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

Abstract

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.

Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

“…This value is very close to the predicted molecular weight of 171.2 KDa, which is the sum of 2 x 48.8 KDa (6xHis-FEZ1) and 2 x 36.8 KDa (GST-SCOCO). However, we must consider the dimerization of GST in this analysis [37-39]. To access exclusively the oligomeric state of the GST-SCOCO molecule when interacting with FEZ1 protein is not trivial because FEZ1 protein has many regions intrinsically disordered and most methods that allow us to estimate the molecular mass use globular proteins as standards.…”

Possible tetramerisation of the proteasome maturation factor POMP/proteassemblin/hUmp1 and its subcellular localisation

“…Hydrophobic interactions are known to be the most common motif for multimerisation of proteins. GST itself is known to stabilise its dimeric form through a hydrophobic lock-and-key motif [32]. Furthermore, several chaperones act as dimers, tetramers or oligomers, e.g.…”

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).…”

“…A combination of evidence from size exclusion chromatography and multiangle static light scattering, a well known method for measurement of absolute molecular weight of macromolecules in solution (27,39), revealed that all three mutants were successfully destabilized to form monomers in solution (Figs. 2 and 3).…”