Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin

Abstract

The bacterial type III secretion system, or injectisome, is a syringe shaped nanomachine essential for the virulence of many disease causing Gram-negative bacteria. At the core of the injectisome structure is the needle complex, a continuous channel formed by the highly oligomerized inner and outer membrane hollow rings and a polymerized helical needle filament which spans through and projects into the infected host cell. Here we present the near-atomic resolution structure of a needle complex from the prototypical Salmonella Typhimurium SPI-1 type III secretion system, with local masking protocols allowing for model building and refinement of the major membrane spanning components of the needle complex base in addition to an isolated needle filament. This work provides significant insight into injectisome structure and assembly and importantly captures the molecular basis for substrate induced gating in the giant outer membrane secretin portal family.

Fig. 1

Cryo-EM structures of the injectisome needle complex and isolated needle. a Needle complex C1 reconstruction (low pass filtered from 7.4 Å reconstruction to highlight overall features) cut away at the mid-section. The domain annotation of PrgH, PrgK, and InvG is overlaid on the left. Boxed regions indicate the periplasmic region of the export apparatus and the rod/needle filament. b Central slice view of needle complex reconstruction (gray) overlaid with the 6.3 Å basal body reconstruction (EMD-8400) (pink). c Reconstructions for the 24-fold averaged IM rings (green; 3.6 Å resolution), the 15-fold averaged secretin (blue; 4.1 Å resolution) and the isolated needle (magenta; 3.3 Å resolution). High-resolution reconstructions overlaid on C1 reconstruction shown as central slice (black). The needle was fit into the needle complex C1 map using Chimera and agrees well with the wider part of the needle complex filament. d Refined structures for InvG_34–557 (blue), PrgH_171–364 (green), PrgK_20–203 (green), and PrgI_3–80 (magenta). One monomer encompassing InvG_34–557 is colored according to structural domains: N0-N3 domains (blue); outer β-sheet (cyan); inner β-sheet (green); secretin domain lip (orange); S domain (red)

Fig. 2

The export apparatus forms an asymmetric substructure composed of SpaP and SpaQ and SpaR. a The region of the needle complex C1 reconstruction corresponding to the export apparatus and putative rod colored yellow. Slabbed remainder of reconstruction colored gray. b The region of previous C1 basal body reconstruction corresponding to the export apparatus colored purple. The map features a flat, symmetrical pore, averaged out by the signal of the 24-mer IM rings (24-mer repeating features evident on upper surface). Slabbed remainder of reconstruction colored gray. c Slabbed view of overlay of a and b showing export apparatus region from needle complex map (yellow) is asymmetric compared to the same region from the basal body map (purple) (lower box). The 3.3 Å needle map (magenta) fits well (correlation coefficient = 0.95) into the wider part of the filament density. We propose the boxed narrower region between the base of the filament and the export apparatus represents the PrgJ rod, which has been proposed to form a short adaptor between export apparatus and needle. d The recent structure of the Salmonella flagellar export apparatus FliP (blue)/Q (red)/R (yellow)) forms a complex with stoichiometry of 5:4:1 and overlays very well with the needle complex export apparatus substructure. The subunits form a helical assembly ideal as a structural foundation for the assembly of the rod and needle filament. Atomic model figure reproduced under the CC-BY 4.0 International license

