Journal of Molecular Biology
Volume 366, Issue 1, 9 February 2007, Pages 193-206
Journal home page for Journal of Molecular Biology

Solution Structure and Backbone Dynamics of the Reduced Form and an Oxidized Form of E. coli Methionine Sulfoxide Reductase A (MsrA): Structural Insight of the MsrA Catalytic Cycle

https://doi.org/10.1016/j.jmb.2006.11.042Get rights and content

Abstract

Methionine sulfoxide reductases (Msr) reduce methionine sulfoxide (MetSO)-containing proteins, back to methionine (Met). MsrAs are stereospecific for the S epimer whereas MsrBs reduce the R epimer of MetSO. Although structurally unrelated, the Msrs characterized so far display a similar catalytic mechanism with formation of a sulfenic intermediate on the catalytic cysteine and a concomitant release of Met, followed by formation of at least one intramolecular disulfide bond (between the catalytic and a recycling cysteine), which is then reduced by thioredoxin. In the case of the MsrA from Escherichia coli, two disulfide bonds are formed, i.e. first between the catalytic Cys51 and the recycling Cys198 and then between Cys198 and the second recycling Cys206. Three crystal structures including E. coli and Mycobacterium tuberculosis MsrAs, which, for the latter, possesses only the unique recycling Cys198, have been solved so far. In these structures, the distances between the cysteine residues involved in the catalytic mechanism are too large to allow formation of the intramolecular disulfide bonds. Here structural and dynamical NMR studies of the reduced wild-type and the oxidized (Cys51-Cys198) forms of C86S/C206S MsrA from E. coli have been carried out. The mapping of MetSO substrate-bound C51A MsrA has also been performed. The data support (1) a conformational switch occurring subsequently to sulfenic acid formation and/or Met release that would be a prerequisite to form the Cys51-Cys198 bond and, (2) a high mobility of the C-terminal part of the Cys51-Cys198 oxidized form that would favor formation of the second Cys198-Cys206 disulfide bond.

Introduction

Reactive oxygen species (ROS), that originate from various sources such as aerobic respiration, are involved in various physiological processes including signal transduction and immune response.1 Due to their high chemical reactivity, ROS can damage cellular targets like proteins. Methionine (Met) residues in proteins are one of the most sensitive amino acid targets of oxidation by ROS. Its oxidation leads to formation of methionine sulfoxide (MetSO), which in turn can lead to inactivation or modulation of the protein activity. Methionine sulfoxide reductases (Msrs) are able to reduce MetSO, back to Met. Msrs of class A are stereospecific for the S epimer, whereas MsrBs reduce the R epimer of the sulfoxide function.2 Although MsrAs3., 4., 5., 6. and MsrBs7., 8. are structurally unrelated, all of the Msrs characterized so far, display a similar catalytic mechanism involving at least three successive steps including: (1) formation of a sulfenic acid on the catalytic cysteine with the concomitant release of 1 mol of methionine per mol of Msr, (2) formation of an intramolecular disulfide bond and, (3) reduction of the oxidized form of Msr by thioredoxin (Trx). This is the case, for instance, for MsrAs from Neisseria meningitidis and Mycobacterium tuberculosis and for MsrBs from Escherichia coli and N. meningitidis. In E. coli and Bos taurus MsrAs, the presence of one supplementary recycling cysteine residue leads to formation of two successive intramolecular disulfide bonds, the first one between the catalytic Cys51 and the recycling Cys198, followed by the second one between Cys198 and the second recycling Cys206 (Figure 1). Both disulfide bonds are reduced by Trx but with a higher catalytic efficiency for the second one.7

Three X-ray structures of MsrAs from E. coli (MsrAE.coli, PDB code 1FF3),6 B. taurus (MsrAB.tau, PDB codes 1FVA and 1FVG)4 and M. tuberculosis (MsrAM.tub, PDB code 1NWA)5 have been reported. In all of the X-ray structures, the active site is occupied by a molecule that is covalently bound or unbound, to the catalytic Cys51. In the case of the MsrAM.tub, a Met residue belonging to a neighboring monomer is in the active site, while for the MsrAE.coli and the MsrAB.tau, a dimethyl arsenate and a DTT molecule are covalently bound to the catalytic Cys51, respectively. The X-ray structures show that in all the three structures, the distances between Cys51 and Cys198 are too large to allow the formation of intramolecular disulfide bonds. This is also the case for the distances between Cys198 and Cys206 in the MsrAE.coli and MsrAB.tau.3., 9. This apparent contradiction could be explained either by the fact that the crystalline state does not reflect the solution conformation or by a conformational change occuring concomitantly or after formation of the sulfenic acid intermediate with the release of methionine. This would be a prerequisite to bring closer in space both the catalytic Cys51 and the recycling Cys198. To achieve greater insight into the understanding of the formation of the intramolecular disulfide bonds Cys51/Cys198 and Cys198/Cys206 in the E. coli MsrA, we have determined the solution structure of two forms of E. coli MsrA by high resolution NMR spectroscopy, i.e. the reduced apo form (MsrARed) with an active site free from any bound molecule, and the oxidized C51-C198 MsrA form (MsrAOx) in which Cys86 and Cys206 have been substituted by Ser. Furthermore, we have quantified backbone mobility for both forms via the 15N longitudinal (R1) and transverse (R2) relaxation rates, and the steady-state heteronuclear 15N nuclear Overhauser effect (NOE) value measurements. We have also compared MsrARed solution structure with known MsrA X-ray structures, to assess conformational changes occurring upon the substrate binding. For this purpose, the mapping of MetSO substrate-bound C51A MsrA has been performed.

Section snippets

Solution structure of the reduced wild-type MsrA and of the C86S/C206S oxidized (Cys51-Cys198) MsrA

The NMR solution structures of MsrARed and MsrAOx were determined with the aim to obtain more structural information for Cys51-Cys198 and Cys198-Cys206 disulfide bond formation. The reasons for substituting Cys86 and Cys206 by Ser in the MsrAOx are (1) Cys206 would lead, in the presence of excess MetSO, to the formation of an oxidized MsrA with Cys51 under the sulfenic acid state and, Cys198 and Cys206 under disulfide state, (2) Cys86, located on the protein surface, can lead, in the absence of

Discussion

The examination of MsrARed solution structure reveals that, in the reduced state, the distances between cysteine residues are too large to allow the formation of any of the disulfide bonds (Cys51-Cys198 and Cys198-Cys206 as well). The NMR dynamics study also shows that the free active site undergoes mobility on the picosecond to nanosecond time scale, generating a conformational adaptability that may facilitate substrate binding. The structural comparison between the MsrARed structure in

Sample preparation

The E. coli strain used for production of E. coli wild-type MsrA and C86S/C206S mutated MsrA was BL21-DE3 transformed with a plasmid construction containing the corresponding coding sequence under the T7 promoter. Site-directed mutagenesis was performed as described.3 15N and 15N/13C-labeled samples were prepared by growing cells at 37 °C in M9 minimal media, supplemented with 15NH4Cl as the sole nitrogen source and with 13C-labeled glucose, as the unique carbon source. MsrA production was

Acknowledgements

This research was supported by the CNRS, the Universities of Nancy I and INPL, the IFR 111 Bioingénierie, the Association pour la Recherche sur le Cancer (ARC-No 5436) and the French Ministry of Research (ACI BCMS047). M.A. gratefully thanks the French Ministry of Research for financial support. Accesses to the Bruker DRX 600 (NMR facilities of the Service Commun de Biophysicochimie des Interactions, Nancy I) and to the Bruker DRX 800 (NMR facilities of the Laboratoire de RMN à haut champ,

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