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Structural insights into the secretin translocation channel in the type II secretion system

Abstract

The secretin GspD of the type II secretion system (T2SS) forms a channel across the outer membrane in Gram-negative bacteria to transport substrates from the periplasm to the extracellular milieu. The lack of an atomic-resolution structure of the GspD channel hinders the investigation of substrate translocation mechanism of T2SS. Here we report cryo-EM structures of two GspD channels (1 MDa), from Escherichia coli K12 and Vibrio cholerae, at 3 Å resolution. The structures reveal a pentadecameric channel architecture, wherein three rings of GspD N domains form the periplasmic channel. The secretin domain constitutes a novel double β-barrel channel, with at least 60 β-strands in each barrel, and is stabilized by S domains. The outer membrane channel is sealed by β-strand-enriched gates. On the basis of the partially open state captured, we proposed a detailed gate-opening mechanism. Our structures provide a structural basis for understanding the secretin superfamily and the mechanism of substrate translocation in T2SS.

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Figure 1: Top-view class averages and overall structures of T2SS secretin GspD channels.
Figure 2: Protomer structure and domain assignment.
Figure 3: Interaction networks of periplasmic N domains and flexible N0 domains.
Figure 4: β-barrel structure and its interactions with S domains of Vc_GspD.
Figure 5: The structure of the Vc_GspD channel and conformational changes in the partially opened state.
Figure 6: Partially opened central gate and proposed detailed mechanism of gate opening.

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Acknowledgements

We acknowledge J. Wang for providing support of model building and H. Wang for discussion about this project. We acknowledge Tsinghua University Branch of China National Center for Protein Sciences Beijing for providing facility supports in Cryo-EM and computation. This work was supported by funds from Advanced Innovation Center for Structural Biology (to X.L.), Tsinghua-Peking Joint Center for Life Sciences (to X.L.), the National Key Research and Development Program (2016YFA0501102 and 2016YFA0501902 to X.L.), National Natural Science Foundation of China (31570730 to X.L. and 81322024, 81561130162 and 81530068 to Y.Z.) and One-Thousand Talent Program by the State Council of China (to X.L.).

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Contributions

Z.Y., M.Y. and X.L. designed all experiments. Z.Y. and M.Y. performed all the Cryo-EM and biochemical experiments. D.X. and Y.Z. provided suggestions and assistance in part of functional validation. Z.Y. and X.L. built the models. Z.Y., M.Y., Y.Z. and X.L. wrote the manuscript. All authors contributed to the data analysis and manuscript revision.

Corresponding authors

Correspondence to Yongqun Zhu or Xueming Li.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Purification of GspD complexes of Escherichia coli K12 and Vibrio cholerae.

(a, b) Schematic diagrams of GspD proteins for Escherichia coli K12 (a) and Vibrio cholerae (b). The strep tag (blue) was fused to C terminus or N terminus to validate the integrity of GspD N0 domain. (c-f) Size-exclusion chromatograms of the GspD complexes from Escherichia coli K12 (c, d) and Vibrio cholerae (e, f). GspD complexes with strep tag at C terminus (c, e) and N terminus (d, f) presented similar main peak position. Fractions were analyzed by SDS-PAGE and Coomassie blue staining.

Supplementary Figure 2 Cryo-EM images and 2D classification analysis.

(a,b) Representative cryoEM raw images of K12_GspD (a) and Vc_GspD (b). Top view and side view particles were shown in blue and red cycles, respectively. (c, d) 2D class averages of K12_GspD particles (c) and Vc_GspD particles (d). Side views (above) and top views (below) showed clear secondary structural features. Red arrows pointed to smeared densities, indicating flexibility of N0 domain. (e) Side views of K12_GspD, which exhibited slightly tilted N1-N2 domains (left) compared with other class averages (right).

Supplementary Figure 3 Local resolutions and Fourier shell correlation (FSC) curves.

(a, b) Local resolution cryoEM density maps for K12_GspD (a) and Vc_GspD (b) colored according to resolutions estimated by ResMap. The outer (left) and inner (right) surfaces are displayed for each map. Unstructured densities of cap gate, central gate and N3 constriction site were emphasized with white, red and yellow ellipses, respectively. (c, d) Gold standard FSC curves (cyan) of whole K12_GspD channel (c) and Vc_GspD channel (d). FSC curves between the atomic model and the cryoEM density map are shown in red. (e, f) Cross-validations for K12_GspD (e) and Vc_GspD (f). Whole model versus summed map (black), whole model versus half map 1 (red, work) and whole model versus half map 2 (cyan, free). See Methods for details.

Supplementary Figure 4 Model building strategies and cryo-EM densities at different regions.

(a, b) Model building strategies for different regions of K12_GspD (a) and Vc_GspD (b). Atomic models of N3 domain, secretin domain and S domain (cyan) were generated by de novo building and atomic models for N1 domain and N2 domain (magenta) were based on homology model building. (c-f) The representative cryoEM densities at different regions. Shown here were the cryoEM densities for N3 domain (c), S domain (d), outer barrel subdomain (e) and inner barrel subdomain (f). The figures were prepared using PyMol at the 6σ contour level of the density maps.

Supplementary Figure 5 Unstructured density in the central gate and cross-linking assay.

