Determination of the solution three dimensional structure of chemically-synthesized native 113Cd3S9 domain of Lobster Metallothionein by NMR Spectroscopy Amalia Muñoz*, F. Holger Forsterling, C. Frank Shaw III+ and David H. Petering * Biochemistry, University of Wisconsin - Madison Chemistry, University of Wisconsin - Milwaukee & Marine and Freshwater Biomedical Sciences Center + Chemistry, University of Eastern Kentucky

Abstract

Lobster metallothionein is a rich protein that binds six Cd²⁺ ions forming two separate ß-domains, each of which includes a Cd₃S₉ cluster of the same structure. The metal-cysteine connectivities and reactivity also differ between the two domains (ß_C and ß_N). Thus, in order to study structure-function relationships, the native lobster ß-domains have been synthesized and characterized by UV-, CD- and NMR-spectroscopies. The distribution of the cysteine residues in the sequence of each domain is highly conserved but different. The 3D-structure of the Cd₃ß_C domain was calculated using 249 proton-proton distance restraints from NOE data, 35 restraints for the theta and cysteine chi-1 dihedral angles derived from ³J_HN/Ha and ³J_Ha/Hb and 5 restraints for the Cys C^aC^bS^gCd angle chi-2 from heteronuclear ³J_Hb/113Cd. The metal clusters were defined from the 2D-heteronuclear ¹H-¹¹³Cd HSQC-TOCSY experiment; a 80 ms delay allowed the signal to be transfer to the amide protons, which simplified the identification of the bridging and terminal cysteines coordinated to each cadmium. From these results the only elements of regular secondary structure are a short segment of helix 3-10 between residues 41-45 and one type 1 of ß-turn, due to the folding of the polypeptide chain. The more stable structure of ß_C in comparison with ß_N as shown by the NMR studies is reflected in this slower reactivity toward DTNB (5,5'-dithio-bis-(2-nitrobenzoic) acid) or EDTA when compared with the ß_C-domain. This appears to be due to the smaller solvent accessibility of the cluster sulfhydryl groups in ß_C, although the structure of the Cd₃S₉ cluster is the same in each domain. The detailed folding and sequence of polypeptide chain about the cluster core determine its reactivity and possibly its biological function.

This research was supported by the US NIH grants ES 04026, ES 04184 and DK51308.