Intra- and inter-protein couplings of backbone motions underlie protein thiol-disulfide exchange cascade

Wenbo Zhang,Changwen Jin,Jienv Ding,Xiaogang Niu,Yunfei Hu

doi:10.1038/s41598-018-33766-4

Abstract

The thioredoxin (Trx)-coupled arsenate reductase (ArsC) is a family of enzymes that catalyzes the reduction of arsenate to arsenite in the arsenic detoxification pathway. The catalytic cycle involves a series of relayed intramolecular and intermolecular thiol-disulfide exchange reactions. Structures at different reaction stages have been determined, suggesting significant conformational fluctuations along the reaction pathway. Herein, we use two state-of-the-art NMR methods, the chemical exchange saturation transfer (CEST) and the CPMG-based relaxation dispersion (CPMG RD) experiments, to probe the conformational dynamics of B. subtilis ArsC in all reaction stages, namely the enzymatic active reduced state, the intra-molecular C10–C82 disulfide-bonded intermediate state, the inactive oxidized state, and the inter-molecular disulfide-bonded protein complex with Trx. Our results reveal highly rugged energy landscapes in the active reduced state, and suggest global collective motions in both the C10–C82 disulfide-bonded intermediate and the mixed-disulfide Trx-ArsC complex.

Highlights

Protein thiol-disulfide exchange reactions play fundamental roles in living systems, represented by the thioredoxin (Trx) and glutaredoxin (Grx) systems that maintain the cytoplasmic reducing environment, the protein DsbA that catalyzes the formation of protein disulfide bonds in bacterial periplasm, as well as the protein disulfide isomerase (PDI) proteins that facilitate correct disulfide bonding[1,2,3,4,5]
Since the re-arsenate reductase (ArsC) structure requires oxyanion binding to become stabilized[19,20,25], the available solution structure of re-ArsC, which was determined in the presence of sulfate, should be regarded as a mimic of the enzyme non-covalently bound with the substrate
The re-ArsC·sulfate sample was prepared with ArsC concentration of 1.8 mM and sulfate concentration of 20 mM. (E) Comparison between the Δω values extracted from chemical exchange saturation transfer (CEST) experiment of re-ArsC·sulfate and the chemical shift differences Δδfree − sulfate for residues showing sulfate concentrationdependent CEST profiles

Summary

Introduction

Protein thiol-disulfide exchange reactions play fundamental roles in living systems, represented by the thioredoxin (Trx) and glutaredoxin (Grx) systems that maintain the cytoplasmic reducing environment, the protein DsbA that catalyzes the formation of protein disulfide bonds in bacterial periplasm, as well as the protein disulfide isomerase (PDI) proteins that facilitate correct disulfide bonding[1,2,3,4,5]. Via combined efforts from both X-ray crystallography and solution NMR spectroscopy, the structures of Trx-coupled ArsC (either B. subtilis ArsC or S. aureus pI258 ArsC, abbreviated as Bs_ArsC and Sa_ArsC) have been elucidated in various reaction stages, including the transiently formed Trx-ArsC mixed-disulfide complex[15,16,17,19,20]. We use a combination of the Carr-Purcell-Meiboom-Gill (CPMG)-based R2 relaxation dispersion (CPMG RD) and chemical exchange saturation transfer (CEST) NMR methods[21,22,23,24] to investigate the conformational dynamics of Trx-coupled ArsC during the catalytic cycle, and to gain further understanding of protein thiol-disulfide exchanges. All states of the ArsC sample in the manuscript refer to Bs_ArsC

Methods

Results

Conclusion