Structure-Reactivity Realtionships among Metallothionein Three-metal Domains:Role of Non-Cysteine Amino Acid Residues in Lobster Metallothionein and Human Metallothionein-3

摘要

Metallothionein (MT) domains of different origins, exhibiting distinct, highly conserved cysteine positions. show~ differences in metal—cysteine coordination and reactivity. Lobster MT, which includes two Cd3S9 /3 domains, was chosen as a basic model to study the structure—function relationship among the clusters. The possible influen~ of (I) the position of the cysteine residues and (2) the steric and electrostatic effects of neighboring amino acids on the folding and stability of MT clusters have been examined with the native lobster /3c and /3N domains, each having nine cysteines and binding three M2t ions, and a modified domain /3C—.N, in which the cysteines of the C-terminal domain are relocated so they are spaced as in the N-terminal domain. Each has been synthesized and characterized by UV, CD, “3Cd NMR, and NMR spectroscopies. The synthetic native domains (Cd3/3c and Cd3/3N) displayed spectroscopic properties. metal-binding affinities, and kinetic reactivity similar to those of the bob protein. In contrast, the modified Cd3I3c—.N domain was unusually reactive and, in the presence of Chelex, a metal-ion chelating resin, was converted to a Cd5Q3c—.N) dimer. These differences in structure and reactivity demonstrate that the requirements for formation of a stable type-B, Cd3S9, /3 cluster are more stringent than simply the sequential positions of the cysteines along the peptide chain and include specific interactions with neighboring amino acids. Molecular mechanics calculations suggest that changes of even a single amino acid in lobster Cd,/3N toward lobster Cd3/3C—N or in mammalian MT1 or MT2 toward Cd3/3-MT3 (GIF) can destabilize their structures.