Direct interaction of a chaperone-bound type III secretion substrate with the export gate

Abstract

Several gram-negative bacteria employ type III secretion systems (T3SS) to inject effector proteins into eukaryotic host cells directly from the bacterial cytoplasm. The export gate SctV (YscV in Yersinia) binds substrate:chaperone complexes such as YscX:YscY, which are essential for formation of a functional T3SS. Here, we present structures of the YscX:YscY complex alone and bound to nonameric YscV. YscX binds its chaperone YscY at two distinct sites, resembling the heterotrimeric complex of the T3SS needle subunit with its chaperone and co-chaperone. In the ternary complex the YscX N-terminus, which mediates YscX secretion, occupies a binding site within one YscV that is also used by flagellar chaperones, suggesting the interaction's importance for substrate recognition. The YscX C-terminus inserts between protomers of the YscV ring where the stalk protein binds to couple YscV to the T3SS ATPase. This primary YscV-YscX interaction is essential for the formation of a secretion-competent T3SS.

Fig. 1. Overall structure of the YscX:YscY heterodimer.

a Cartoon representation of YscX (cyan) and YscY (red). YscX binds to YscY at two independent sites. b, c Surface hydrophobicity gradient of YscY shown from hydrophobic (red) to hydrophilic (white) using the normalized consensus hydrophobicity scale. b YscX binds the groove of the TPR protein YscY via the hydrophobic side of a short amphipathic helix. c Two leucine-rich helices of YscX interact with the largely hydrophobic surface of the N-terminal TPR in YscY. d Superposition of YscX₅₀:YscY (cyan and red) and YscX₃₂:YscY (blue and pink) models. The alignment was calculated using the Cα atoms of YscY. Zoom-in shows the YscX N-terminus before α1 for both structures when aligned using the Cα atoms of YscX.

Fig. 2. Sequence alignment of SctX and SctY.

a Alignment of SctX. b Alignment of SctY. Alignments were calculated using ClustalW and visualized with ESPript3 (espript.ibcp.fr,). SctX and SctY sequences from Yersinia enterocolitica (YscX: AAD16820.1, YscY: AAK69217.1), Aeromonas hydrophila (AscX: AAS91814.1, AscY: AAS91815.1), Pseudomonas aeruginosa (PscX: AAY17109.1, PscY: AAY17110.1), Photorhabdus luminescens (LscX: CAE16135.1, LscY: CAE16134.1), and Vibrio parahaemolyticus (VscX: UJX28051.1, VscY: EDM61220.1) were used to generate the alignment. Definition of colors and symbols: red box, white character: strict identity; red character: similarity in a group; blue frame: similarity across groups; gray star: residues with alternate conformations.

Fig. 3. Overall structure of the ternary YscV_C:YscX₃₂:YscY complex.

a Side View of the two stacked nonameric rings of YscV_c (orange, three shades for clarity). Half of the chains are shown as gray surface. YscX₃₂ (cyan) and YscY (red) bind on the periphery of the nonamer. b View on the membrane-distal side of one nonamer. The C-terminal helix of YscX protrudes into the ring between two adjacent YscV_C protomers.

Fig. 4. Interactions of YscV, YscX, and YscY.

One YscV_C protomer is highlighted (orange surface) within the nonameric ring (gray surface). The cartoon representation of YscV_C is divided into the four subdomains: SD1 (red, residues 372–438 and 493–513), SD2 (yellow, residues 439–492), SD3 (green, residues 514–600), and SD4 (blue, residues 601–704). For clarity, YscX (cyan) and YscY (dark red) are shown only for this heterotrimer and are depicted as ribbon in (b–d). a The overall structure of the heterotrimer within the assembly shows that all interactions, except for the insertion of YscX’s C-terminus, are confined within one heterotrimer, i.e., YscY does not interact with a neighboring YscV subunit. b YscY interacts with SD4 via the loop of its first TPR (site A) and c with SD2 via the face of its C-terminal TPR (site B). d In chains FB, GB, KB, and LB, YscX exhibits density for an extended N-terminus which binds to SD1 and SD2 via a conserved ⁴⁶RLYP⁴⁹ motif (site C). Note that the exact positions of the side chains could not be determined at this resolution and are only included for completeness. e The C-terminus of YscX is inserted into the SD4–SD4 cleft. Both involved YscV_C subunits are shown as cartoon colored by subdomain. YscX is shown as ribbon with sidechains starting from L118.

