Structural insights into the effector-immunity system Tse1/Tsi1 from Pseudomonas aeruginosa

Abstract

During an interbacterial battle, the type-6-secretion-system (T6SS) of the human pathogen Pseudomonas aeruginosa injects the peptidoglycan(PG)-hydrolase Tse1 into the periplasm of gram-negative enemy cells and induces their lysis. However, for its own benefit, P. aeruginosa produces and transports the immunity-protein Tsi1 into its own periplasm where in prevents accidental exo- and endogenous intoxication. Here we present the high-resolution X-ray crystal structure of the lytic enzyme Tse1 and describe the mechanism by which Tse1 cleaves the γ-D-glutamyl-l-meso-diaminopimelic acid amide bond of crosslinked PG. Tse1 belongs to the superfamily of N1pC/P60 peptidases but is unique among described members of this family of which the structure was described, since it is a single domain protein without any putative localization domain. Most importantly, we present the crystal structure of Tse1 bound to its immunity-protein Tsi1 as well and describe the mechanism of enzyme inhibition. Tsi1 occludes the active site of Tse1 and abolishes its enzyme activity by forming a hydrogen bond to a catalytically important histidine residue in Tse1. Based on our structural findings in combination with a bioinfomatic approach we also identified a related system in Burkholderia phytofirmans. Not only do our findings point to a common catalytic mechanism of the Tse1 PG-hydrolases, but we can also show that it is distinct from other members of this superfamily. Furthermore, we provide strong evidence that the mechanism of enzyme inhibition between Tsi1 orthologues is conserved. This work is the first structural description of an entire effector/immunity pair injected by the T6SS system. Moreover, it is also the first example of a member of the N1pC/P60 superfamily which becomes inhibited upon binding to its cognate immunity protein.

Figure 1. Crystal structure of Pseudomonas. aeruginosa Tse1.

Crystal structure of P.aeruginosa Tse1. The central antiparallel β sheet (red) is surrounded by six α-helices (blue). Cys30, located at the beginning of helixB, is shown as a stick model.

Figure 2. Model of Tse1 fitting through the Hcp1 ring of the T6SS injection needle.

The hexameric Hcp1 ring (PDB: 1Y12) is shown as surface representation colored according to the electrostatic surface potential (contouring from +15 kT/e in blue to −15 kT/e in red). The ribbon representation of Hcp1 polypeptide chains is illustrated in gray underneath. The best-fit model of Tse1 is illustrated as ribbon representation using the color scheme from Figure 1.

Figure 3. The active site of Pseudomonas. aeruginosa Tse1.

Close-up of the active site of Tse1, indicating the catalytic diad Cys30-His91, as well as Ile113, Gly111 which are proposed to be catalytically important, and the water molecules W1, which is surmised to play a role in cysteine deprotonation, as well as W2, located in the oxyanion hole. Hydrogen bonds are shown as black dashed lines, and the refined 2mF_o-DF_c electron density map is shown at a contour level of 2.0 σ. Also shown is Cys110, in the position where a third catalytic residue was proposed for N1pC/P60 papain-like cysteine peptidases to which Tse1 is structural related.

Figure 4. Amino acid sequence alignment of Tse1 and Tsi1 of Pseudomonas aeruginosa and Burkolderia phytofirmans.

Amino acid sequence alignment of P. aeruginosa and B. phytofirmans Tse1 (A) and Tsi1 (B). Helices and β-strands in Tse1 are colored blue and red, respectively. Helices in Tsi1 are colored in green and β-strands are shown in orange. Red stars show either residues of Tse's active site or residues in Tsi1 which are important for catalytic inhibition of Tse1. A grey stars indicates Cys110 located at the structurally equivalent position of the histidine residue in the catalytic triad of N1pC/P60 papain-like cysteine peptidases. Below the sequence alignments, residues which connect Tse1 and Tsi1 in our crystal structure by either salt bridges and hydrogen bonds (black triangles) or hydrophobic interactions (green pentagons) are indicated. Dashed lines and solid lines represent disulfide bond (DSB) formation. Conservation of amino acids goes from dark green (high conservation) to orange (low conservation).

Figure 5. Crystal structure of the Tse1/Tsi1 complex and inhibition of Tse1 by Tsi1.

(A) Crystal structure of the Pseudomonas aeruginosa Tse1/Tsi1 complex using the color scheme of figure 1 for Tse1. Tsi1 is formed by three antiparallel β-sheets (orange) arranged as a partial β-propeller, and a short C-terminal α-helix (green). The N-terminal β-sheet consists of the β-strands β1, β2, β3, β5, and β6. The central β-sheet is made of the β-strands β1, β8, β9, β10, and β11. The C-terminal β-sheet is formed by the β-strands β12, β13, and β14. Residues and disulfide bonds (DSB) in Tse1 (DSB2) as well as in Tsi1 (DSB1 and 3) are depicted as sticks. (B) Close-up of the interaction between Tse1 and Tsi1 at the Tse1 active site. Tsi1 inserts the Ser109 side chain into the Tse1 active site, forming a hydrogen bond to the catalytic His91, keeping it from deprotonating Cys30.

Figure 6. Surface representation of Pseudomonas aeruginosa Tse1 and Tsi1 showing their electrostatic potential, interaction and conservation.

“Open-Book view” of the Tse1/Tsi1 complex showing surface representations of Tse1 (left) and Tsi1 (right). (A). Electrostatic surface potential of Tse1 and Tsi1 contoured over a range of ±15 kT/e (blue/red). (B) Amino acid sequence conservation (Figure 3) mapped onto the molecular surfaces in identical view as in (A). (C) Tse1/Tsi1interacting surface patch colored in red. The highly conserved interaction patches of Tsi1 and Tse1 are highlighted with black circles in (A), (B), and (C).