Bi(III) Binding Stoichiometry and Domain-Specificity Differences Between Apo and Zn(II)-bound Human Metallothionein 1a

Abstract

Bismuth is a xenobiotic metal with a high affinity to sulfur that is used in a variety of therapeutic applications. Bi(III) induces the cysteine-rich metallothionein (MT), a protein known to form two-domain cluster structures with certain metals such as Zn(II), Cd(II), or Cu(I). The binding of Bi(III) to MTs has been previously studied, but there are conflicting reports on the stoichiometry and binding pathway, which appear to be highly dependent on pH and initial metal-loading status of the MT. Additionally, domain specificity has not been thoroughly investigated. In this paper, ESI-MS was used to determine the binding constants of [Bi(EDTA)]^- binding to apo-MT1a and its individual αMT fragment. The results were compared to previous experiments using βMT1a and βαMT3. Domain specificity was investigated using proteolysis methods and the initial cooperatively formed Bi₂MT was found to bind to cysteines that spanned across the traditional metal binding domain regions. Titrations of [Bi(EDTA)]^- into Zn₇MT were performed and were found to result in a maximum stoichiometry of Bi₇MT, contrasting the Bi₆MT formed when [Bi(EDTA)]^- was added to apo-MT. These results show that the initial structure of the apo-MT determines the stoichiometry of new incoming metals and explains the previously observed differences in stoichiometry.

Keywords: bismuth; electrospray ionization mass spectrometry; metallothionein; protein structure.