Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

Abstract

Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.

Catalysis of Peroxide Reduction by Fast Reacting Protein Thiols

“…223−225 It seems that the reduction step of 2-Cys Prx can be accomplished by any Trx, independently of the species, thus the standard Prx activity assays usually employ the systems of Trx coupled to TR from Escherichia coli 226 or Saccharomyces cerevisiae. 227 The kinetics of reduction of 2-Cys Prx are also understudied, only a few systems of partner Prx and Trx have been thoroughly studied, most notably the reduction of Prx5 by Trx2, both human mitochondrial enzymes, 126 the reduction of TPx by Trx1 from E. coli, 228,229 and the reduction of PrxQ B by TrxC from Mycobacterium tuberculosis. 230 The reductions of tryparedoxin peroxidase by tryparedoxin (a Trx-like protein) from trypanosomatids, 175 and of AhpC by AhpF from Salmonella typhimurium, a flavo-enzyme present in several bacteria, 219,231 have been also characterized.…”

“…The kinetics of reduction of 2-Cys Prx are also understudied, only a few systems of partner Prx and Trx have been thoroughly studied, most notably the reduction of Prx5 by Trx2, both human mitochondrial enzymes, the reduction of TPx by Trx1 from E. coli, , and the reduction of PrxQ B by TrxC from Mycobacterium tuberculosis . The reductions of tryparedoxin peroxidase by tryparedoxin (a Trx-like protein) from trypanosomatids, and of AhpC by AhpF from Salmonella typhimurium, a flavo-enzyme present in several bacteria, , have been also characterized.…”

Kinetic studies reveal a key role of a redox-active glutaredoxin in the evolution of the thiol-redox metabolism of trypanosomatid parasites

“…However, the reaction of Grx1S 2 with DTT is not specific and occurs with a moderate rate constant (690 Ϯ 6 M Ϫ1 s Ϫ1 , Fig. S3) in the expected range for the reduction of any disulfide by this dithiol (6,45) and is mainly driven by the entropic effect of the formation of a six-membered cyclic disulfide of DTT (trans-4,5-dihydroxy-1,2-dithiane).…”

Exploring the conformational transition between the fully folded and locally unfolded substates ofEscherichia colithiol peroxidase

“…The specificity constant of Ec TPx for H 2 O 2 ( k cat /K M = 8.9 × 10 4 M -1 s -1 , [16] ) is in the range observed for other TPx-Prx subfamily members [17] . The kinetic parameters for the individual steps in the reduction of H 2 O 2 by TPx (catalyzed by TRX) have been reported [8] , k cat = 76 s -1 (H 2 O 2 ), K M = 1.7 mM (H 2 O 2 ) and K M = 25.5 µM (TRX), and indicate that the most likely rate-limiting step of the cycle is the peroxidation (Reaction I, Figure 1) since large concentrations of H 2 O 2 would be needed to saturate the enzyme.…”

“…Remarkably, this behavior contrasts with that observed for Ec TRX enzyme, the oxidoreductase partner of Ec TPx in the catalytic cycle (see Figure 1B). When both redox states of the Ec TRX are compared no conformational change are present and only subtle changes in internal motions are observed [16,35,36] , whereas the difference in the free energy of unfolding for oxidized state is significantly higher than the corresponding to the reduced state (Δ G o NU, H2O ox -Δ G o NU, H2O red = 3.5 kcal mol -1 at 25°C, [37,38] ). More experiments should be done to understand why such different behaviors were selected through evolution and to elucidate if there is a thermodynamic connection between both proteins.…”