Domain-domain interface packing at conserved Trp-20 in class alpha glutathione transferase impacts on protein stability

The folding and assembly of the dimeric glutathione transferases (GST) invo lves the association of two structurally distinct domains per subunit. A pr ominent and conserved domain-domain interaction in class alpha GSTs is form ed by the packing of the indole side chain of Trp-20 from domain I into a h ydrophobic pocket in domain II. Stability studies have shown that partial d issociation of the domains near Trp-20 occurs as an initial fast-event duri ng the unfolding kinetics of human GSTA1-1 (Wallace et al., Biochemistry 37 (1998) 5320-5328; Wallace et al., Biochem. J. 336 (1998) 413-418). The con tribution of Trp-20 toward stabilising the domain-domain interface was inve stigated by mutating it to either a phenylalanine (W20F) or alanine (W20A) and determining the functionality (catalysis and non-substrate ligand bindi ng) and stability (thermal- and urea-induced denaturation) of the mutant pr oteins. The replacement of Trp-20 did not impact on the protein's gross str uctural properties. Functionally, the W20F was non-disruptive, whereas the cavity-creating W20A mutation was. Both mutants destabilised the native sta te with W20A exerting the greatest effect. Reduced m-values as well as the protein concentration dependence of the urea unfolding transitions for W20F GSTA1-1 suggest the presence of a dimeric intermediate at equilibrium that is not observed with wild-type protein. Unfolding kinetics monitored by st opped-flow tyrosine fluorescence was mono-exponential and corresponded to t he global unfolding of the protein during which the dimeric intermediate un folds to two unfolded monomers. The similar unfolding kinetics data for wil d-type and W20F AI-l indicates that the global unfolding event was not affe cted by amino acid replacement. We propose that the packing interactions at the conserved Trp-20 plays an important role in stabilising the intrasubun it domain I-domain II interface of class a GSTs. (C) 2000 Elsevier Science B.V. All rights reserved.