The intersubunit lock-and-key motif in human glutathione transferase A1-1:: role of the key residues Met51 and Phe52 in function and dimer stability

The dimeric structure of certain cytosolic GSTs (glutathione S-transferases) is stabilized by a hydrophobic lock-and-key motif at their subunit interface. In hGSTA1-1 (human class Alpha GST with two type-1 subunits), the key consists of two residues, Met(51) and Phe(52), that fit into a hydrophobic cavity (lock) in the adjacent subunit. SEC (size-exclusion chromatography)-HPLC, far-V CD and tryptophan fluorescence of the M51 and M51/F52S mutants indicated the non-disruptive nature of these mutations on the global structure. While the M51 mutant retained 80% of wild-type activity, the activity of the M51A/52S was markedly diminished, indicating the importance of Phe(52) in maintaining the correct conformation at the active site. The M51 and M51/F52S mutations altered the binding of ANS (8-anilinonaphthalene-1sulphonic acid) at,the H-site by destabilizing helix 9 in the C-terminal region. Data from urea unfolding studies show that the dimer is destabilized by both mutations and that the dimer dissociates to aggregation-prone monomers at low urea concentrations before global unfolding. Although not essential for the assembly of the dimeric structure of hGSTA1-1, both Met(51) and Phe(52) in the intersubunit lock-and-key motif play important structural roles in maintaining the catalytic and ligandin functions and stability of the GST dimer.