The three-dimensional structure of a glutathione S-transferase from the Mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-.ANG. resolution

Abstract

The crystal structure of a mu class glutathione S-transferase (EC 2.5.1.18) from rat liver (isoenzyme 3-3) in complex with the physiological substrate glutathione (GSH) has been solved at 2.2-A resolution by multiple isomorphous replacement methods. The enzyme crystallized in the monoclinic space group C2 with unit cell dimensions of a = 87.98 A, b = 69.41 A, c = 81.34 A, and beta = 106.07 degrees. Oligonucleotide-directed site-specific mutagenesis played an important role in the solution of the structure in that the cysteine mutants C86S, C114S, and C173S were used to help locate the positions of mercuric ion sites in nonisomorphous derivatives with ethylmercuric phosphate and to align the sequence with the model derived from MIR phases. A complete model for the protein was not obtained until part of the solvent structure was interpreted. The dimer in the asymmetric unit refined to a crystallographic R = 0.171 for 19,298 data and I > or = 1.5 sigma (I). The final model consists of 4150 atoms, including all non-hydrogen atoms of 434 amino acid residues, two GSH molecules, and oxygen atoms of 474 water molecules. The dimeric enzyme is globular in shape with dimensions of 53 x 62 x 56 A. Crystal contacts are primarily responsible for conformational differences between the two subunits which are related by a noncrystallographic 2-fold axis. The structure of the type 3 subunit can be divided into two domains separated by a short linker, a smaller alpha/beta domain (domain I, residues 1-82), and a larger alpha domain (domain II, residues 90-217). Domain I contains four beta-strands which form a central mixed beta-sheet and three alpha-helices which are arranged in a beta alpha beta alpha beta beta alpha motif. Domain II is composed of five alpha-helices. Domain I can be considered the glutathione binding domain, while domain II seems to be primarily responsible for xenobiotic substrate binding. The active site is located in a deep (19-A) cavity which is composed of three relatively mobile structural elements: the long loop (residues 33-42) of domain I, the alpha 4/alpha 5 helix-turn-helix segment, and the C-terminal tail. GSH is bound at the active site in an extended conformation at one end of the beta-sheet of domain I with its backbone facing the cavity and the sulfur pointing toward the subunit to which it is bound.(ABSTRACT TRUNCATED AT 400 WORDS)

UCS15A, a novel small molecule, SH3 domain-mediated protein–protein interaction blocking drug

“…Crystallographic analyses suggest that GST has the potential to dimerize (Parker et al, 1990;Reinemer et al, 1991;Ji et al, 1992). Therefore, it was important to ensure that the observed interaction in vitro was mediated by SH3, and not by GST.…”

MIF proteins are theta‐class glutathione S‐transferase homologs

“…The recent pi-and mu-class GST X-ray structures solved with bound substrate (Reinemer et al, 1991Ji et al, 1992) indicate that 81% of the residues directly contacting glutathione in domain 1 are found in the first 26 residues. This allowed us to focus on this region to develop class-specific patterns to describe the N-terminal glutathione-binding motif.…”

Fluorescence characterization of Trp 21 in rat glutathione S‐transferase 1–1: Microconformational changes induced by S‐hexyl glutathione

“…et al, 1991W Wang et ai. et al, , 1992Ji et al, 1992). NMR studies also have demonstrated the similarity in structure between these classes of GSTs (Penington & Rule, 1992).…”