Localization of the C-terminus of rat glutathione S-transferase A1-1: Crystal structure of mutants W21F and W21F/F220Y

Twelve C-terminal residues of human glutathione S-transferase Al-1 form a h elix in the presence of glutathione-conjugate, or substrate alone, and part ly cover the active site. According to X-ray structures, the helix is disor dered in the absence of glutathione, but it is not known if it is helical a nd delocalized, or in a random coil conformation. Mutation to a tyrosine of residue 220 within this helix was previously shown to affect the pK(a) of Tyr-9 at the active site, in the apo form of the enzyme, and it was propose d that an on-face hydrogen bond between Tyr-220 and Tyr-9 provided a means for affecting this pK(a). In the current study, X-ray structures of the W21 F and of the C-terminal mutation, W21F/F220Y, with glutathione sulfonate bo und, show that the C-terminal helix is disordered (or delocalized) in the W 21F crystal but is visible and ordered in a novel location, a crystal packi ng crevice, in one of three monomers in the W21F/ F220Y crystal, and the pr oposed hydrogen bond is not formed. Fluorescence spectroscopy studies using an engineered F222W mutant show that the C-terminus remains delocalized in the absence of glutathione or when only the glutathione binding site is oc cupied, but is ordered and localized in the presence of substrate or conjug ate, consistent with these and previous crystallographic studies. Proteins 2001;42:192-200, (C) 2000 Wiley-Liss, Inc.