Crystal Structure of the Oxidized Form of the Periplasmic Mercury-binding Protein MerP from Ralstonia metallidurans CH34

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Abstract

In Ralstonia metallidurans CH34, the gene merP encodes for a periplasmic mercury-binding protein which is capable of binding one mercury atom. The metal-binding site of MerP consists of the highly conserved sequence GMTCXXC found in the family that includes metallochaperones and metal-transporting ATPases. We purified MerP from R. metallidurans CH34 and solved its crystal structure under the oxidized form at 2.0 Å resolution. Superposition with structures of other metal-binding proteins shows that the global structure of R. metallidurans CH34 oxidized MerP follows the general topology of the whole family. The largest differences are observed with the NMR structure of oxidized Shigella flexneri MerP. Detailed analysis of the metal-binding site suggests a direct role for Y66 in stabilizing the thiolate group of C17 during the mercury-binding reaction. The metal-binding site of oxidized MerP is also similar to the metal-binding sites of oxidized copper chaperone for superoxide dismutase and Atx1, two copper-binding proteins from Saccharomyces cerevisiae. Finally, the packing of the MerP crystals suggests that F38, a well-conserved residue in the MerP family may be important in mercury binding and transfer. We propose a possible mechanism of mercury transfer between two CXXC motifs based on a transient bi-coordinated mercury intermediate.

Section snippets

Redox state analysis of R. metallidurans CH34 MerP

As the reduced form of MerP can react with iodoacetamide, alkylation was used as a probe for checking the redox state of MerP. Immediately after purification, MerP was incubated with iodoacetamide. The mass spectrum of this solution (Figure 1A) shows two species with a mass difference of 114 Da corresponding to the addition of two alkylating groups. The lowest value (7519 Da) matches the theoretical molecular mass deduced from the amino acid sequence and the highest (7633 Da) matches the reduced

Conclusion

Our work describes the first crystal structure of the periplasmic mercury-binding protein MerP from R. metallidurans CH34. The architecture of the consensus sequence region in the crystallographic model of oxidized MerP is similar to oxidized superoxide dismutase chaperone and Atx1, two eukaryotic copper transporters. This model proposes that the side-chain of Y66, a conserved residue in MerP family, and the main-chain amide of C14 are good candidates to maintain C17 under its anionic form in

Bacterial strains and plasmids

The transposon Tn4378 from R. metallidurans CH34 was previously fully transferred on plasmid R1CM (ChloramphenicolR). The resulting plasmid R1CM∷Tn4378 was then used to transform a derivative of the E. coli strain CSH23 [recA1 spcR supE thisrl∷Tn10 Δpro lac]. The resulting strain E. coli CSH23 (R1CM∷Tn4378) is thus HgR, CmR, and TcR (unpublished results) and is a gift from Pr. Max Mergeay (CEN/SCK, Mol, Belgium).

Construction of expression plasmid and purification of recombinant MerP

R1CM∷Tn4378 was used as a template for PCR amplification of the merP gene. The primer

Acknowledgements

We thank David Lemaire and David Lascoux for their help in mass spectroscopy experiments, M. Mergeay for providing us the mer-containing bacteria strain and Corinne Vivès for her suggestions in the manuscript preparation. E.R. is a recipient of a PhD fellowship from the Région Rhône-Alpes.

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