Conformational analysis of oxidized peptide fragments of the C-terminal redox center in thioredoxin reductases by NMR spectroscopy

Abstract

Vicinal disulfide rings (VDRs) occur when a disulfide bond forms between adjacent cysteine residues in a protein and results in a rare eight-membered ring structure. This eight-membered ring has been found to exist in four major conformations in solution, divided between cis and trans conformers. Some selenoenzymes use a special type of VDR in which selenium replaces sulfur, generating a vicinal selenosulfide ring (VSeSR). Here we provide evidence that this substitution reduces ring strain, resulting in a strong preference for the trans conformation relative to cis in a VSeSR (cis:trans – 9:91). This was determined by using the “γ-gauche effect” which makes use of both 1H-NMR and two-dimensional (2D) NMR techniques for determining the amide bond conformeric ratio. The presence of selenium in a VSeSR also lowers the dihedral strain energy (DSE) of the selenosulfide bond relative to the disulfide bond of VDRs. While cis amide geometry decreases strain on the amide bond, it increases strain on the scissile disulfide bond of the VDR found in thioredoxin reductase from D. melanogaster (DmTR). We hypothesize that the cis conformation of the VDR is the catalytically competent conformer for thiol/disulfide exchange. This hypothesis was investigated by computing the DSE of VDR and VSeSR conformers, the structure of which was determined by 2D NMR spectroscopy and energy minimization. The computed values of the VDR from DmTR are 16.5 kJ/mol DSE and 14.3 kJ/mol for the C+ and T− conformers, respectively, supporting the hypothesis that the enzyme uses the C+ conformer for thiol/disulfide exchange.

The Marine Neurotoxin Brevetoxin (PbTx-2) Inhibits Karenia brevis and Mammalian Thioredoxin Reductases by Targeting Different Residues

“…A possible explanation as to why Kb TrxR can reduce GSSG while mammalian TrxR cannot is that Kb TrxR may bind GSSG similarly to the way in which glutathione reductase (GR) does. When bound to GR, GSSG is in a highly strained conformation, which facilitates GSSG reduction . TrxR and GR are evolutionarily and structurally related, with both having a conserved N-terminal CVNVGC redox center and a bound flavin .…”

Conformation dynamics of cyclic disulfides and selenosulfides in CXXC(U) (X = Gly, Ala) tetrapeptide redox motifs

“…The 14‐membered rings of oxidized CXXC cyclic peptides have wider variation in available conformations than the smaller CC/U motif . In multi‐μs MD simulations, CGGC has multiple regions of varying root mean square (RMSD) (Figure ) indicating a high degree of ring flexibility.…”

“…Conformations of the CC(U) ring, having the most ring strain and the least number of available conformations have been studied extensively. [31][32][33][34] Density functional theory (DFT) calculations showed that the observed 77 Se NMR spectrum of the CU motif was consistent with multiple conformations of the eight-membered ring coexisting in solution rather than different selenium functionalities or oxidation states. 35,36 In larger, more flexible cyclic CXXC(U) motifs, various groups have used molecular modeling to explore the conformation space of the oxidized peptides.…”

“…Smaller motifs would be expected to have a higher reduction potential related to the loss of strain energy with the breaking of the disulfide or selenosulfide bond. Conformations of the CC(U) ring, having the most ring strain and the least number of available conformations have been studied extensively . Density functional theory (DFT) calculations showed that the observed 77 Se NMR spectrum of the CU motif was consistent with multiple conformations of the eight‐membered ring coexisting in solution rather than different selenium functionalities or oxidation states .…”

“…2 | RESULTS AND DISCUSSION The 14-membered rings of oxidized CXXC cyclic peptides have wider variation in available conformations than the smaller CC/U motif. 32,33,44 In multi-μs MD simulations, CGGC has multiple regions of varying root mean square (RMSD) ( Figure 2 and i+2 residues determined the type of β-turn in a series of oxidized CXXC peptides. 38 Heatmaps for the backbone ϕ i + 1,2 ,ψ i + 1,2 dihedrals are shown in ( Figure S1).…”