Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein

Abstract

The type VI secretion system (T6SS) is a bacterial protein-export machine that is capable of delivering virulence effectors between Gram-negative bacteria. The T6SS of Pseudomonas aeruginosa transports two lytic enzymes, Tse1 and Tse3, to degrade cell-wall peptidoglycan in the periplasm of rival bacteria that are competing for niches via amidase and muramidase activities, respectively. Two cognate immunity proteins, Tsi1 and Tsi3, are produced by the bacterium to inactivate the two antibacterial effectors, thereby protecting its siblings from self-intoxication. Recently, Tse1-Tsi1 has been structurally characterized. Here, the structure of the Tse3-Tsi3 complex is reported at 1.9 Å resolution. The results reveal that Tse3 contains a C-terminal catalytic domain that adopts a soluble lytic transglycosylase (SLT) fold in which three calcium-binding sites were surprisingly observed close to the catalytic Glu residue. The electrostatic properties of the substrate-binding groove are also distinctive from those of known structures with a similar fold. All of these features imply that a unique catalytic mechanism is utilized by Tse3 in cleaving glycosidic bonds. Tsi3 comprises a single domain showing a β-sandwich architecture that is reminiscent of the immunoglobulin fold. Three loops of Tsi3 insert deeply into the groove of Tse3 and completely occlude its active site, which forms the structural basis of Tse3 inactivation. This work is the first crystallographic report describing the three-dimensional structure of the Tse3-Tsi3 effector-immunity pair.

Keywords: calcium binding; effectors; immunity; interaction; muramidases; peptidoglycan.

Figure 1

The overall structure and model quality of the Tse3–Tsi3 complex. (a) Ribbon diagram showing the binary complex, in which Tse3 is coloured yellow and Tsi3 cyan. The Ca²⁺ ions present in Tse3 are represented by magenta balls. (b) Representative 2F _obs − F _calc density map contoured at 1.0σ of the interface between Tse3 and Tsi3. The protein stick model is coloured by element: carbon, yellow (Tse3) or cyan (Tsi3); oxygen, red; nitrogen, blue.

Figure 2

The structure of Tse3 and its catalytic domain in comparison with representative structures containing the SLT domain (PF01464 in the Pfam database). (a) Ribbon model of the overall structure of Tse3. The N-terminal domain and the C-terminal catalytic domain are coloured red and cyan, respectively, while the linker segment is shown in yellow. The loop that is not visible in the electron-density map (residues 13–19) is schematically represented by a green dashed curve. (b) Ribbon diagram of the C-terminal SLT-like domain of Tse3. (c–g) Representative SLT domains of the lytic transglycosylases MltE (PDB entry 2y8p) (c), Slt35 (PDB entry 1qus) (f) and Slt70 (PDB entry 1qsa) (g) from E. coli and the goose-type lyzosymes from goose egg (PDB entry 153l) (d) and Atlantic cod (PDB entry 3gxr) (e). Catalytic glutamic acids and metal ions are highlighted as stick models and magenta spheres, respectively.

Figure 3

Stereoviews of the three Ca²⁺-binding sites in the structure of Tse3. (a) The adjacent non-EF-hand binding sites for Ca1 and Ca2 at a position surrounded by two helices (α17 and α18) and a two-stranded β-sheet. (b) The EF-hand binding site for Ca3 coordinated by amino acids on a loop that connects α27 and α28. Ca²⁺ ions are represented by magenta spheres and the ligated amino acids are shown as stick models.

Figure 4

Sequential and structural properties of the potential active site of Tse3. (a) Three conserved motifs identified from structure-based sequence alignment between Tse3 and other muramidases containing an SLT domain. Amino-acid conservation is depicted using WebLogo (Crooks et al., 2004 ▶). (b) Stereoview of structural superimposition of MltE (PDB entry 2y8p; purple) and gLYS (PDB entry 3gxr; green) onto Tse3 (cyan). The invariant residues are highlighted as stick models with element colour indices. (c)–(e) Electrostatic potential maps calculated on the surface of Tse3 including the three calcium ions (c), MltE (d) and gLYS (e). Positive and negative charges are shown in blue and red colours, respectively. The substrate-binding groove in Tse3 is indicated by a black arrow, while those in the other structures are indicated by bound glycan.

Figure 5

The structure of Tsi3 and comparison with structural neighbours obtained from a DALI search (Holm & Rosenström, 2010 ▶). (a) Ribbon representation of the structure of Tsi3; (b) ribbon models of structures adopting a similar β-sandwich fold. The corresponding PDB codes are given below the structural representations. Loops, α-helices and β-strands are coloured orange, cyan and red, respectively.

Figure 6

Tse3–Tsi3 interactions. (a) Insertion of three loops of Tsi3 (ribbon model in cyan), including two β-hairpins bridging β4–β5 and β7–β8 and a loop connecting β9 and α1, into the substrate-binding groove of Tse3 (surface representation in yellow). (b) The hydrogen-bond network formed between Tse3 (yellow) and Tsi3 (cyan) around the catalytic Glu residue in Tse3. The amino acids involved in hydrogen-bond formation are represented as stick models coloured by element.

Figure 7

Sensorgrams from surface plasmon resonance experiments measuring the interactions of wild-type and mutant Tsi3 with Tse3. Raw data are represented by magenta curves, while the data fitted 1:1 to the Langmuir binding model are shown as black curves.