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

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 β_N domain in the native holoprotein. Time- and temperature-dependence studies of the ¹¹³Cd and ¹H 1D-NMR spectra indicated that the β_N 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 H^N...S hydrogen bond interactions were also determined from a long-range optimized ¹H^N-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 Cd₃β_N 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.