Extended Data Fig. 10. Validation of the VirB4-VirB11 interaction, mutational analysis of the VirB2-binding and -assembly sites on VirB6, and conformational changes required or not required for the pilus to pass through the Arches (not required), the I-layer (not required) and the O-layer (required).

a, Assessment of the TraB/VirB4 and TraG/VirB11 ALPHAFOLD models against known individual structural homologues. Left: superposition of the ALPHAFOLD model of TraB/VirB4 (in yellow) with the cryo-EM model of TrwK/VirB4_central (in dark blue; this work). Right: superposition of the ALPHAFOLD model of TraG/VirB11 (in yellow) with the crystal structure of B. suis VirB11³⁸ (2GZA, in magenta). RMSD in Cα position are 1.26 Å and 1.23 Å, respectively. b, The VirB4-VirB11 structural model and the two independent methods used for its validation. Left: ALPHAFOLD structure of a complex of TraB/VirB4 (dark blue ribbon) bound to TraG/VirB11 (magenta ribbon). Middle: first validation of the complex model using the mapping of the TrROSETTA top-scoring (70% threshold) co-evolving pairs (listed in green in Supplementary Table 4; see Methods). Right: location of the interface residues mutated to provide a second independent validation of the ALPHAFOLD complex model. c, Pull-down of the TraB/VirB4-TraG/VirB11 wild-type and mutated complexes. Pull-downs were performed by taking advantage of a His-Tag at the C-terminus of TraB/VirB4. Samples were loaded so as to equalise as much as possible the amount of TraB/VirB4. The coomassie-stained gel is shown in upper panel, while the Western blots using anti-His antibodies to detect TraB/VirB4 (middle panel) or anti-Strep antibodies to detect TraG/VirB11 (lower panel) are shown below. Mutants are indicated as well as the positions of some molecular markers (MW), and those of TraB/VirB4 and TraG/VirB11. All mutated proteins expressed as well as wild-type and were equally soluble (not shown). Although similar amounts of TraB/VirB4 are loaded, the TraG/VirB11 band is less intense in the mutants, indicating weaker interactions; moreover, the inability of interface mutants to bind TraG/VirB11 also results in a slight degradation of TraB/VirB4 which is not observed in the wild-type interaction. For gel source data, see Supplementary Fig. 1c and corresponding legend in SI guide. n = 3 independent experiments. d, PATCHDOCK docking of the TraA/VirB2 and the TrwI/VirB6 pentamers. The docking is based on shape complementarity. The top-scoring model is shown in ribbon representation colour-coded black and red for TraA/VirB2 and TrwI/VirB6, respectively. The TrROSETTA analysis of VirB2-VirB6 did not detect any pairs involving VirB6 residues in this putative assembly site. This is not surprising: transient interactions are known to provide only weak co-evolutionary pressure⁷⁵ and one indeed expects VirB2 subunits to make only transient and weak interactions with the assembly site so as to not prevent incoming VirB2 subunits from displacing already assembled subunits at the base of the pilus. e, Model of the T4SS with bound F pilus and ALPHAFOLD TrwD/VirB11 model. Proteins are shown in ribbon colour-coded as in Fig. 1d, with the pilus in blue-white and TrwD/VirB11 in magenta. The O-layer is in the open conformation as shown in panel k. f, Overall view of the TrwI/VirB6 residues mutated in this study. Inset at left: overall stalk view in red (TrwI/VirB6) and orange (TrwJ/VirB5) ribbon representation. The dashed line box indicates the zoomed-in region at right. Residues mutated are shown in ball-and-stick representation. Mutations increasing conjugation are shown in green while those decreasing conjugation are shown in blue. The sites are labelled. For the VirB2- binding/recruitment site (labelled “recruitment site”), residues were mutated to bulky residues (T41F or G48I) or to acidic residue (V60E), all three anticipated to interfere with VirB2-binding. For the VirB2-assembly site, 3 pairs of double mutations, one to acidic residues, the other to hydrophobic, were designed with the intention of potentially increasing the affinity of the site for VirB2 subunits, thereby potentially increasing the binding of the pilus to its base. The R388 and pKM01 pilus are known to only weakly attach to the cell surface and thus, mutations increasing its affinity to its VirB6 base might increase its residency time, thereby affecting conjugation. Note that none of the mutants in the assembly site overlaps with the TrwJ/VirB5 binding site. Finally, For the Trp-blocks, 3 double mutations to W were implemented, with the intention to create obstacles preventing VirB2 subunits from reaching the assembly site while translocating from their binding site. g, Details of residues mutated in the assembly site (left), the recruitment/binding site (middle) and to generate the Trp blocks (right). Residues and TrwI/VirB6 are as in panel e. Residues are labelled. h, Western blot analysis of wild-type and mutated TrwI/VirB6 in the membrane (see Methods). All mutated TrwI/VirB6 proteins express similarly and all locate to the IM to the same extent as wild-type. Note that Redzej et al. (2017)⁹ have shown that deletion of TrwI/VirB6 does not affect the integrity of the T4SS except for TrwJ/VirB5 which is lost. However, none of the mutations described here are within the TrwI/VirB5 binding site and thus, we can safely conclude that the T4SS assembly is not affected in any of the mutants. For gel source data, see Supplementary Fig. 1d and corresponding legend in SI guide. n=3 independent experiments. i, Position of the VirB6 residues involved in the 50 top-scoring co-evolution pairs listed in Supplementary Table 4 for the VirB2/VirB6 interaction. All locate in the α1 helix, making this helix a strong candidate for VirB2 binding and recruitment. Inset at left: overall TrwI/VirB6 pentamer structure in red ribbon representation. Dashed line box indicates the zoomed-in region at right. The Cα atom of each residue is shown in sphere representation coloured in green. j, Position of the VirB3 residues involved in the 50 top-scoring co-evolution pairs listed in Supplementary Table 4 for the VirB2/VirB3 interaction. All locate in the α1 helix, making this helix another strong candidate for VirB2 binding. The Cα atom of each residue is shown in sphere representation coloured in blue white. k, Conformational change needed to open up the O-layer OM channel. As shown in Extended Data Fig. 8a, c, the heterotrimeric unit of the O-layer contains a 2-helix bundle that traverses the OM. 14 of these bundles form the OM channel. For the pilus to go across the OM through this OM channel, the 14 helical bundles need to open up, a conformational change that only requires a hinge motion along the two linkers that connect each of the helical bundles to the TrwE/VirB10_CTD β-barrel. Left panel: superposition of the heterotrimeric unit of the open (in green) and partially closed (in grey) O-layer. The partially closed state is that seen in the T4SS structure solved here. The open state is the one modelled here as described in Methods. Right panel: superposition of the entire open and partially closed O-layer states. The dimension of the open channel is shown. l, Cut-away of the structure with pilus shown in panel e. Colour-coding for the various proteins are indicated by labels, except for the pilus which is as in panel e. Left: cut-away at the level of the Arches. Middle: cut-away at the level of the I-layer. Right: cut-away at the level of the O-layer in its open conformation. These panels illustrate the fact that no conformational change is needed in the Arches or the I-layer for the pilus to pass through during pilus biogenesis but is needed to pass through the O-layer. We hypothesize that the Arches and the I-layer provide scaffolding rings through which the pilus is directed.