Application of 113Cd NMR to metallothioneins

Metallothioneins constitute a class of ubiquitously occurring low molecular mass proteins (6-7 kDa) possessing two cysteine thiolate-based metal clusters usually formed by the preferential binding of d10 metal ions such as ZnII and CdII. The three-dimensional solution structure of mammalian proteins has been determined by two-dimensional NMR spectroscopy of 113Cd7-metallothionein. The structure shows two protein domains encompassing the M3(CysS)9- and M4(CysS)11-cluster with each metal ion being tetrahedrally coordinated by thiolate ligands. The application of 113Cd NMR proved to be indispensable in the structural studies of metallothioneins. Thus, both homonuclear 113Cd decoupling studies and 113Cd-113Cd COSY of 113Cd7-metallothionein established the existence of two metal-thiolate clusters in this protein. The identification of sequence specific cysteine-cadmium coordinative bonds came from heteronuclear 113Cd-1H COSY experiments. Independently, the 113Cd NMR characterization of the intermediate metal-protein complexes, leading to the cluster structure in 113Cd7-metallothionein, revealed a stepwise cluster formation process with the Cd4(CysS)11-cluster being formed first. The recent demonstration of a Karplus-like dependence between the heteronuclear3 J(113Cd, 1H) coupling constants for the cysteine C beta protons and the H beta-C beta-S gamma -Cd dihedral angles should allow to derive the geometry of the CD-(S-Cys) centers in various metallothioneins and related metalloproteins. A possible application of 113Cd NMR to the study of metallothioneins in the environment is discussed.