Structure elucidation of the metal-binding sites in metallothionein by ¹³³Cd NMR

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 cystein 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 four 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.