Heavy metal transport by the CusCFBA efflux system

Abstract

It is widely accepted that the increased use of antibiotics has resulted in bacteria with developed resistance to such treatments. These organisms are capable of forming multi-protein structures that bridge both the inner and outer membrane to expel diverse toxic compounds directly from the cell. Proteins of the resistance nodulation cell division (RND) superfamily typically assemble as tripartite efflux pumps, composed of an inner membrane transporter, a periplasmic membrane fusion protein, and an outer membrane factor channel protein. These machines are the most powerful antimicrobial efflux machinery available to bacteria. In Escherichia coli, the CusCFBA complex is the only known RND transporter with a specificity for heavy metals, detoxifying both Cu(+) and Ag(+) ions. In this review, we discuss the known structural information for the CusCFBA proteins, with an emphasis on their assembly, interaction, and the relationship between structure and function.

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

Figure 1

Crystal structure of the CusA transporter. (a) Ribbon diagram of a protomer of CusA. Each domain of CusA is labeled with a different color (cyan, transmembrane helices; yellow, PN1; green, PN2; red, PC1; pink, PC2; orange, DN; slate, DC). (b) Ribbon diagram of the CusA trimer. Each monomer of CusA is labeled with a different color. Subdomains DN, DC, PN2, PC1, and PC2 are labeled for the front subunit (blue), while PN1 is occluded from view.

Figure 2

The methionine relay network of CusA. (a) Stereo view of the trimeric CusA pump. Each subunit of CusA independently forms a methionine relay network for exporting metal ions. The methionine relay network is included in the front subunit of the trimer. (b) The methionine cluster (M573, M623, and M672) and four methionine pairs (M271-M755, M401-M501, M403-M486, and M391-M1009) of each monomer of CusA form the metal transport pathway. These residues are depicted as spheres (green, C; orange, O; blue, N; yellow, S). Heavy-metal substrates can enter this pathway through the periplasmic cleft between PC1 and PC2 or through the cytoplasm, illustrated by red arrows.

Figure 3

Superposition of apo-CusA (purple) and CusA-Cu(I) (green). The bound Cu(I) is blue. Binding induces a conformational change in the subdomains PC1 and PC2, which creates a 30° opening in the metal-bound structure. The transition from the resting state to the binding state is illustrated by the black arrow.

Figure 4

The conformational change induced by binding in the horizontal helix and TM8 of CusA. The apo-CusA structure (yellow) and Cu(I)-bound (green) CusA are superimposed, together with the bound Cu(I) (pink). The blue mesh indicates the anomalous signal of bound Cu(I) (contoured at 8 σ). In the proximity of copper, residues M573, M623, and M672 are closely coordinated and this transient binding site is revealed to the periplasm.

Figure 5

Crystal structure of the CusB membrane fusion protein. (a) Two distinct conformations of CusB were observed in the crystal structure (gold, purple). (b) Each CusB monomer can be divided into four domains: three β-stranded and one α-helical.

Figure 6

Co-crystal structure of the CusBA adaptor-transporter complex. Three monomers of CusA (red) and six molecules of CusB (blue, molecule A; green, molecule B) form the CusBA efflux complex. The subdomains DC, DN, PC1, PC2, and PN2 of CusA are labeled for the front monomer, while PN1 is occluded from view.

Figure 7

Predicted assembly of the complete CusCBA efflux complex. The pump is shown as a surface rendering of trimeric CusA (gold), hexameric CusB (cyan), and trimeric CusC (purple). The tripartite pump completely spans the inner- and outermembranes of E. coli to expel toxic Cu(I) and Ag(I).

Figure 8

Crystal structures of the CusBA-Cu(I) efflux complex. (a) Form Ia of the crystal structure has been designated as the “pre-extrusion 1” state (cyan), with bound copper (purple). (b) Form Ib of the crystal structure has been designated as the “pre-extrusion 2” state (orange), with bound copper (red). (c) Form II of the crystal structure has been designated as the “pre-extrusion 2” state (green), with bound copper (red). (d) Form III of the crystal structure has been designated as the “extrusion” state (magenta), with bound copper (blue). Molecules of CusB are not shown. In each state, the opening of the periplasmic cleft is depicted by the dashed line.

Figure 9

Superimposition of each crystal structure of the CusBA-Cu(I) complex. (a) Superimposition of subdomains PC2 and TM8 of CusA. The 30° rotation of PC2 upon transition from the “pre-extrusion 1” state (blue), through the “pre-extrusion 2” state (green) to the “extrusion” (red) state is depicted by the black arrow. (b) Superimposition of subdomains PC1 and the horizontal helix of CusA. The C-terminal end of the horizontal helix is observed to rotate by 21° upon binding (gray, apo-CusBA; yellow, CusA-Cu(I); blue, form Ia; green, form II; magenta, form III). The C-terminal residues 391 to 400 of molecule 1 of CusB (orange) are also included. (c) Superimposition of TM5 and TM6 of CusA. Upon binding, these subdomains are observed to shift toward the periplasm by approximately one turn (gray, apo-CusBA; yellow, CusA-Cu(I); blue, form Ia; green, form II; red, form III). The structure of form Ib, which is nearly identical to that of form II, has not been included.

Figure 10

Schematic representation of the metal transport pathway of a subunit of CusA. The conserved charged residues R83, E567, D617, E625, R669, and K678 of a subunit of CusA are labeled. This figure also includes the three-methionine binding site formed by M573, M623, and M672 (dashed red circle) within the subunit. The direction of metal transport is indicated by the black arrows.

Figure 11

Sequential conformational changes of the CusBA complex. To transport metal ions, this pump transitions from the “resting” state through the “extrusion” state (a, “resting” state; b, “binding” state; c, “pre-extrusion 1” state; d, “pre-extrusion 2” state; e, “extrusion” state). Subdomains PC1, PC2 and the helices TM5, TM6, and TM8 of CusA are shown for each state with the bound copper ion (blue). The change in conformation from the previous state is depicted by the black and blue arrows.

Figure 12

Crystal structures of the CusC outer membrane channel. (a) One monomer of the CusC trimer is shown in rainbow colors, while the other two monomers are depicted in gray. (b) Each CusC monomer (rainbow) can be divided into four β-strands and nine α-helices. The CusC protomer is acylated (red) through the residue C1 to anchor to the outer membrane. (c) Structure of the monomeric ΔC1 mutant. (d) Structure of the monomeric C1S mutant. The mutant structures are seen to adopt a dramatically different, partially folded conformation, compared with the wild-type CusC.

Figure 13

Superimposition of a monomer of wild-type CusC onto that of the ΔC1 mutant. The structures of the wild-type CusC and ΔC1 protomers are colored green and purple, respectively. The arrows indicate the drastic changes in positions and secondary structures when comparing the conformations of the wild-type and ΔC1 CusC.