complex, and for this reason named CaCd/CuMT, but the poor level of expression and the lack of metal response of its encoding gene suggested a residual role, if any, in the snail metabolism of metals, either physiological or xenobiotic. The examination of the similarities and differences between the three CaMT protein sequences promptly revealed that the comparative analysis of their metal binding abilities -and also in relation to those of the H. pomatia MTs (HpCdMT and HpCuMT, already extensively analyzed [11,15]) -could yield invaluable information about the determinants of metal specificity in metallothioneins. As a rule-of-thumb, we have unequivocally shown that recombinant synthesis of an MT peptide as a complex with its cognate metal produces unique, energetically optimized complexes, while when an MT is forced to coordinate noncognate metals, it yields a mixture of complexes of different stoichiometry and/or folding, none of which achieves a definite energy well.…”

“…In further studies using recombinantly synthesized metalcomplexes, it was clearly established that these two H. pomatia MTs had achieved their metal binding specificity by protein sequence diversification, through the different amino acid residues located between their fully conserved cysteines [11]. More recently, further characterization of the each snail MT binding behavior towards either cognate or non-cognate metal ions has shown that, as a rule of thumb, MT synthesis in the presence of their cognate metal ions leads to unique, energetically optimized complexes that define the MT metal specificity or preference, while with non-cognate metal ions, they render a mixture of complexes, none of them with a conformation compatible with a clear energy well, and thus principally dependent on the amount of metal ions present in solution [15]. Obviously, all MT peptides, with independence of their metal specificity or preference, are able to bind both divalent (Zn 2+ , Cd 2+ ) and monovalent (Cu + ) metal ions because MTs behave as "conventional" polydentate thiolate ligands [15].…”

“…More recently, further characterization of the each snail MT binding behavior towards either cognate or non-cognate metal ions has shown that, as a rule of thumb, MT synthesis in the presence of their cognate metal ions leads to unique, energetically optimized complexes that define the MT metal specificity or preference, while with non-cognate metal ions, they render a mixture of complexes, none of them with a conformation compatible with a clear energy well, and thus principally dependent on the amount of metal ions present in solution [15]. Obviously, all MT peptides, with independence of their metal specificity or preference, are able to bind both divalent (Zn 2+ , Cd 2+ ) and monovalent (Cu + ) metal ions because MTs behave as "conventional" polydentate thiolate ligands [15]. Therefore the strength of the formed metal-SCys bonds follows the wellknown rules or the Irwing-Williams series (i.e.…”

Copper metallothioneins

“…In HpCd 6 MT, Cd(II) ions are bound in tetrahedral coordination each by four thiolate ligands in the form oligonuclear metal thiolate clusters, for which the structure remains to be determined. In Cu 12 ‐HpCuMT, spectroscopic analysis indicate that Cu(I) is organized in the form of oligonuclear Cu(I) thiolate clusters with features very similar to mammalian Cu 12 ‐MT in which metals are bound predominantly in trigonal coordination . Stability of the expressed Cd 6 ‐HpCdMT and Cu 12 ‐HpCuMT complexes was demonstrated by their metal exchange inertness.…”