A conserved "hydrophobic staple motif" plays a crucial role in the refolding of human glutathione transferase P1-1

The specific (i, i+5) hydrophobic staple interaction involving a helix resi due and a second residue located in the turn preceding the helix is a recur rent motif at the N terminus of alpha-helices, This motif is strictly conse rved in the core of all soluble glutathione transferases (GSTs) as well as in other protein structures. Human GSTP1-1 variants mutated in amino acid I le(149) and Tyr(154) Of the hydrophobic staple motif of the alpha 6-helix w ere analyzed, In particular, a double mutant cycle analysis has been perfor med to evaluate the role of the hydrophobic staple motif in the refolding p rocess. The results show that this local interaction, by restricting the nu mber of conformations of the alpha 6-helix relative to the al-helix, favors the formation of essential interdomain interactions and thereby accelerate s the folding process, Thus, for the first time it is shown that the hydrop hobic staple interaction has a role in the folding process of an intact pro tein. In P-i class GSTs, Tyr(154) appears to be of particular structural im portance, since it interacts with conserved residues Leu(21), Asp(24), and Gln(25) of the adjacent alpha 1-helix which contributes to the active site. Human GSTP1-1 variants L21A and Y154F have also been analyzed in order to distinguish the role of interdomain interactions from that of the hydrophob ic staple. The experimental results reported here suggest that the strict c onservation of the hydrophobic staple motif reflects an evolutionary pressu re for proteins to fold rapidly.