Letter to the Editor: 1H, 13C and 15N Resonance Assignment of the Reduced Form of Methionine

1 13 15 H, C and N resonance assignment of an oxidized form (Cys51-Cys198) of Methionine Sulfoxide Reductase A from Escherichia coli

Nicolas Coudevyllea, Mathias Antoineb, Sandrine Boschi-Mullerb, Guy Branlantb and Manh- Thong Cunga, * aLCPM, UMR 7568 CNRS-INPL, 1 rue Grandville, B.P. 20451, 54001 Nancy Cedex, France; bMAEM, UMR CNRS-UHP 7567, Faculté des Sciences, Bld des Aiguillettes, B.P. 239, 54506 Vandoeuvre-lès-Nancy Cedex, France. *To whom correspondence should be adressed. E-mail: [email protected] nancy.fr Key words: methionine sulfoxide reductase A, Escherichia coli, resonance assignment

Biological context

Reactive oxygen species (ROS) are generated by cellular metabolism and involved in various physiological processes as signal transduction or immune response. Upon oxidative stress conditions, methionines (Met) included in proteins, are easily oxidized into Met-(R)-SO and Met-(S)-SO. In many cases, Met oxidation leads to protein functional alteration.

Methionine Sulfoxide Reductases (Msr) are enzymes able to reduce MetSO back to Met. Msr of class A is stereospecific for the S absolute configuration of MetSO (Moskovitz et al., 2000;

Olry et al., 2002). The E. coli MsrA exhibits an original catalytic mechanism in three steps involving three cysteine residues (Boschi-Muller et al., 2000). First, the attack of the catalytic cysteine (Cys51) on the sulfoxide function of MetSO leads to the formation of a sulfenic acid intermediate and the liberation of one mole of Met per mole of enzyme. Then, two successive intra disulfide bonds are formed, first between Cys51 and Cys198, and after, between Cys198 and

Cys206. The last step consists of reducing the Cys198-Cys206 disulfide bond by thioredoxin. As shown for the MsrAs which only have the Cys198 as a recycling cysteine, the Cys 51-Cys198 disulfide bond in a E. coli MsrA in which the C206 has been replaced, is also efficiently reduced by Trx as the Cys198-Cys206 disulfide bond is in the wild type (unpublished results). In a first approach, we recently reported the backbone and side-chain assignment of the reduced form of MsrA from E. coli (Coudevylle et al., 2004). We now report the nearly complete backbone and side-chain assignment of the first oxidized form occurring in the catalytic cycle and containing the Cys51-Cys198 disulfide bond.

Methods and experiments

E. coli MsrA contains four cysteines at positions 51, 86, 198 and 206. C206 was replaced by a serine residue in order to form only the Cys 51-Cys198 disulfide bond while C86, which is located on the surface of MsrA, was substituted by a serine to avoid any protein aggregation. The C86S/C206S double mutant was obtained by site-directed mutagenesis. The

E. coli strain used for MsrA production was BL21-DE3 transformed with a plasmidic construction containing the coding sequence under the T7 promoter. 15N and 15N/ 13C samples

15 labeled were prepared by growing cells in a minimal media with ( NH4)Cl as the sole nitrogen source and with glucose, 13C-labeled or not, as the only carbon source. MsrA production was induced at an OD600 of 0.6 by addition of 1 mM IPTG and harvested after 4 h. for the 15N/13C protein and 16 h for the 15N protein. Purification was done as described by

Boschi-Muller et al. (2000). The oxidation was performed by incubation of the labeled MsrA with excess of MetSO. The sample was then purified by gel filtration. Purity of the oxidized form of the mutant and its molecular mass were checked by SDS-PAGE and electrospray mass spectrometry, respectively.

The NMR sample contained 1 mM protein concentration (95% H2O, 5% D2O) in 10 mM phosphate buffer (pH 7.1). Spectra were acquired at 298 K on a Bruker DRX 600 MHz spectrometer equipped with a 3-axis gradient TXI probe and on a Bruker DRX 800 MHz spectrometer equipped with a cryoprobe. Spectra were processed using the program

XWINNMR (Bruker) and analyzed with the program XEASY (Bartels et al., 1995).

Backbone 1HN, 15N, 13Cα, 1Hα, 13C’, and side chain 1H, 13C resonances were assigned using 1H-

15N HSQC, HNCO, HN(CA)CO, HNCA, HN(CO)CA, CBCANH, CBCA(CO)NH and HNHA experiments. HCCH-TOCSY, H(CCCO)NH, CC(CO)NH and 15N-, 13C- 3D NOESY spectra were also recorded with the aim of side-chain assignments.

Extent of assignments and data deposition

1 15 The H- N TROSY spectrum for this oxidized form (Cys51-Cys198) of E. coli MsrA is shown in Figure 1. 88% of backbone HN, N, C, C' and C nuclei were assigned, as the large majority of the 1H, 13C side-chain nuclei. Almost of the unassigned residues are located in the

122-132 segment, likely poorly structured and undergoing a conformational exchange, as revealed by linewidth increase for residues up and down it (certainly due to exchange contribution to transverse relaxation). Chemical shifts were deposited in the BioMagResBank under the access number BMRB-6786 (http://www.bmrb.wisc.edu).

Acknowledgements

This research was supported by the CNRS, the Universities of Nancy I and INPL, the

IFR 111 Bioingénierie and the Association pour la Recherche sur le Cancer (ARC-No 4393).

Accesses to the Bruker DRX 600 of the SCBIM, Nancy I, and to the Bruker DRX 800 of the

ISCN, CNRS, NMR facilities were deeply appreciated.

References

Boschi-Muller, S., Azza, S., Sanglier-Cianferani, S., Talfournier, F., Van Dorsselear, A., Branlant, G. (2000) A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli, J Biol Chem 275, 35908-13.

Coudevylle, N., Thureau, A., Azza, S., Boshi-Muller, S., Branlant, G., Cung, M. T. (2004) (1)H, (13)C and (15)N resonance assignment of the reduced form of methionine sulfoxide reductase A from Escherichia coli, J Biomol NMR 30, 363-4.

Grimaud, R., Ezraty, B., Mitchell, J.K., Lafitte, D., Briand, C., Derrick, P.J. and Barras, F. (2001) Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase. J. Biol. Chem., 276, 48915-20.

Moskovitz, J., Poston, J.M., Berlett, B.S., Nosworthy, N.J., Szczepanowski, R. and Stadtman, E.R. (2000) Identification and characterization of a putative active site for peptide methionine sulfoxide reductase (MsrA) and its substrate stereospecificity. J. Biol. Chem., 275, 14167-72. Olry, A., Boschi-Muller, S., Marraud, M., Sanglier-Cianferani, S., Van Dorsselear, A., Branlant, G. (2002) Characterization of the methionine sulfoxide reductase activities of PILB, a probable virulence factor from Neisseria meningitidis, J Biol Chem 277, 12016-22. 1 15 15 Figure 1: 2D H- N TROSY spectrum of N labeled oxidized form (Cys51-Cys198) of E. coli MsrA at 600 MHz, 298 K, pH 7.1.