(a) Unstructured density and β-strands in the central gate region of Vc_GspD. The subscripts, 1 and 2, denote different subunits. (b) Model of an asymmetry subunit. The structure was divided to two parts: before (cyan) and after (magentas) the connection region between β16 and β17. If β161 connects to its adjacent β171, these two parts will constitute one protomer. Otherwise, for example, β161 connects to non-adjacent β172, these two parts will belong to different protomers. (c) Cysteine crosslinking site enlarged from the rectangle region in (b). The residues to be cross-linked, A318 and R587, were labeled. (d) The result of crosslinking assay. R587C&A318C cross-linked monomer without DTT treatment run faster than that with DTT treatment. Since circular peptide reduces the resistance and run faster in 4-12% NuPAGE (Invitrogen) than linear peptide (similar to that closed circular DNA runs faster than linear DNA in agarose gel), this result indicated that the monomer was intra-connected and hence formed circular peptide. This result supports that β16 connects to its adjacent β17 as the monomer presented in (a).

Supplementary Figure 6 Docking and structural comparisons of various N-domain models.

(a, b) Structural comparisons of Vc_GspD N3 domain with E.coli ETEC N1 domain (a; PDB code 3EZJ) and N2 domain (b; PDB code 3EZJ). Both of them adopt KH domain folding pattern (β-α-β-β-α). (c, d) Crystal structures of N1-N2 domains from E.coli ETEC (c; PDB code 3EZJ) and Pseudomonas aeruginosa (d; PDB code 4E9J) were docked into N1 and N2 domains of low-pass filtered K12_GspD map. (e, f) Crystal structures of N0-N1 domains from E.coli ETEC (e; PDB code 3EZJ) and Pseudomonas aeruginosa (f; PDB code 4E9J) were docked into N1 domain of low-pass filtered K12_GspD map. N2 domain, N1 domain and N0 domain are drawn in green, cyan and dim gray, respectively.

Supplementary Figure 7 Assembly assay and bile salt sensitivity assay.

(a) An enlarged view of a part of double-β-barrels of Vc_GspD. Hydrophobic residues are colored with orange on the β-strands. Five residues to be mutated are labeled and shown with orange side chains. (b) Assembly assay result of five mutants shown in (a). All mutations disrupted the formation of the complex. (c) Assembly assay result of glycine mutations. Only G453A mutant can assemble into multimers whereas G481A and G481A&G453A can’t. (d) Bile salt sensitivity assay. ∆cap&G453A mutant presented remarkable sensitivity (lower plating efficiency) to bile salt compared with WT and ∆cap. Data were shown as mean with s.e.m.

Supplementary Figure 8 Possible interactions between S domain and GspS.

(a) Hydrophobic interactions between α12 (cyan) and α11 (pink) of Vc_GspD (electrostatic surface on background). (b) Possible interactions between GspS and GspD S domain. PA3611 (PDB code 3NPD), a homolog of GspS, exhibits a hydrophobic crevice on the surface (left panel). In the presence of GspS, α12 will bind to the hydrophobic crevice (right panel), helping GspD position onto the outer membrane.

Supplementary Figure 9 Comparison of several core complexes in secretion systems and several β-barrel structures.

(a) Cap gate structures. The cap gate of Vc_GspD is composed of 30 β-strands, forming a small β-barrel (left, orange). The cap of T4SS core complex (PDB code 3JQO) is composed of 28 α-helixes (right, orange), different from that of Vc_GspD. (b) Domain architectures of secretins. Shown here are domain architectures of Vc_GspD (Vibrio cholerae), K12_GspD (Escherichia coli K12), Pa_XcpQ (Pseudomonas aeruginosa) and Ko_PulD (Klebsiella oxytoca), St_InvG (Salmonella typhimurium) and Nm_PilQ (Neisseria meningitidis). They all shared similar N terminal domains, C terminal secretin domain and S domain. (c) CryoEM structures of secretin channels. Shown here from left to right are T3SS St_InvG (EMD-1224), T2SS K12_GspD, T2SS Vc_GspD, T2SS Ko_PulD (EMD-2628) and T4PBS Nm_PilQ (EMD-2105). They all shared cylindrical structures. (d) Comparison of several β-barrel structures. Vc_GspD exhibits more complicated β-barrel structure than those of OmpF (PDB code 1PHO), BamA (PDB code 4K3B), CsgG (PDB code 4UV3) and TolC (PDB code 1EK9), including much larger size, partial β-barrel transmembrane and novel double-β-barrel architecture.

Supplementary Figure 10 Cryo-EM density maps of Δcap and G453A of Vc_GspD.

(a) Density maps of Δcap presented in side view (left) and top view (right). (b) Gold standard FSC of Δcap. (c) Density maps of G453A presented in side view (left) and top view (right). (d) Gold standard FSC of G453A. (e) Wild type Vc_GspD model was well fitted into the Δcap map, which means that the cap deletion doesn’t cause any conformational change to other part of the channel.

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Yan, Z., Yin, M., Xu, D. et al. Structural insights into the secretin translocation channel in the type II secretion system. Nat Struct Mol Biol 24, 177–183 (2017). https://doi.org/10.1038/nsmb.3350

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