Fig. 5. The C-terminus of YscX binds between two YscV protomers.

a Surface hydrophobicity of YscV (left) and the neighboring protomer (right) from hydrophobic (red) to hydrophilic (white) with a ribbon and stick representation of YscX (cyan). Helix α3 inserts between two YscV molecules and binds via hydrophobic interactions. The C-terminus is pointing towards a positively charged patch of the neighboring subunit, which likely stabilizes the carboxylate. b 2mF_O-DF_C density contoured at 1σ at the C-Terminus of YscX suggests positioning of the carboxylate towards R551 of YscV (orange). The density of the arginines is exemplary and varies between the chains.

Fig. 6. Recognition of YscXY by YscV_C depends on the C-terminus of YscX as well as SD3 and SD4 of YscV_C.

a Coomassie-stained SDS-PAGE of MBP pull-downs. N- and C-terminally truncated MBP-YscX constructs were co-expressed with His₆-YscY and YscV_C. Lysates were loaded onto amylose resin and the washed matrix loaded onto the gel. The pull-downs generated consistent results over three replications. Source data are provided as a Source Data file. b Analytical size exclusion chromatography with 10 µM YscV_C, YscV_SD12 (both blue), YscX₃₂:YscY (black), or mixtures (red) of the proteins. Absorbance at 280 nm shows a shift in retention volume for a mixture of YscV_C with YscX₃₂:YscY, but not YscV_SD12 with YscX₃₂:YscY. Chromatographies involving YscV_SD12 or YscV_C were performed twice or four times, respectively, with consistent results. A SDS-PAGE and Western blot analyzing contents of gel filtration peaks are shown in Supplementary Fig. 8.

Fig. 7. Concentration-dependent nonamerization of YscV_C.

For comparability, absorbances were normalized to the highest peak. From top to bottom: YscV_C, a ternary complex of YscV_C co-expressed with YscX₅₀ and His₆-YscY, and the same ternary complex but with full-length YscX (YscX_fl) were loaded at 2.5 µM (pink) to 100 µM (blue) onto a Superdex 200 column. For reference, the expected retention volumes for the monomeric species (or 1:1:1 heterotrimer) and nonameric species (assuming 9:9:9 stoichiometry and a globular shape) are shown as dashed gray lines. The weak auto-nonamerization of YscV_C is enhanced by the presence of YscX₅₀ but severely impeded by full-length YscX. Experiments were performed three times with consistent results.

Fig. 8. Comparison of YscXY with other T3S substrate:chaperone complexes.

a Superposition of YscX₅₀:YscY with YscEFG (PDB: 2P58). The alignment was done using the chaperones YscY (red) and YscG (pink) with an r.m.s.d. of 5.736 Å. The substrates YscX (cyan) and YscF (blue) as well as the co-chaperone YscE (green) are also shown. b Superposition of YscXY with the complex of class II chaperone AcrH (pink) and the major translocator protein AopB (blue), both from Aeromonas (PDB: 3WXX). The alignment was calculated using the first TPR of YscY (residues 7–39) and the first TPR of AcrH (residues 30–64) with an r.m.s.d. of 0.687 Å to obtain the right register. For clarity, sticks are shown for side chains of AopB where it binds the TPR groove of AcrH.

Fig. 9. Superposition of export gate complexes with YscVXY.

YscV, YscX, and YscY are shown in orange, cyan, and red, respectively. a Superposition with the cryo-EM structure of YscV_C (gray, PDB: 7ALW), which is shown as cartoon representation. YscV_C protomers are aligned with an r.m.s.d. of 0.797 Å. b, c Superposition with FlhA (gray) bound to the flagellar chaperone:substrate complexes (green:light green). b FlhA:FliS:FliC (PDB: 6CH3). c FlhA:FliT:FliD (PDB: 6CH2). Alignments were obtained using SD3 of YscV_C (residues 514–600) with an r.m.s.d. of 1.194 Å (PDB: 6CH2) and 1.207 Å (PDB: 6CH3). d YscVXY superimposed with Chlamydia pneumoniae CdsV in complex with the stalk protein CdsO (PDB: 6WA9). The alignment was calculated using SD3 of YscV_C with an r.m.s.d. of 0.854 Å. Two subunits of YscV and CdsV (gray) are shown, CdsO is colored in green.