Structure of the 113Cd3β domains from Homarus americanus metallothionein-1: hydrogen bonding and solvent accessibility of sulfur atoms

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Abstract
The three-dimensional structures of the isolated Cd3β domains from Homarus americanus metallothionein have been determined by NMR methods in order to establish a set of β-domain structures for comparative analysis. First, it was determined that the Cd-cysteine connectivities forming the Cd3S9 metal center were identical to those observed for the βN domain in the native holoprotein. Time- and temperature-dependence studies of the 113Cd and 1H 1D-NMR spectra indicated that the βN domain undergoes slow conformational changes before reaching an equilibrium structure. In addition to structural information provided by the metal-to-cysteine connectivities, Φ, χ1 and χ2 angle constraints, three HN...S hydrogen bond interactions were also determined from a long-range optimized 1HN-113Cd HMQC experiment. A simulated annealing protocol was applied to the distance and angle constraints obtained from the 2D-NMR experiments to calculate the three-dimensional structure of the synthetic Cd3βN domain of lobster metallothionein. Structure-reactivity relationships are proposed for the reactions of Cd3β domains with 5,5′-dithiobis(2-nitrobenzoate), based on comparisons of surface exposure of sulfur atoms of the lobster and rabbit Cd3β domain structures. Finally, the surface exposure of the β domains of lobster is compared with β domains from mammalian metallothioneins. Electronic supplementary material to this paper, comprising four tables and three figures, can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-002-0345-3.

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