Quantifying Escherichia coli Glutaredoxin-3 Substrate Specificity Using Ligand-induced Stability

Abstract

Traditionally, quantification of protein-ligand affinity is performed using kinetic or equilibrium measurements. However, if the binding reaction proceeds via a stable covalent complex, these approaches are often limited. By exploiting the fact that the conformational stabilization of a protein is altered upon ligand binding due to specific interactions, and using an array of selectively chosen ligand analogs, one can quantify the contribution individual interactions have on specificity. We have used ligand-induced stability as a basis to dissect the interaction between glutaredoxin-3 (Grx3) and one of its native substrates, the tripeptide glutathione. Taking advantage of the fact that Grx3 can be trapped in a covalent mixed disulfide to glutathione or to selected synthetic glutathione analogs as part of the natural catalytic cycle, individual contributions to binding of specific molecular groups can be quantified by changes in ligand-induced stability. These changes in conformational stability are interpreted in terms of interaction energies (i.e. specificity) of the particular groups present on the ligand analog. Our results illustrate that although Grx3 recognizes glutathione predominantly through independent and additive ionic interactions at the N-and C-terminal of glutathione, van der Waals interactions from the unique ␥-glutamate moiety of glutathione also play an important role. This study places us closer to understanding the complex task of accommodating multiple substrate specificities in proteins of the thioredoxin superfamily and underscores the general applicability of ligand-induced stability to probe substrate specificity.In the era of structural genomics, a large number of biomolecules will have their tertiary structure determined. However, to fully appreciate the potential of these structures, it is fundamental to determine how these molecules interact with other molecules. Therefore, when exploring the structural basis of observed biological activity it is of great importance to investigate the details of intermolecular interactions. One way of attaining a deeper insight into these interactions is by quantifying ligand affinity, usually performed by kinetic or equilibrium measurements of the binding reaction. Specific interactions between a ligand and a protein can be investigated further by modifying specific residues within the protein using site-directed mutagenesis. The relative binding constants of a wildtype and a mutant protein to a particular ligand then provide information about specificity. However, protein mutagenesis sometimes results in secondary effects as intrinsic properties of the protein may be altered, making subsequent analysis complex. Furthermore, if the interaction between two molecules involves a relatively stable covalent intermediate, standard methods of measuring affinity are often of limited value.Several studies have demonstrated that the free energy of non-covalent binding of a ligand to a protein results in a change in conformational stabilization with respect to...

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

“…Interestingly, in E. coli Grx2, which has the highest activity of the three bacterial dithiol Grxs toward 2-ME-SSG, the Lys is replaced by a Tyr. This supports previous reports that the characteristic ␥-glutamate moiety is more important for GSH recognition than the glycine part (43,44). In T. brucei Grx1 and Grx2, these substitutions probably support the interaction with T(SH) 2 (see below), in which the glycine carboxylate does not carry a negative charge but is engaged in an amide bond with the spermidine bridge.…”

Mono- and Dithiol Glutaredoxins in the Trypanothione-Based Redox Metabolism of Pathogenic Trypanosomes

“…However, it should be stressed that in E. coli Grx2, which has the highest HED reductase activity of the three bacterial dithiol Grxs (E. coli 2-C-Grx 1 to 3), the Lys is replaced by a Tyr. This supports previous reports that the characteristic c-Glu-Cys moiety is more important for GSH recognition than the glycine carboxylate (23,81). As will be outlined in this review, the trypanosomal 2-C-Grxs are involved in reactions with both GSH and/or T(SH) 2 .…”

Mechanistic and Kinetic Details of Catalysis of Thiol-Disulfide Exchange by Glutaredoxins and Potential Mechanisms of Regulation

“…The carboxylate of the glycyl moiety of the glutathionyl adduct is stabilized by H-bond donors or a positively charged lysine or both in most Grx-SSGs; however, a recent study of hGrx1 in which the interacting Lys was mutated to Leu or Gln resulted in retention of glutathionyl substrate specificity (58), suggesting that this specific ionic interaction is not required for reaction of Grx with the glutathionyl sulfur of the GS-containing mixed disulfide substrate. Moreover, analysis of the stability of mixed disulfides between E. coli Grx3 and various GSH analogues (28) suggested that the interaction between Grx and the glycyl group of the GS moiety contributes little to stabilization of the glutathionyl moiety in the Grx-SSG mixed disulfide intermediate.…”