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Metailothioneins are small cysteine-rich proteins capable of binding heavy metal ions such as Zn2+ and Cd2+. They are ubiquitous tissue components in higher organisms, which tentatively have been attributed both upe protective functions against toxic metal ions and highly specific roles in fundamental zinc-regulated cellular processes. In this paper a detailed comparison of the NMR solution structure [Schultze, P., Worgitter, E., Braun Mammalian metallothioneins (MTs) are small proteins with 61 or 62 amino acid residues that have the ability to accommodate metal ions of different size and chemical reactivity without compromising the overall molecular architecture (1-4). This observation has been used to support the hypothesis that the primary function of MTs is to shield cellular structures from the harmful influence of toxic metals such as cadmium, mercury, platinum, bismuth, silver, and gold by limiting the intracellular concentration of these heavy metal ions (5, 6). In addition to such rather unspecific protective functions, recent findings imply that MTs have a more specific major role in fundamental zinc-related cellular processes, with thionein supplied by controlled biosynthesis regulating the flow of zinc(II) within the cell and thereby modulating the action ofzinc-dependent processes in response to signals for cell activation in proliferation and differentiation (4,(7)(8)(9)). An assessment of these suggested MT functions on the molecular level has so far been limited by the fact that a MT structure determined by x-ray diffraction in single crystals (10) had a different molecular architecture from that in MT structures determined by NMR spectroscopy in solution (2,11,12). Subsequently it was found that the crystal structure needed to be revised, and a new crystal structure of rat MT2 was presented (3) that has the same overall architecture as the NMR structure (2). To provide a reliable basis for investigations on structure-function correlations in MTs, this paper now describes a detailed comparison of the structures of rat MT2 in crystals and in solution. 11 METHODSThe NMR solution structure of rat heptacadmium(II) metallothionein II ([Cd7]-MT2) (2) was characterized in the usual way (13) by a group of 10 conformers obtained from the NMR data with distance geometry calculations (14-16). The structure consists of two domains containing, respectively, residues 1-30 and three Cd2+ ions (I domain) and residues 31-61 and four Cd2+ ions (a domain). The two domains are connected by a flexible polypeptide segment, and their relative positions have not been determined. Therefore, all structure comparisons have been performed separately for the a and ,3domains. The quality of the structure determination as indicated by the closeness of coincidence of the 10 conformers is defined by the root-mean-square deviations (rmsd) between the individual NMR conformers and the mean solution structure (Table 1). The new x-ray crystal structure of [Cd5, Zn2]-MT2 (containing four Cd2+ ions in the a domain and two Zn2+...