Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

Abstract

Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1-11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1-11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein-protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.

Keywords: VirD4; bacterial conjugation; structure; type 4 secretion system.

Figure 1. SDS–PAGE analysis of purified T4SS _3‐10+D4 complex, comparison with the T4SS _3‐10 complex, stoichiometry measurement and purification of T4SS _3‐10+D4 subcomplexes after deletion of single T4S system components

1. SDS–PAGE analysis of purified T4SS_3‐10+D4 complex. Left panel: Coomassie‐stained SDS–PAGE gel of the T4SS_3‐10+D4 complex. Right panel: Sypro Ruby‐stained SDS–PAGE gel of the T4SS_3‐10m and T4SS_3‐10+D4 complexes. Molecular weights of MW markers are shown on the left. Identification of bands is shown in the middle. *Indicates TrwK_/VirB4 degradation products. **Indicates minor contaminants OmpA and OmpC.

2. Stoichiometry measurement. Fluorescence scan of an SDS–PAGE gel analysis of the T4SS_3‐10+D4 complex where cysteine residues were reacted with Alexa Fluor 633 C₅ maleimide. Signals corresponding to the proteins coupled to Alexa Fluor 633 were detected at a wavelength of 633 nm. Fluorescent Trw protein bands are labelled, and the derived stoichiometry for TrwB_/VirD4 is indicated.

3. SDS–PAGE analysis of complexes purified from cell expressing deletion mutants of the pBADM11_trwN _/virB1 ‐trwE _/virB10Strep _trwD _/virB11 _ _His trwB _/virD4 constructs termed pBADM11_ΔtrwX _/ΔvirBY (see main text). The same molecular weight marker has been used on this gel as in the gel presented in panel (A). *Indicates degradation products of TrwK_/VirB4.

Source data are available online for this figure.

Figure EV1. Western blot analysis aimed at ascertaining that all proteins are expressed

1. Western blot visualizing the expression levels of TrwB_/VirD4 within different deletion constructs demonstrating that there is no polar effect observed for the expression of TrwB_/VirD4 due to gene deletion and that similar quantities of materials were loaded on the gel.

2. Western blot visualizing expression of components which are lost during purification of the T4SS_3‐10+D4 complex upon deletion of single T4S secretion components. The construct used for expression is shown at the top of each lane while the FLAG‐tagged protein detected by anti‐FLAG tag antibodies is shown under the panel.

Source data are available online for this figure.

Figure 2. Interaction between TrwB_/VirD4 and TrwE_/VirB10

Left panel: Coomassie‐stained SDS–PAGE gel of the T4SS_3‐10+D4 complex (lane 1), T4SS_{3‐10+D4:TrwEΔN42} complex (lane 2) and T4SS_{3‐10+D4:TrwEΔN64} complex (lane 3). Middle panel: Western blot analysis using αStrep antibodies to detect the TrwE_/VirB10 protein within the T4SS_3‐10+D4 complex and its variants. Right panel: Western blot analysis using αHis antibodies to detect the TrwB_/VirD4 protein within the T4SS_3‐10+D4 complex and its variants. Source data are available online for this figure.

Figure 3. NS‐EM of the T4SS _3‐10+D4 complex

1. NS‐EM micrograph of the T4SS_3‐10+D4 complex. A few representative particles of the T4SS_3‐10+D4 complex are circled in yellow. Scale bar 50 nm.

2. Representative class averages of T4SS_3‐10+D4 complex obtained after initial alignment and classification.

3. The upper row represents typical class averages of the T4SS_3‐10+D4 IMC complex (i.e. with the outer membrane core complex (OMC) masked out). The second row shows projections of the T4SS_3‐10+D4 IMC complex in the same directions determined for the classes above. The third row displays projections of the T4SS_3‐10 IMC complex (EMD‐2567) in the same directions. The bottom row shows the differences between the projections above (plus or minus TrwB_/VirD4) corresponding to positions of the TrwB_/VirD4 protein.

4. FSC of the electron density map of the T4SS_3‐10+D4 complex. The 0.5 line crosses the FSC at a resolution of 28 Å.

Source data are available online for this figure.

