3-DIMENSIONAL SOLUTION STRUCTURE OF CALLINECTES-SAPIDUS METALLOTHIONEIN-1 DETERMINED BY HOMONUCLEAR AND HETERONUCLEAR MAGNETIC-RESONANCE SPECTROSCOPY

Metallothionein is a cysteine-rich metal-binding protein whose biosynt hesis is closely regulated by the level of exposure of an organism to zinc, copper, cadmium, and other metal salts. The metallothionein from Callinectes sapidus is known to bind six divalent metal ions in two s eparate metal-binding clusters. Heteronuclear H-1-Cd-113 and homonucle ar H-1-H-1 NMR correlation experiments have been used to establish tha t the two clusters reside in two distinct protein domains. The three-d imensional solution structure of the metallothionein has been determin ed using the distance and angle constraints derived from these two-dim ensional NMR data sets and a distance geometry/simulated annealing pro tocol. There are no interdomain short distance (less than or equal to 4.5 Angstrom) constraints observed in this protein, enabling the calcu lation of structures for the N-terminal, beta domain and the C-termina l, ct domain separately. A total of 18 structures were obtained for ea ch domain. The structures are based on a total of 364 experimental NMR restraints consisting of 277 approximate interproton distance restrai nts, 12 chi(1) and 51 phi angular restraints, and 24 metal-to-cysteine connectivities obtained from H-1-Cd-113 correlation experiments. The only element of regular secondary structure in either of the two domai ns is a short segment of helix in the C-terminal alpha domain between Lys42 and Thr48. The folding of the polypeptide backbone chain in each domain, however, gives rise to several type I beta turns. There are n o type II beta turns.