How thioredoxin dissociates its mixed disulfide

Abstract

The dissociation mechanism of the thioredoxin (Trx) mixed disulfide complexes is unknown and has been debated for more than twenty years. Specifically, opposing arguments for the activation of the nucleophilic cysteine as a thiolate during the dissociation of the complex have been put forward. As a key model, the complex between Trx and its endogenous substrate, arsenate reductase (ArsC), was used. In this structure, a Cys29(Trx)-Cys89(ArsC) intermediate disulfide is formed by the nucleophilic attack of Cys29(Trx) on the exposed Cys82(ArsC)-Cys89(ArsC) in oxidized ArsC. With theoretical reactivity analysis, molecular dynamics simulations, and biochemical complex formation experiments with Cys-mutants, Trx mixed disulfide dissociation was studied. We observed that the conformational changes around the intermediate disulfide bring Cys32(Trx) in contact with Cys29(Trx). Cys32(Trx) is activated for its nucleophilic attack by hydrogen bonds, and Cys32(Trx) is found to be more reactive than Cys82(ArsC). Additionally, Cys32(Trx) directs its nucleophilic attack on the more susceptible Cys29(Trx) and not on Cys89(ArsC). This multidisciplinary approach provides fresh insights into a universal thiol/disulfide exchange reaction mechanism that results in reduced substrate and oxidized Trx.

Figure 1. In the structure of the Trx-ArsC mixed disulfide complex the functionally key disulfide between Cys29^Trx and Cys89^ArsC is formed.

The Bs_Trx-ArsC complex (2IPA) with the side chains of residues Cys29^Trx, Cys32^Trx, Trp28^Trx, Arg16^ArsC, Cys82^ArsC and Cys89^ArsC in stick representation is shown. The Trx α1-helix is shown in blue; the ArsC looped-out redox helix between Cys82^ArsC and Cys89^ArsC in pink.

Figure 2. Bs_Trx reduces Bs_ArsC via an intermediate Trx-ArsC complex.

A Cys29^Trx of reduced Bs_Trx (2GZY) nucleophilically attacks Cys89^ArsC of the Cys82^ArsC-Cys89^ArsC disulfide of oxidized Bs_ArsC (1Z2E), leading to the formation of the mixed Cys29^Trx-Cys89^ArsC disulfide (2IPA). (B and C) Cys32^Trx performs a nucleophilic attack on Cys29^Trx, leading to the release of reduced Bs_ArsC (1Z2D) and oxidized Bs_Trx (2GZZ).

Figure 3. Experimental pKa’s are correlated with the calculated NPA charge.

NPA-charge-pKa calibration curve (black circles) obtained A for a series of small thiolate molecules (the experimental pKa's are obtained from ref. [74]) and B for a series of cysteine residues of Trx and ArsC systems. The data points of the calibration curve B (a–g) are tabulated in Table S1 in Supplemental Data and are also indicated on curve A. trifluoromethanethiol (1), methanethiol (2), mercaptoethanol (3), cysteine (4), trifluoroethanethiol (5), benzenemethanethiol (6) and thioacetic acid (7). Cys89 S. aureus ArsC (a), Cys77 B. subtilis resA , (b), Cys10 S. aureus ArsC (c), Cys32 E. coli Trx1 (d), Cys29 S. aureus P31T C32S Trx1 (e), Cys73 R. capsulatus Trx2 (f) and Cys35 E. coli Trx1 (g).

Figure 4. Model systems of Bs_Trx, Bs_ArsC and Bs_Trx-ArsC complex for pKa and reactivity analysis.

A Reduced Bs_Trx (2GZY) modelled by the Trp28^Trx-Cys32^Trx active site and the Lys33-Glu45 α1-helix (blue). B Oxidized Bs_ArsC (1Z2E) represented by the Cys82^ArsC-Cys89^ArsC looped-out redox helix (ArsC_ox). C Model of Bs_Trx-ArsC (Trx_ArsC_1), in which no hydrogen bond interactions with Cys32^Trx nor with Cys82^ArsC are present, represented by the Trp28^Trx-Cys32^Trx Trx active site and the Cys82^ArsC-Cys89^ArsC ArsC redox helix (pink) and Thr11^ArsC. D Model of Bs_Trx_ArsC (Trx-ArsC_2), in which the Cys82^ArsCS_γ—Arg16^ArsCN_η1 and the Cys32^TrxS_γ—Cys29^TrxN hydrogen bonds are present. The Trp28^Trx-Cys32^Trx Trx active site and the Cys82^ArsC-Cys89^ArsC ArsC redox helix (pink) and Thr11^ArsC and Arg16^ArsC are included. E Trx_ArsC_1_trunc and Trx_ArsC_2_trunc model systems including the Trp28^Trx-Cys32^Trx active site of Trx and Cys89^ArsC of the ArsC. F Trx_ArsC_2_trunc+helix and Trx_ArsC_1_trunc+helix model systems including everything described under (E.) and the Trx Lys33^Trx-Glu45^Trx α1-helix (blue).