Fig. 3

Structure of the PrgI needle filament at 3.3 Å defines a spiral groove for effector secretion. a, b Side and top views of the helical packing of the PrgI needle with 5.7 monomers per turn and a helical rise of 4.33 Å. The ssNMR structure of PrgI (PDB 2LPZ; gray transparent) is overlaid showing the subtle differences in helical parameters create a small but accumulating change in subunit packing. Side chains, resolved in the reconstruction here, are shown in b highlighting the constriction of the interior channel. c Representative density (3.3 Å resolution). d Zoomed in view showing interface of i, i−6, and i−11 monomers formed around the N-terminal loop of i. The ssNMR structure of PrgI (gray transparent) is overlaid. The variable N-terminal loop here is packed closer to the kinked helix of monomer i−6. The kink (residues 20–23) adopts a different conformation compared to the ssNMR structure. e Zoomed view of the interaction network around the C-terminal Arg₈₀. The ssNMR structure is overlaid on monomer i showing the Arg₈₀ side chain and carboxylate in a flipped orientation. Here, the Arg₈₀ side chain guanidinium group interacts with Gln₇₇ and Asn₇₈ on monomer i–1 while the carboxylate interacts with Lys₆₆ on i–5. f The interior channel is significantly more conserved than the outer surface (conservation colored from cyan (low) to maroon (high)). The cluster of conserved residues around the C-terminus as in e define the raised edge of a right handed spiral that extends the length of the lumen. The groove is lined by repeating deeper pockets defining the path of effector secretion. g Same view as in f colored according to residue type: hydrophobic–gray, aromatic–light pink, polar–light cyan, positive–blue, negative–red, cysteine–light yellow, proline–light green, glycine–green. The raised edge of the groove is demarcated by charged residues while the groove itself is predominantly polar and hydrophobic. h Surface corresponding to the interior needle lumen highlighting the right handed spiral and dimensions of the channel

Fig. 4

Structure of the InvG secretin pore in the open state. InvG_34–557 secretin pore structure viewed from (a) side slab, (b) top (OM perspective) and (c) three monomers highlighting inter-domain and intra-domain packing of monomers i, i + 1 and i + 2. One monomer colored according to structural features as per Fig. 1. Secretin domain β-strand numbers as per our previous closed InvG structure indicated in c

Fig. 5

Structural changes involved in InvG gate opening. a Comparison of the InvG secretin gate open state (colored as per Fig. 1) and the closed state (gray transparent). The major structural changes are the ordering of the N3 domain variable loop (residues 217–226 and 252–265, disordered in other secretin structures) and accompanying change in N3 domain position, the opening of the periplasmic gate involving the repositioning of GATE1 and GATE2, and the more elevated lip region caused by interactions with GATE1 and the insertion of the assembled needle filament. b and c–side view–and d and e inside view–compare the interface between outer and inner β-barrels in the open and closed states, respectively with accompanying interface areas of 2111 Ų and 1153 Ų, residues forming the open and closed interfaces shown as sticks in b and c. The core interface at the base of the β-sandwich formed by the outer and inner β-barrels is predominantly hydrophobic while the region formed by the extension of the GATE1 and GATE2 hairpins is mostly polar in nature. Key interactions defining the open and closed conformations between the N3 domain β-INSERTION, the inner β-barrel GATE1 and GATE2 motifs, and the upper outer β-barrel and lip are labeled and shown as ball and stick. The middle subunit is colored gray in d and e to define the outer and inner β-barrels. The closed gate conformation is supported by interactions of the N3 β-INSERTION, specifically Arg₁₉₈, with the kinked regions of the GATE1 (Asp₃₈₄) and GATE2 (Glu₄₂₉) hairpins and further supported by the surrounding network of interactions. In the open gate conformation, the N3 β-INSERTION interface is disrupted and the gate forming GATE1 hairpin is now extended toward the lip and packed against the outer β-barrel with Ile₃₉₄ at the tip packing in a hydrophobic notch formed by the side chains of Arg₃₂₀, Asn₃₄₀, Asn₃₅₇, and Leu₃₅₉. The GATE2 hairpin undergoes a significant rotation with residues stabilizing the closed gate—Leu₄₄₇, Pro₄₄₈, Glu₄₄₉, and Val₄₅₀—packing against the outer β-barrel wall. A salt bridge between GATE2 Glu₄₄₉ and GATE1 Arg₃₈₇ is maintained between closed and open conformations

Fig. 6

Proposed secretin gating mechanism. a Closed secretin. b Initial rod/needle polymerization within the secretin lumen contacts the N3 domain triggering the ordering of the variable N3 loop^a providing lateral stability and altering the N3 domain-inner β-barrel interface^b. This in turn disrupts the interactions of the N3 β-INSERTION with the GATE1 and GATE2 kinks and unlocks the gate^c. c Continued needle polymerization sterically pushes the gate open^d. d Final assembled needle with fully open gate