Three-dimensional solution structure of mouse [Cd-7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy

Sequential H-1-NMR assignments of mouse [Cd-7]-metallothionein-1 (MT1) have been carried out by standard homonuclear NMR methods and the use of an acc ordion-heteronuclear multiple quantum correlation (HMQC) experiment for est ablishing the metal, Cd-113(2+) to cysteine connectivities. The three-dimen sional structure was then calculated using the distance constraints from tw o-dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys-Cd connectivities as input for a distance geometry-dynamical simulated annealing protocol in X-PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two separate domains, eac h containing one metal cluster. Because there were no interdomain constrain ts, the structure calculation for the N-terminal beta- and the C-terminal a lpha-domain were carried out separately. The structures are based on 409 NM R constraints, consisting of 381 NOEs and 28 cysteine-metal connectivities. The only elements of regular secondary structure found were two short stre tches of 3(10) helices along with some half-turns in the alpha-domain. Stru ctural comparison with rat liver MT2 showed high similarity, with the beta- domain structure in mouse MT1 showing evidence of increased flexibility com pared to the same domain in MT2. The latter was reflected by the presence o f fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium-cadmium exchange rates found in the beta-domain of MT1.