Role of the C-Terminal Helix 9 in the Stability and Ligandin Function of Class α Glutathione Transferase A1-1

Abstract

Helix 9 at the C-terminus of class alpha glutathione transferase (GST) polypeptides is a unique structural feature in the GST superfamily. It plays an important structural role in the catalytic cycle. Its contribution toward protein stability/folding as well as the binding of nonsubstrate ligands was investigated by protein engineering, conformational stability, enzyme activity, and ligand-binding methods. The helix9 sequence displays an unfavorable propensity toward helix formation, but tertiary interactions between the amphipathic helix and the GST seem to contribute sufficient stability to populate the helix on the surface of the protein. The helix's stability is enhanced further by the binding of ligands at the active site. The order of ligand-induced stabilization increases from H-site occupation, to G-site occupation, to the simultaneous occupation of H- and G-sites. Ligand-induced stabilization of helix9 reduces solvent accessible hydrophobic surface by facilitating firmer packing at the hydrophobic interface between helix and GST. This stabilized form exhibits enhanced affinity for the binding of nonsubstrate ligands to ligandin sites (i.e., noncatalytic binding sites). Although helix9 contributes very little toward the global stability of hGSTA1-1, its conformational dynamics have significant implications for the protein's equilibrium unfolding/refolding pathway and unfolding kinetics. Considering the high concentration of reduced glutathione in human cells (about 10 mM), the physiological form of hGSTA1-1 is most likely the thiol-complexed protein with a stabilized helix9. The C-terminus region (including helix9) of the class alpha polypeptide appears not to have been optimized for stability but rather for catalytic and ligandin function.

Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding

“…Although the exact location of the ANS binding site in the hGSTA1 subunit is unknown, a fluorescence resonance energy-transfer study indicated binding of ANS at the V-shaped cleft along the dimer interface [11]. The C-terminus of helix 9, also located near the dimer interface, is adjacent to the ANS site and modulates ANS binding [28]. Other non-substrate ligands that bind the V-shaped cleft include aflatoxin B " [12], oestradiol disulphate [13] and 5-o[2-[(acetyl)amino]ethyl]aminoq-naphthalene-1-sulphonic acid (AEDANS) [42].…”

“…that is implicated in ligandin function [28]. The role of the ultimate residue Phe-222, located near the cleft at the dimer interface, in ligandin function was also addressed.…”

“…The purity of the protein was confirmed by SDS\PAGE [31] and size-exclusion chromatography\HPLC. The dimeric protein concentration was calculated spectrophotometrically using a molar absorption coefficient (ε) of 38 200 M −" :cm −" at 280 nm [28]. ANS was purchased from Sigma and its concentration in solution was determined spectrophotometrically at 350 nm using ε l 4950 M −" :cm −" .…”

“…Proteins were excited at 295 nm, to obtain tryptophan fluorescence spectra. The binding of ANS to protein was monitored using enhanced fluorescence of the dye when excited at 390 nm [28].…”

cDNA cloning and mRNA expression of four glutathione S-transferase (GST) genes from Mytilus galloprovincialis

“…Using the CD-Search in NCBI's CDD, the predicted protein sequence of MgGSTa cDNA was matched to the GST_N_alpha_like and GST_C_alpha_like, containing an H-site which is a substrate binding site in the C-terminal [14]. The longer alpha C-terminal also forms an a-helix, which is thought to be important to dimer stabilization and affects both the GSH-binding rate and the ionization state of the catalytically essential residue Tyr [15,16]. The sigma class GSTs are lacking in both components of the ball-and-socket interface, and rely on a Tyr residue for GSH stabilization in the G-site [12], which was also found in the sequence of MgGSTS1, MgGSTS2 and MgGSTS3.…”