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, Met51 and Phe52, that fit into a hydrophobic cavity (lock) in the adjacent subunit. SEC (size-exclusion chromatography)-HPLC, far-UV CD and tryptophan fluorescence of the M51A and M51A/F52S mutants indicated the non-disruptive nature of these mutations on the global structure. While the M51A mutant retained 80% of wild-type activity, the activity of the M51A/F52S was markedly diminished, indicating the importance of Phe52 in maintaining the correct conformation at the active site. The M51A and M51A/F52S mutations altered the binding of ANS (8-anilinonaphthalene-l-sulphonic 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 Met51 and Phe52 in the intersubunit lock-and-key motif play important structural roles in maintaining the catalytic and ligandin functions and stability of the GST dimer.
Alves
Carla S
CS
Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
Kuhnert
Diane C
DC
Sayed
Yasien
Y
Dirr
Heini W
HW
D020816:Amino Acid Motifs; D019281:Dimerization; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D008715:Methionine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation
D007527:Isoenzymes; D010649:Phenylalanine; D008715:Methionine; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha