A PUTATIVE GLUTATHIONE-BINDING SITE IN CDZN-METALLOTHIONEIN IDENTIFIED BY EQUILIBRIUM BINDING AND MOLECULAR-MODELING STUDIES

Glutathione (GSH) has been found to form a complex with both vertebrat e and invertebrate copper-metallothionein (CuMT) [Freedman, Ciriolo an d Peisach (1989) J. Biol. Chem. 264, 5598-5605; Brouwer and Brouwer-Ho exum (1991) Arch. Biochem. Biophys. 290, 207-213]. In this paper we re port on the interaction of GSH with CdZnMT-I and CdZnMT-II from rabbit liver and with CdMT-I from Blue crab hepatopancreas. Ultrafiltration experiments showed that all three MTs combined with GSH. The measured binding data for the three MTs could be described by a single binding isotherm. The GSH/MT stoichiometry was 1.4 +/- 0.3 and K(diss) = 14 +/ - 6 muM. Partially Zn-depleted MT does not significantly bind GSH, ind icating that the GSH-binding site is located on MT's Zn-containing N-t erminal domain. The putative GSH-binding site on rabbit liver MT was i nvestigated using molecular-graphics analysis. A cleft on the MT's N-t erminal domain, which has the labile Zn-2 at its base, could easily ac commodate GSH. Cysteine-ligand exchange between the terminal (non-brid ging) Cys-26, bound to Zn-2, and the cysteine in GSH is stereochemical ly possible. Based on these considerations a model of MT-GSH was built in which GSH's cysteine replaces Cys-26 as a terminal Zn-2 ligand. Th is complex was energy-minimized by molecular-mechanics calculations, t aking into account computed partial electrostatic charges on all atoms , including Cd and Zn. These calculations showed that the MT-GSH compl ex was thermodynamically more stable than MT, due to favourable non-bo nded, electrostatic and van der Waals interactions. Six hydrogen bonds can form between GSH and MT. The average pairwise root-mean-square de viations (RMSD) of the metals in energy-minimized MT and MT-GSH, compa red with the metals in the crystal structure, were 0.0087 +/- 0.0028 m m (0.087 +/- 0.028 angstrom) and 0.0168 +/- 0.0087 nm (0.168 +/- 0.087 angstrom) respectively. The RMSD values for the polypeptide-backbone a, carbons were 0.0136 +/- 0.0060 nm (0.136 +/- 0.060 angstrom) and 0. 0491 +/- 0.0380 nm (0.491 +/- 0.380 angstrom) respectively. No other d ocking sites for GSH were found. The energy-minimized structure of an MT-2-mercaptoethanol complex was somewhat less stable than the native MT domain, attesting to the specificity of the MT-GSH interaction. The possible physiological significance of the MT-GSH interaction is disc ussed.