Structure of a bacterial type IV secretion core complex at subnanometre resolution

Abstract

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane-spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O-layer inserted in the outer membrane and the I-layer inserted in the inner membrane. While the structure of the O-layer has been solved by X-ray crystallography, there is no detailed structural information on the I-layer. Using high-resolution cryo-electron microscopy and molecular modelling combined with biochemical approaches, we determined the I-layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.

Figure 1

CC_elastase purification. (A) Schematic representation of the regions of TraN/VirB7, TraO/VirB9 and TraF/VirB10 present in the CC_elastase complex. Domains corresponding to the VirB9-binding domain (B9BD), the signal peptide (SP), the N-terminal TM helix and the C-terminal domains (CTD) are shown in darker colours. (B) SDS–PAGE of the CC_elastase and the FLCC complexes. For the CC_elastase complex, the first aa in the bands identified using Edman degradation are indicated. The CC_elastase complex is composed of three major bands: full-length TraN/VirB7 and TraO/VirB9 and cleaved TraF/VirB10. In addition two minor byproducts are observed, both consisting of cleaved TraO/VirB9. Molecular weight standards (Std) are indicated. (C) Size exclusion chromatography of the CC_elastase complex using a Superose 6 10/300 (GE Healthcare). The mobility of the pKM101 FLCC and the OL complex is also indicated by blue arrows in the size exclusion chromatography plot.

Figure 2

Cryo-EM structures of the CC_elastase and FLCC complexes. (A, B) Side view and top view, respectively, of the cryo-EM structure of the CC_elastase complex. (C) Central section of the cryo-EM map shown in A and B. The density corresponding to the middle platform is indicated in purple circles. (D–E) Side view and top view, respectively, of the cryo-EM structure of the FLCC. (F) Central section of the FLCC cryo-EM map. The new density corresponding to the inner wall is outlined with red ellipses. (G) Cutaway view of the superposition of the cryo-EM maps of the FLCC (blue) and CC_elastase (orange) complexes. (H) Difference map between the FLCC and CC_elastase cryo-EM maps. (I) Cutaway view of the superposition of the difference map shown in H (green) and the cryo-EM structure of the CC_elastase complex (orange). In H and I, arrows indicate the extra ring at the base of the FLCC (‘base’) not present in the CC_elastase map.

Figure 3

Modelling and fitting of atomic data in the cryo-EM map of the CC_elastase complex. (A) Central vertical slice showing the fitting of the OL atomic structure and the atomic model obtained for TraO/VirB9_NT. The densities corresponding to the middle platform and two α-helices α2 and α3 of TraF/VirB10_CT are indicated with purple and black dashed circles, respectively. The dashed line corresponds to the equivalent cutting plane perpendicular to the vertical axis of the complex shown in panel D. (B) Detailed view of the crystal structure fitting in the OL. In the cap, there is clear extra density not accounted for by helices α2 and α3 (in a black dashed circle). The plausible location of the lipidic moiety covalently bound to TraN/VirB7 is indicated with black ovals. (C, D) Fitting of the model obtained for TraO/VirB9_NT in the IL. In panel D, the cutting plane shown is in a perpendicular direction to the vertical axis of the CC_elastase cryo-EM map shown in panel A. (E) Superposition of one subunit of the original atomic structure (grey) of the OL, and the corresponding subunit of the atomic model after flexible fitting (TraN/VirB7, TraO/VirB9 and TraF/VirB10 are shown in green, blue and red, respectively). The RMSD between the two models is 1.21 Å. On the right, the N-terminal α-helix present in the lever arm (red) is shown fitted in the equivalent density of the CC_elastase cryo-EM map. The symmetry-related subunits are shown in blue. The same helix of the original atomic structure of the OL is shown in grey.

Figure 4

Modelling and fitting of atomic models in the cryo-EM map of the FLCC. (A) Central slice of the FLCC with fitted OL atomic structure and atomic models obtained for TraO/VirB9_NT and the α-helices predicted in TraF/VirB10_NT. (B) Schematic model for the organisation of TraF/VirB10 in the FLCC. Four TraN/VirB7+TraO/VirB9_CT+TraF/VirB10_CT subunits of the 14-mer present in the OL atomic structure are shown. One of the subunits is shown in dark blue. The tentative docking of the three TraF/VirB10_NT α-helical regions modelled with high confidence together with a plausible connection with TraF/VirB10_CT is shown in dashed lines. The density for one subunit in the difference map is shown in transparent green. This subunit is located immediately below the lever arm of TraF/VirB10_CT shown in dark blue. The three α-helical regions of TraF/VirB10_NT correspond to αA (purple, from K₄₀ to F₅₃), which transverses the IM, αB (green, from A₁₀₇ to A₁₁₃) and αC (red, from P₁₃₈ to R₁₄₆). The connections are shown in dashed lines together with the corresponding number of aa.

Figure 5

NB labelling of the FLCC, CC_elastase and OL complexes. (A) Selection of NBs preferentially binding the CC_elastase (see also Supplementary Figure 6). The diagram reports the comparative affinity of three NBs for the FLCC (green), the CC_elastase (blue) and the OL (red) complexes. NBCA4271 and NBCA4304 target the IL while NBCA4296 binds to the OL, a region present in the three complexes used for the ELISA experiment. NBCA4304 was chosen for the labelling experiments. The right panel represents the schematic organisation of the complexes used in the ELISA experiments coloured correspondingly. (B) SDS–PAGE analysis and western blot of the labelled FLCC. The different lanes correspond to the FLCC, the NBCA4304, the purified complex containing the FLCC and the NB (FLCC+NBCA4304), and the final purified complex containing the FLCC, the NB and the anti-His antibody (CC+NBCA4304+anti-His). This final complex was used to prepare negative stain grids. The right panel shows a western blot of the FLCC+NBCA4304+anti-His fraction in the presence or absence (+/− DTT) of reducing agent. The western blot was performed using only the secondary antibody HRP-conjugated. Molecular weight standards (Std) are indicated in both cases. (C) Gallery of negatively stained single particles with a clear extra density emerging from the CC. (D) Representative top view class averages of the FLCC+NBCA4304+anti-His sample. The scale bar corresponds to 10 nm.