Structure-function studies of glutaredoxins and related oxidoreductases

The members of the ubiquitous group of thiol-disulfide oxidoreductases are characterized by a conserved tertiary structure, the thioredoxin (Trx) fold, and by having a consensus -C-X-X-C- active site sequence motif. By utilizing the active site cysteines, these proteins participate in redox reactions by catalyzing reversible disulfide oxidation and reduction. The glutaredoxin (Grx) subgroup and the group of protein disulfide oxidoreductases (PDOs) found in hyperthermophilic prokaryotes, provide intriguing examples of variations on the common thioredoxin theme. The glutaredoxins being effective catalysts of protein-glutathione mixed disulfides, having a high specificity to the γGlu-Cys-Gly tripeptide, glutathione, and the PDOs being atypical in the sense of having two Trx fold domains each having the -C-X-X-C- active site sequence.

To some extent, the members of the Trx superfamily have overlapping activities. However, regardless of their mechanistic and structural similarities, they also have distinct activities. These differences are to some extent due to the extrinsic milieu, however, they are largely dependent on the intrinsic properties of these enzymes such as active site disulfide standard state redox potential ( E°' ) and active site cysteine thiol pKa values, in addition to their various substrate specificities.

The central theme of this thesis has been to acquire a deeper knowledge about the determinants of substrate specificity of the glutaredoxins, using Grx3 from E. coli as a model system. This required the development of an approach which uses ligand- induced stability to quantify substrate specificity. This work has also included the investigation of redox properties of active site and nonactive site disulfides in the human glutaredoxins (hGrx1, hGrx2, and hGrx5) as well as two PDO proteins from the hyperthermophilic organisms A. aeolicus and P. furiosus.

The results provide details about substrate specificity of the glutaredoxins. In particular, the individual interactions between Grx3 and glutathione have been quantified demonstrating the importance of ionic and van der Waals interactions in Grx3 substrate recognition. Furthermore, additional insight into the structural basis of activity is obtained from experiments designed at uncovering the origin of Grx1-like activity in a M43V mutant of Grx3. The results presented here underscore the importance of Grx1 conformational flexibility in the recognition of substrate proteins. The characterization of redox properties of human Grx1 and Grx2, and the two PDO proteins from A. aeolicus and P. furiosus provides data related to their biological function. Evidence is presented that the human dithiol glutaredoxins as well as the active sites of the two PDO C-terminal Trx-fold domains have redox potentials in the order of that reported for many other glutaredoxins (∼ -220-240 mV). The ∼50 mV redox potential difference of the PDO N-terminal domains, being more reducing, demonstrate the nonequivalence of the two active sites in each PDO protein. Furthermore, the identification and determination of redox potential ( E°' = -317 mV) of a nonactive site disulfide in hGrx2 shows that this disulfide is present in all but the most reducing conditions, conferring nearly 5 kcal mol-1 in stability to this protein.

List of scientific papers

I. Elgán TH, Berndt KD (2008). "Quantifying Escherichia coli glutaredoxin-3 substrate specificity using ligand-induced stability." J Biol Chem 283(47): 32839-47. Epub 2008 Aug 29
https://pubmed.ncbi.nlm.nih.gov/18757366

II. Sagemark J, Elgán TH, Bürglin TR, Johansson C, Holmgren A, Berndt KD (2007). "Redox properties and evolution of human glutaredoxins." Proteins 68(4): 879-92
https://pubmed.ncbi.nlm.nih.gov/17546662

III. Elgán TH, Planson AG, Beckwith J, Güntert P, Berndt KD (2008). "Investigating the determinants of activity in glutaredoxins: an in vitro evolved Grx1-like variant of E. coli Grx3." (Submitted)

IV. Elgán TH, Ladenstein R, Berndt KD (2008). "Redox properties of two hyperthermophilic protein disulfide oxidoreductases and comparison to protein disulfide isomerase." (Manuscript)