Double Mutation at the Subunit Interface of Glutathione Transferase rGSTM1-1 Results in a Stable, Folded Monomer

Abstract

Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A mutants indicate that a reorganization of the loop structure between helix alpha2 and strand beta3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha4 and alpha5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A mutant suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.

Engineered Monomeric Human Histidine Triad Nucleotide-binding Protein 1 Hydrolyzes Fluorogenic Acyl-adenylate and Lysyl-tRNA Synthetase-generated Lysyl-adenylate

“…1D). The substitution of charged residues, such as Asp, Arg, and Glu into a protein dimer or oligomer interface has been shown to be a viable approach for preparing monomeric enzymes (25,(37)(38)(39).…”

Catalytically Active Monomer of Glutathione S-Transferase π and Key Residues Involved in the Electrostatic Interaction between Subunits

“…Although it has been suggested that the hydrophobic lock and key interaction is the major interaction between subunit interfaces in all GSTs (7,14,15,25,34,35), the electrostatic attractions definitely enhance the interaction between subunits and play an important role at the dimer interface (22,23,25,36). Electrostatic interactions have also been shown to be involved in both the catalytic reaction and the subunit interactions of the ␦ class GST of Anopheles dirus (37,38) and of the cephalopod class GST (39).…”

A Rationally Designed Monomeric Variant of Anthranilate Phosphoribosyltransferase from Sulfolobus solfataricus is as Active as the Dimeric Wild-type Enzyme but Less Thermostable

“…Along these lines, amino acid substitutions at the contact interface lead to the dissociation of oligomeric enzymes into subunits with more or less reduced reaction rates and conformational stabilities. [7][8][9][10][11] The members of the phosphoribosyltransferase (PRT) enzyme class catalyze the transfer of a phosphoribosyl group to an aromatic base. PRTs have an important role in the metabolism of nucleotides and amino acids; for example, as key components of the purine salvage and the histidine and tryptophan biosynthetic pathways.…”