Functional Role of the Lock and Key Motif at the Subunit Interface of Glutathione Transferase P1-1

Abstract

The glutathione transferases (GSTs) represent a superfamily of dimeric proteins. Each subunit has an active site, but there is no evidence for the existence of catalytically active monomers. The lock and key motif is responsible for a highly conserved hydrophobic interaction in the subunit interface of pi, mu, and alpha class glutathione transferases. The key residue, which is either Phe or Tyr (Tyr 50 in human GSTP1-1) in one subunit, is wedged into a hydrophobic pocket of the other subunit. To study how an essentially inactive subunit influences the activity of the neighboring subunit, we have generated the heterodimer composed of subunits from the fully active human wild-type GSTP1-1 and the nearly inactive mutant Y50A obtained by mutation of the key residue Tyr 50 to Ala. Although the key residue is located far from the catalytic center, the k cat value of mutant Y50A decreased about 1300-fold in comparison with the wild-type enzyme. The decrease of the k cat value of the heterodimer by about 27-fold rather than the expected 2-fold in comparison with the wild-type enzyme indicates that the two active sites of the dimeric enzyme work synergistically. Further evidence for cooperativity was found in the nonhyperbolic GSH saturation curves. A network of hydrogen-bonded water molecules, found in crystal structures of GSTP1-1, connects the two active sites and the main chain carbonyl group of Tyr 50 , thereby offering a mechanism for communication between the two active sites. It is concluded that a subunit becomes catalytically competent by positioning the key residue of one subunit into the lock pocket of the other subunit, thereby stabilizing the loop following the helix ␣2, which interacts directly with GSH.

Catalytically Active Monomer of Glutathione S-Transferase π and Key Residues Involved in the Electrostatic Interaction between Subunits

“…Although it has been suggested that the hydrophobic lock and key interaction is the major interaction between subunit interfaces in all GSTs (7,14,15,25,34,35), the electrostatic attractions definitely enhance the interaction between subunits and play an important role at the dimer interface (22,23,25,36). Electrostatic interactions have also been shown to be involved in both the catalytic reaction and the subunit interactions of the ␦ class GST of Anopheles dirus (37,38) and of the cephalopod class GST (39).…”

Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells

“…Under cellular stress, such as that from oxidative metabolism, however, GSTP1 dissociates from the GSTP1-JNK1 complex, leading subsequently to the activation of JNK (30). This intracellular switch from the monomeric to the dimeric form of GSTP1 and the associated altered JNK signaling provides a mechanism of cellular response to and protection from oxidative and other potentially deleterious stress (50,51).…”

“…Structural factors that contribute to GSTP1 dimer formation include the hydrophobic lock and key interaction between subunit interfaces and the electrostatic attractions associated with the interactions at the dimer interface (50). Under cellular stress, such as that from oxidative metabolism, however, GSTP1 dissociates from the GSTP1-JNK1 complex, leading subsequently to the activation of JNK (30).…”

Structure and Antioxidant Catalytic Function of Plant Glutathione Transferases

“…The lock-and-key motif is a common feature of GSTs of the tau, phi, alpha, mu and pi classes [67,75,76]. Only subunits with the same interfacing type appear to be compatible for dimerization.…”

“…The interactions that are involved in assembling the quaternary structure of GSTs include salt bridges, hydrogen bonds, hydrophilic and hydrophobic interactions, including a lock-and-key motif that physically anchors the two subunits together [75][76][77]. The lock-and-key motif is a common feature of GSTs of the tau, phi, alpha, mu and pi classes [67,75,76].…”