Structure and Operation of Bacterial Tripartite Pumps

Supplementary Data

• Download Supplemental Tables 1-2 (PDF).

  Download Supplemental Figures 1-5 (PDF). Figures and captions also reproduced below.

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  Supplemental Figure 1

  

  Structural repeats in TolC and AcrB. Trimeric TolC and AcrB are both built from structurally homologous N-terminal and C-terminal halves, shown schematically in panels a and b, respecitvely. For each half, the N- and C-terminal repeats in one protomer are indicated on a thumbnail of the trimeric assemblies (inset boxes). These are colored blue and red for the N- and C-terminal halves of TolC and green and orange for the N- and C-terminal halves of AcrB. The breaks introduced between the C-terminal half and the N-terminal half for display purposes are indicated by dotted lines between backbone positions (white circles). To show the structural equivalences, the halves of both proteins are presented in a similar orientation. These ribbon representations show increasing depth of their corresponding color through each half. To match the presentation in the complete protomer (inset boxes), the C-terminal halves (C termini labeled) are to the left and the N-terminal halves (N termini labeled) are to the right. Equivalent main structural domains are labeled for each from top to bottom (dotted line ranges). Within TolC the equatorial domain (center label) is unique in combining elements from each half. The equivalent pairs of TolC helices (H3, H4, H7, and H8) in each of the coiled-coil domains are shown, and for AcrB the internal subdomains are also indicated (PN1, PN2, PC1, and PC2 for the pore domain; DN and DC for the docking domain). Owing to distinct rotations compared with the other subdomains, DN and DC present different aspects but are structurally equivalent and are superimposable, but in a distinct reference frame.

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  Supplemental Figure 2

  

  Comparison of the proposed models of assembled tripartite pumps. (a) Data-driven model of the Escherichia coli tripartite RND drug efflux pump to an estimated resolution of ∼8 Å (15). The assembled AcrA (green)-AcrB (blue)-TolC (yellow) complex is based on complete high-resolution crystal structures of the separate components and extensive in vivo site-specific cross-linking, utilizing a multidomain docking approach (15). (b) CusC (yellow) docked on the crystal structure of the CusBA subcomplex (green and blue, respectively) based on our AcrA-AcrB-TolC model shown in panel a (14). (c) Low-resolution cryo-electron tomography bipartite structure of MexA complexed with OprM from Pseudomonas aeruginosa. It is postulated this is an intermediate step of assembly and that binding of the IM antiporter MexB triggers a shift in helical-helical interactions of MexA-OprM, leading to a tripartite complex that is 170 Å between membranes (i.e., as in panels a and b) (17). MexA (yellow) is docked on TolC (red) and is overlaid on the gray isosurface representation of the averaged tomogram density (from 17). (d) Speculative models of two TolC-dependent tripartite machineries. (Left) The E. coli macrolide trasporter, MacA-MacB-TolC, extrapolated from the crystal packing of MacA (19, 20, 23). (Right) The Pseudomonas MexA-MexB-OprM drug efflux machinery (18) extrapolated from the MacA-MacB-TolC model.

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  Supplemental Figure 3

  

  Width of the periplasm: comparing known structures of secretion system complexes. The calculated size of the periplasm (distance in Å between IM and OM) is derived from the known size of the complexes and is indicated to the left of each structure. (Left) Assembled structure of the AcrA-AcrB-TolC drug efflux pump from Escherichia coli (surface rendered and colored as in Figure 4) based on 2.1–3.2 Å resolution crystal structures of the separate components and extensive in vivo site-specific cross-linking data to ∼8 Å resolution (15). (Center) 17 Å resolution cryomicroscopy structure of the Type III secretion needle complex from Salmonella typhimurium (8). (Right) 15 Å resolution cryo-electron microscopy structure of a type IV secretion system core complex from E. coli (4).

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  Supplemental Figure 4

  

  The observed substrate binding sites of AcrB. An overview of the proposed small-molecule binding sites of AcrB built on data from eight studies in which AcrB has been soaked and crystallized with various efflux substrates represented by colored spheres. The AcrB surface is rendered in light orange, red, and transparent gray for each protomer. (a) Side view of AcrB with the front protomer removed for clarity. Small molecules binding within the central cavity (green): ampicillin (16), ciproflaxin (21, 22), dequalinium (22), ethidium (21, 22), nafcil (21), PABN (21), rodamine (21, 22). Binding to the pore domain (yellow): bile (2), copper (7), ciproflaxin (21), ethidium (21), PABN (21), rhodamine (21), silver (7), nafcil (21). Binding in a multisite access pocket (orange): doxorubicin (3), erythromycin (10), rifampicin (10). Binding deeper into the periplasmic cleft (blue): doxorubicin (9), minocycline (3, 9). No drugs have been observed in the funnel of the TolC-docking domain. (b) View of AcrB (with bound drugs) from the cytoplasm into the central cavity. The DN subdomain projects an intermonomer connecting loop into the neighboring protomer, stabilizing the trimeric state.

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  Supplemental Figure 5

  

  Inhibition sites on assembled pumps. (Left) Assembled tripartite pump (colored as in Figure 4) with sites of inhibition revealed from liganded crystal structures. The screening for natural and synthetic inhibitors is reviewed in 1, 5, 11, 12. (Upper right) The hexamine cobalt ([Co(NH₃)₆]³⁺)-blocked α-barrel periplasmic entrance of TolC, where the bound ligand (ball and stick) forms hydrogen bonds with Asp374 at the pore constriction (6). (Lower right) The two designed ankyrin repeat protein (DARPin, magenta) binding sites on AcrB (blue) (13).