Crystal Structure and Metal-Binding Specificity of ZneB, the Periplasmic Adaptor Protein of A Heavy Metal Resistance System from Cupriavidus Metallidurans CH34

Abstract

In Gram-negative bacteria, tripartite efflux systems are involved in the transport of a broad range of toxic compounds such as drugs or heavy metals out of the cell. 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. The periplasmic adaptor plays an important role in the recruitment of the two integral membrane partners and for the assembly of a functional transport complex. It has been proposed that this component could also contribute to the binding of the substrate. We report here on the characterization of ZneB, the MFP of a Heavy Metal Efflux-RND system from Cupriavidus metallidurans CH34. Using mass spectrometry, we have demonstrated that ZneB has a high specificity for zinc binding with a metal stoichiometry of 1:1 to the protein. The protein was crystallized in the presence of zinc and the apo- and metallated-forms were detected in the same asymmetric unit. The involvement of two histidine and a glutamate residues in the metal ion coordination site was confirmed by site-directed mutagenesis. The comparison of apo- and Zn-bound conformations based on the crystal structures and on data obtained in solution reveals important conformational changes upon zinc binding, suggesting an active role of the MFP in the efflux mechanism. The characterization at the molecular level of the efflux system proteins and the comparison with their counterparts in homologous RND-type transport systems involved in multidrug resistance will allow a better understanding of the resistance mechanisms.