Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

Abstract

Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed "type VII." How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE-PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.

Keywords: antigenic variation; host–pathogen interactions; protein secretion; tuberculosis; virulence factor.

Fig. 1.

Crystal structure of EspG_3Mt reveals a novel fold. (A) Overall fold and secondary structure assignment of EspG_3Mt. The β-strands and α-helices of the N-terminal subdomain are denoted β1–β5 and α1–α4, whereas the structurally equivalent elements from the C-terminal subdomain are labeled β1′–β5′ and α1′–α4′. (B) EspG_3Mt exhibits pseudo-twofold rotational symmetry about an axis perpendicular to the center of the β-sheet. The two pseudosymmetrical subdomains of EspG are colored differently along the axis of pseudosymmetry. There is structural similarity between the N-terminal and C-terminal EspG subdomains (C) and the most closely related protein structure identified in the Protein Data Bank in a Dali Database search (D; PDB ID code 1TU1, a protein of unknown function from Pseudomonas aeruginosa). Cartoon depictions (Left) and topology diagrams (Right) are included. Structurally equivalent elements are depicted in the same color.

Fig. 2.

Structure of the EspG_5Mt–PE25–PPE41 ternary complex. (A) Overview of the EspG_5Mt–PE25–PPE41 complex, with PE25 in yellow, PPE41 in red, and EspG_5Mt in purple. (B) Surface representation of the PE25-PPE41 with EspG_5Mt bound. The EspG_5Mt contacts on PPE41 are colored red, with key residues indicated. (C) Detailed view of the EspG_5Mt contact surface on PPE41 (red), with key interacting residues labeled. The structure is rotated 90° with respect to the representation in B. (D) Contact surface between PPE41 and EspG_5Mt, oriented to show the shallow basin on EspG_5Mt (white surface with contact residues in purple), which binds PPE41 (red). (E) Four distinct elements on EspG_5Mt that make up the PPE binding surface. EspG_5Mt is oriented similarly in D and E. (F) Interaction between the long tongue of EspG_5Mt (peach) and the α5 helix from PPE41 (red), which accounts for nearly half of the EspG_5Mt–PPE41 interface.

Fig. 3.

Structural variation between EspGs. (A) Overlay of EspG_3Mt (white) and EspG_3Ms (red). The variable β2-β3 loops and α2 helices are indicated. (B) Overlay of EspG_3Mt (white) and EspG_5Mt (blue). (C) Expanded view of the β2-β3 loop (C) and α1-α2 helices (D) from EspG_3Mt (white), EspG_3Ms (red), and EspG_5Mt (blue). (D) Location of P46, which breaks helix α2 in EspG_5Mt, is indicated.

Fig. 4.

Most PPE proteins bind EspG_5Mt with high affinity. (A) Schematic of chimeric PPE proteins used in binding experiments, with the grafted region highlighted in red. (B) PE25-PPE41 binds to EspG_5Mt with high affinity by biolayer interferometry (BLI). The raw data are plotted in blue, whereas the best global fits are shown in red. The uppermost curve corresponds to PE25-PPE41 binding at 500 nM, with progressively lower responses at 250, 125, 62.5, 31.3, 15.6, and 7.81 nM. (C) Summary of qualitative BLI binding experiments for PPE proteins and EspGs, with (+) or without (−) binding (also SI Appendix, Fig. S11).

Fig. 5.

Putative protein binding site spans the PPE–EspG interface, suggesting a model for EccA-mediated dissociation of the PE–PPE–EspG complex. (A) PPE41 (white surface) bound to EspG_5Mt (purple). Sequence analysis of the PPE surface surrounding the conserved Pro-Pro-Glu motif reveals a patch of conserved residues (orange) that are part of a larger hydrophobic surface (cyan), just adjacent to the β2-β3 loop. (B and C) Close-up views of the hydrophobic region (cyan) and conserved patch (orange) on PPE41 (white surface). (D) Possible model for EccA-mediated dissociation of PE-PPE protein from EspG. Binding of the EccA ATPase across the PPE–EspG interface, perhaps coupled with ATP hydrolysis, may facilitate the release of PE-PPE protein for secretion through ESX and recycling of EspG.

Fig. 6.

Model for the network of interactions between the PPE protein and EspG families in Mtb. PPE proteins encoded within a given ESX cluster generally interact with the EspG from the same cluster (blue box, black lines) and are secreted through the cognate ESX. However, some cluster-encoded PPE proteins can cross-react with EspGs from other clusters, at least in vitro. In contrast, non–ESX-encoded PPE proteins, which account for the majority of PPE genes in Mtb, interact preferentially with EspG_5Mt (pink box, black lines). In all, ∼95% of all PPE proteins from Mtb are predicted to interact with EspG_5Mt, likely leading to their secretion to the cell surface through the ESX-5 secretion system.