Three-Dimensional Solution Structure of Callinectes sapidus Metallothionein-1 Determined by Homonuclear and Heteronuclear Magnetic Resonance Spectroscopy

Abstract

Metallothionein is a cysteine-rich metal-binding protein whose biosynthesis 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 separate metal-binding clusters. Heteronuclear 1H-113Cd and homonuclear 1H-1H NMR correlation experiments have been used to establish that the two clusters reside in two distinct protein domains. The three-dimensional solution structure of the metallothionein has been determined using the distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. There are no interdomain short distance (< or = 4.5 A) constraints observed in this protein, enabling the calculation of structures for the N-terminal, beta domain and the C-terminal, alpha domain separately. A total of 18 structures were obtained for each domain. The structures are based on a total of 364 experimental NMR restraints consisting of 277 approximate interproton distance restraints, 12 chi 1 and 51 phi angular restraints, and 24 metal-to-cysteine connectivities obtained from 1H-113Cd correlation experiments. The only element of regular secondary structure in either of the two domains 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 no type II beta turns.

Effects of heavy metals on the expression of a zinc-inducible metallothionein-III gene and antioxidant enzyme activities in Crassostrea gigas

“…It suggested that CgMT-III was probably a new member of the MT-III protein, distinct from previously identified MT proteins in C. gigas. It has been suggested that MT proteins containing two b domains mainly play a housekeeping function in metal-regulation of physiological metals (Cols et al 1999;Narula et al 1995). Zinc is an important essential metal for the organisms (Klaassen et al 1999), however, excessive zinc can cause toxicity to the marine organisms (Münzinger and Guarducci 1988).…”

Solution Structure of the Oxidized 2[4Fe‐4S] Ferredoxin from Clostridium Pasteurianum

“…The non-bonded interactions were evaluated with a cutoff of 1OOOpm and the pair list was updated every 20 steps. During the calculations, an NOE-weighted and a torsion angle potential function [40] was applied using the SANDER module of AMBER, with a harmonic force constant of 402 kJ mol-' [46]. The mixed linear-harmonic potential is reported elsewhere [I, 401. and indicate dipolar connectivities from the PCH protons of the cysteine and protons belonging to the amino acids where the lines end.…”

Separation and Characterization of the Metal‐Thiolate‐Cluster Domains of Recombinant Sea Urchin Metallothionein

“…tain in an analogous fashion two M:+Cys, clusters (Zhu et al, 1994;Narula et al, 1995). In a recent study with recombinant Cd-containing MTA from the sea urchin Strongylocentrotus purpurutus, we have found using '13Cd-113Cd COSY and homonuclear decoupling ' "Cd-NMR measurements that like the mammalian forms this invertebrate MT also features both a M:' Cys, and a M:+Cys,, cluster.…”