Bacterial multidrug efflux transporters

Abstract

Infections caused by bacteria are a leading cause of death worldwide. Although antibiotics remain a key clinical therapy, their effectiveness has been severely compromised by the development of drug resistance in bacterial pathogens. Multidrug efflux transporters--a common and powerful resistance mechanism--are capable of extruding a number of structurally unrelated antimicrobials from the bacterial cell, including antibiotics and toxic heavy metal ions, facilitating their survival in noxious environments. Transporters of the resistance-nodulation-cell division (RND) superfamily typically assemble as tripartite efflux complexes spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusCFBA complex, which mediates resistance to copper(I) and silver(I) ions, is the only known RND transporter specific to heavy metals. Here, we describe the current knowledge of individual pump components of the Cus system, a paradigm for efflux machinery, and speculate on how RND pumps assemble to fight diverse antimicrobials.

Keywords: CusCFBA efflux system; heavy metal resistance; multidrug resistance; resistance-nodulation-cell division.

Fig. 1. Functional diversity among efflux proteins

Based on mode of transport, energy coupling mechanism and phylogeny (104, 105) these proteins are divided into five superfamilies. The ABC (left) superfamily proteins utilize ATP to transport diverse antimicrobials across the cellular inner-membrane. SMR, MFS and RND proteins function via a H⁺-substrate antiport mechanism. MATE proteins have been found to utilize both H⁺ and Na⁺ as an energy source (106). RND transporters, in particular, are capable of forming multi-protein structures which bridge the inner- and outer-membrane.

Fig. 2. Model of the fully-assembled CusCFBA tetrapartite efflux system

In E. coli, this HME-RND complex is responsible for extruding Cu(I) and Ag(I) ions directly from the cell with use of the proton-motive force. It consists of the inner-membrane transporter CusA (yellow), the membrane-fusion adaptor CusB (blue), the outer-membrane channel CusC (green) as well as the periplasmic metallochaperone CusF (grey). Cu(I) and Ag(I) ions have been found in complex with each of CusA, CusB and CusF.

Fig. 3. Crystal structure of the apo-CusA efflux pump

The CusA protomer found in the asymmetric unit of the crystal lattice is depicted by ribbon diagram (orange). The surface rendering corresponds to the CusA homotrimer, which is formed by symmetry within the crystal. Sub-domains PN1, PN2, PC1 and PC2 form the pore domain, while subdomains DN and DC comprise the docking domain, presumably interacting with the CusC channel.

Fig. 4. Crystal structure of the metal ion-bound CusA efflux pump

a) The ribbon diagram and surface rendering correspond to the Cu(I)- or Ag(I)-CusA subunit (blue). The binding of Cu(I) or Ag(I) by CusA is correlated with a major conformational change in the pump. The separation between the PC1 and PC2 sub-domains of each Cu(I)- and Ag(I)-CusA subunit is exaggerated compared to that of apo-CusA. b) The metal ion binding site of CusA. The bound Ag(I) and Cu(I) ions are closely coordinated by methionine residues M623, M672 and M573.

Fig. 5. Metal ion extrusion pathways of the CusA efflux pump

Four methionine pairs (C, green; O, orange; N, blue; S, yellow) in each protomer of CusA form the path of Cu(I) or Ag(I) transport. Metal ions may enter from the cytoplasmic side of the channel, via M410-M501, or through the periplasmic cleft between the sub-domains PC1 and PC2, via the three-methionine binding site M573, M623 and M672. These pathways are illustrated in black.

Fig. 6. Crystal structures of the CusB adaptor

a) Molecule A (blue) and molecule B (red) of each subunit are depicted by ribbon diagram. The two distinct structures of the elongated CusB molecule suggest the flexible nature of this protein. b) Each protomer of CusB can be divided into four domains. An effective hinge between domains 2 and 3 is responsible for the conformational change between molecule A and molecule B of CusB.

Fig. 7. Co-crystal structure of the CusBA adaptor-transporter complex

The CusBA protomer found in the asymmetric unit of the crystal lattice is depicted by ribbon diagram and the surface rendering corresponds to the CusBA efflux complex. Each subunit of CusBA consists of one CusA molecule (green) and two CusB molecules (magenta and blue). The full structure includes the hexameric CusB adaptor as well as the trimeric CusA transporter.

Fig. 8. Cu(I) binding site and conserved charged residues

This is a schematic representation of the CusA channel. The conserved residues R83, E567, D617, E625, R669 and K678, lining the channel at the periplasmic domain are indicated. The dotted red circle marks the location of the Cu(I) binding site formed by the methionine triad M573, M623 and M672. The paths for metal transport through the periplasmic cleft and transmembrane region are illustrated with black curves.

Fig. 9. Crystal structure of the CusC outer-membrane transporter

The CusC monomer (red) within the asymmetric unit of the crystal is depicted by ribbon diagram. The surface rendering corresponds to the biological CusC trimer, which is created by crystal symmetry. Each subunit of CusC consists of a four β-strands atop nine α-helices, which are arranged as a barrel in the trimeric structure.

Fig. 10. Docking of CusC to CusBA

The α-helical end of CusC interacts with the α-helices (Domain 4) of CusB in the CusBA complex. The surface rendering of the CusC₃-CusB₆-CusA₃ complex is colored as follows: brown, CusC trimer; purple, CusB hexamer; green, CusA trimer.

Fig. 11. Crystal structure of the metal ion-bound CusF metallochaperone

The binding of Cu(I) and Ag(I) is coordinated by M47, M49, W44 and H36, which form a novel metal ion-binding site in CusF.