title: Structure elucidation of the metal-binding sites in metallothionein by 113Cd NMR.
abstract: 113Cd NMR has been used to determine the structures of the multiple metal-binding sites in the two major isoproteins of metallothionein from mammalian livers (rabbits, calf, and human) and from Scylla serrata hepatopancreas. The native protein isolated from the livers of rabbits that had been subjected to repeated injections of 113CdCl2 contains an appreciable amount of Zn2+ in addition to 113Cd2+, ranging from 2 to 3 g-atoms of a total metal content of 7 g-atoms/mol of protein. The native Zn2+ can be replaced in vitro with 113Cd2+ to give a 113Cd NMR spectrum consisting of eight distinct multiplets in the chemical shift range of 604-670 ppm. The multiplet structure is due to 113Cd-113Cd scalar coupling arising from two-bond interactions between 113Cd2+ ions linked to one another by bridging cysteine thiolate ligands. Analysis of the 113Cd spectra by selective homonuclear 113Cd decoupling techniques showed that both isoproteins of rabbit liver metallothionein contain two separate metal clusters, one containing Cd2+ ions (cluster A) and the other containing three (cluster B). Structures for the clusters are proposed that account for the 113Cd chemical shift and spin coupling data and the participation of all 20 cysteine residues in metal ligation. The 113Cd NMR spectrum of 113Cd2+-reconstituted human liver metallothionein is remarkably similar to that of the rabbit and the analysis confirms that it contains the same two-cluster arrangement. Native calf liver metallothionein was found to contain copper and Zn2+ and the in vitro exchange with 113Cd2+ selectively replaces only the Zn2+. The reconstituted protein contains 3.9 g-atoms of 113Cd2+ and 2.6 g-atoms of copper and the 113Cd NMR spectrum showed four major multiplets with identical chemical shifts to the resonances previously assigned to the four-metal cluster A. This result was confirmed by homonuclear decoupling experiments. The 2.6 g-atoms of electron spin resonance-silent copper in this sample is presumably selectively bound to the three-metal cluster B sites. Both isoproteins of metallothionein isolated from 113Cd2+-injected mud crabs (S. serrata) contain only 113Cd2+. The analysis of the 113Cd NMR spectra show that the total of six metals bound per mole of crab metallothionein-1 (MT-1) and MT-2 are arranged in two separate three-metal clusters.
mesh_terms: D000818:Animals; D001665:Binding Sites; D003386:Brachyura; D002104:Cadmium; D008099:Liver; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008667:Metalloproteins; D008668:Metallothionein; D011485:Protein Binding; D011817:Rabbits
chemical_list: D008667:Metalloproteins; D002104:Cadmium; D008668:Metallothionein