Figure 5. Hydrogen bonding of Cys32^Trx in the mixed disulfide of Bs_Trx-ArsC.

Time course in the MD simulation of the distance between A Cys32^TrxS_γ and Trp28^TrxN and between B Cys32^TrxS_γ and Cys29^TrxN, with ionized (black) and neutral (red) Cys82^ArsC. C Example of one MD snapshot with the Cys32^TrxS_γ—Cys29^TrxN and Cys32^TrxS_γ—Trp28^TrxN hydrogen bonds introduced, when Cys82^ArsC is deprotonated.

Figure 6. A conformational change brings Cys32^Trx in contact with Cys29^Trx.

Time course in the MD simulation (ionized Cys82^ArsC) of the distance between Cys29^TrxS_γ and Cys32^TrxS_γ (red) and Cys32^TrxS_γ and Trp28^TrxN (A, black) and Cys32^TrxS_γ and Cys29^TrxN (B, black). C Superposition of the Trx active site (Trx28^Trx-Cys32^Trx) and the ArsC redox helix (Cys82^ArsC-Cys89^ArsC) of the Bs_Trx-ArsC complex at 0 ns (purple) and 14.5 ns (green) simulation time showing the conformational change associated with the approach of Cys32^Trx to Cys29^Trx during the MD simulation (ionized Cys82^ArsC).

Figure 7. Cys32^Trx is primed for nucleophilic attack in the Trx-ArsC activated complex.

Selected snapshots from an MD simulation of the Trx-ArsC complex (ionized Cys82^ArsC), presenting the structural basis of the activation of Cys32^Trx (green carbons, ball and sticks) for its nucleophilic attack onto Cys29^Trx. In these snapshots, Cys32^TrxS_γ is hydrogen-bonded (magenta dotted lines) to the amide NH groups of both Trp28^Trx and Cys29^Trx. Also, the sulphur atoms of Cys32^Trx and Cys29^Trx are within 4.5 Å of each other. The hydrogen bonds satisfy the geometric criteria: Cys32^TrxS_γ—Trp28^TrxN≤4 Å and Cys32^TrxS_γ—Cys29N^Trx≤4 Å, with the corresponding angles between S_γ and the N−H vectors being ≥150 degrees. The hydrogen-bonds donated to the sulphur of Cys32^Trx lower its pKa to 7.4 (Table 4), corresponding to a significant population of the thiolate form of Cys32^Trx. This thiolate being close to Cys29^Trx, it is in effect primed for nucleophilic attack onto Cys29^Trx (black arrow). In combination with the supporting reaction analysis, pKa calculations and complex formation experiments (main text), the conformations and interactions shown here are proposed to underpin the dissociation mechanism of the Tx-ArsC complex.

Figure 8. The putative role of Asp23^Trx in the deprotonation of Cys32^Trx revisited.

A Water is sometimes proposed to assist the deprotonation of Cys32^Trx by Asp23^Trx. The position of the water molecule in the Bs_Trx-ArsC complex was obtained by modeling. B Evaluation of the wild type Sa_Trx-Sa_ArsC^Trip and the Sa_Trx D23A-Sa_ArsC^Trip complex formation on a non-reducing SDS-PAGE (pH 7.5). No stable complex is detected. (1. Sa_Trx+Sa_ArsC^Trip; 2. Sa_Trx D23A+Sa_ArsC^Trip 3. Positive control band of the Sa_Trx-ArsC complex [1]). (C.) Reversed phase-analysis of the reaction products after complex formation between Sa_Trx D23A and Sa_ArsC^Trip. E D Time course in the MD simulation (ionized Cys82^ArsC) of the distance between Asp23^TrxO_δ1 and Cys32^TrxS_γ (red) and Cys32^TrxS_γ and Trp28^TrxN (D, black) and Cys32^TrxS_γ and Cys29^TrxN (E, black).