Determinants of Activity in Glutaredoxins: An in vitro evolved Grx1-like Variant of E. coli Grx3
Résumé
The E. coli glutaredoxins 1 and 3 (Grx1 and Grx3) are structurally similar (37% sequence identity), yet have different activities in vivo. Unlike Grx3, Grx1 efficiently reduces protein disulfides in proteins like ribonucleotide reductase (RR), while being poor at reducing S-glutathionylated proteins. An E. coli strain lacking genes encoding thioredoxins 1 and 2 and Grx1 is not viable on either rich or minimal medium, however, a M43V mutation in Grx3 restores growth under these conditions and results in a Grx1-like protein [Ortenberg, R., et al. (2004) PNAS 101, 7439-44]. To uncover the structural basis of this change in activity, we have compared wild type and mutant Grx3 using CD and NMR spectroscopy. Ligand-induced stability measurements demonstrate that the Grx3(M43V/C65Y) mutant has acquired affinity for RR. Far-UV CD spectra reveal no significant differences, but differences are observed in the near-UV region indicative of tertiary structural changes. NMR 1H-15N HSQC spectra show that approximately half of the 82 residues experience significant (Δδ < 0.03 ppm) chemical shift deviations in the mutant, including 10 residues experiencing extensive (Δδ ≥ 0.15 ppm) deviations. To test if the M43V mutation alters dynamic properties of Grx3, H/D exchange experiments were performed demonstrating that the rate at which backbone amides exchange protons with the solvent is dramatically enhanced in the mutant – particularly in the core of the protein. These data suggest that the Grx1-like activity of the Grx3(M43V/C65Y) mutant may be explained by enhanced intrinsic motion allowing for increased specificity towards larger substrates such as RR.
Domaines
Sciences du Vivant [q-bio]
Origine : Fichiers produits par l'(les) auteur(s)
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