Figure 4. NS‐EM structure of the T4SS _3‐10+D4 complex

1. Side views of the NS‐EM structure of the T4SS_3‐10 (left) and T4SS_3‐10+D4 (right) complexes. The arches, the TrwK_/VirB4 barrels and the TrwB_/VirD4 density are shown in green, yellow and blue, respectively. The boundaries of the three‐tier structure of the TrwK_/VirB4 barrel are indicated in the middle of the panels and each tier is labelled as lower, middle and upper, respectively.

2. Side view of the T4SS_3‐10+D4 structure (upper panel) and derived schematic representation (lower panel). Overall dimensions are indicated.

3. Same as in (B) except that the side view shown is rotated 90 degrees along a vertical axis compared to the view shown in panel (B). Overall dimension is indicated.

4. Bottom view of the NS‐EM structure of the T4SS_3‐10 (upper panel) and the corresponding schematic representation (lower panel). Angle between the axes connecting the TrwK_/VirB4 hexamer pair and the TrwB_/VirD4 dimer pair is shown.

Figure EV2. Difference map, comparison of cross sections of the bottom part of the IMC of the T4SS _3‐10+D4 and the T4SS _3‐10 structures, fitting of the TrwB_/VirD4ΔN70 X‐ray structures into the map of the IMC of the T4SS _3‐10+D4

1. Difference map between the IMC T4SS_3‐10+D4 and the T4SS_3‐10 structures corresponding to TrwB_/VirD4 densities. Left: front view. Middle panel: side view (the structure is rotated on 90 degrees along a vertical axis). Right panel: bottom view.

2. Comparison of the cross sections of the bottom part of the IMC of the T4SS_3‐10 (top), the T4SS_3‐10+D4 (middle) and the difference between them (bottom). Extra density corresponding to TrwB_/VirD4 is indicated by white arrows.

3. Fitting of two TrwB_/VirD4 cytoplasmic domain structures into the blue density of Fig 4B illustrating that this density can accommodate only two of these molecules, not six. One subunit is shown in magenta and another one in dark blue.

Figure 5. Validation of the location of TrwB_/VirD4 in the T4SS _3‐10+D4 complex

1. Location of position 236 of TrwB_/VirD4 where a FLAG tag was introduced for immuno‐labelling of TrwB_/VirD4 within the T4SS_3‐10+D4 complex. Left: structure of hexameric TrwBΔN70_/VirD4ΔN70 (PDB entry code 1GKI). Only two diametrically opposed subunits are shown. The position of residue 236 is shown with a red sphere. Right: region of the T4SS_3‐10+D4 IMC where the location of the residue 236 of TrwB_/VirD4 would be expected (indicated by a red sphere) and schematic representation of an anti‐FLAG antibody bound to that location.

2. Class averages of antibody‐bound T4SS_{3‐10+D4FLAG}. Class averages of the aligned NS‐EM images of the T4SS_{3‐10+D4FLAG} with 3–4 particles per class. Red circles indicate the position of the antibody.

3. Network of spatial restraints identified by XL‐MS mapped onto primary sequences of the TrwB_/VirD4 subunit (blue) and the subunits interacting with it, TrwE_/VirB10 (grey) and TrwK_/VirB4 (yellow). Inter‐molecular and intra‐molecular cross‐links are shown in green and orange, respectively. *Indicates cross‐links mapped in the structure shown in Fig EV3B.

Figure EV3. Confirmation of the localization of TrwB_/VirD4

1. Difference density between class averages. Selected class averages of the T4SS_3‐10+D4 complex (left column), corresponding class averages of the antibody‐bound T4SS_{3‐10+D4FLAG} complex (middle column). The third column represents the difference between column two and column one. The whitest areas indicate the position of the bound antibody.

2. Structure of TrwB_/VirD4 cytoplasmic domain. The structure is shown in ribbon representation coloured in pale green. Lysine residues involved in observed cross‐links are shown as well as the distance separating them. Only one cross‐link is not shown, that between residues 398 and 446 because residue 398 is in a disordered region of the structure.