Structural and Metal-Binding Characterization of the C-terminal Metallochaperone Domain of the Membrane Fusion Protein SilB from Cupriavidus Metallidurans CH34

Abstract

The β-proteobacterium Cupriavidus metallidurans CH34 has an outstanding ability to grow on harsh environments such as heavy-metal contaminated sites. The regulated transport of heavy metal ions out of the cell via tripartite efflux systems is one of the mechanisms used by the bacteria for detoxification. These protein complexes span the entire bacterial cell envelope, and are composed of an inner membrane transporter belonging to the resistance nodulation cell division (RND) family, an outer membrane protein member of the Outer Membrane Factor (OMF) family, and a periplasmic adaptor protein, member of the Membrane Fusion Protein (MFP) family. SilABC is one of the 12 putative efflux systems detected in C. metallidurans CH34 genome and is most probably involved in silver and copper trafficking. We report here on the characterization of the C-terminal domain of the periplasmic adaptor protein SilB. This C-terminal extension exists only in SilB homologs and is not present in other MFPs. A potential Ag(I)/Cu(I) coordination site was detected on the basis of the amino acid sequence and the metal-binding specificity was confirmed by mass spectrometry. NMR solution structure of the apo-form showed that SilB C-terminal domain adopts a β-barrel structure. Comparison of chemical shift data between the apo-and metallated-form demonstrated the implication of two methionine, one histidine and one tryptophan residues in the metal coordination site. Fluorescence quenching and UV-visible data are consistent with a cation-tryptophan π-interaction. With respect to its three-dimensional structure and metal-binding specificity, the SilB C-terminal domain closely resembles CusF, a small periplasmic protein belonging to the CusCFBA efflux system involved in silver and copper resistance in E. coli. Our study suggests that SilB C-terminal domain could function as a metallochaperone to the SilABC system.