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Structure of Tracheal Cytotoxin in Complex with a Heterodimeric Pattern-Recognition Receptor

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Science  24 Mar 2006:
Vol. 311, Issue 5768, pp. 1761-1764
DOI: 10.1126/science.1123056

Abstract

Tracheal cytotoxin (TCT), a naturally occurring fragment of Gram-negative peptidoglycan, is a potent elicitor of innate immune responses in Drosophila. It induces the heterodimerization of its recognition receptors, the peptidoglycan recognition proteins (PGRPs) LCa and LCx, which activates the immune deficiency pathway. The crystal structure at 2.1 angstrom resolution of TCT in complex with the ectodomains of PGRP-LCa and PGRP-LCx shows that TCT is bound to and presented by the LCx ectodomain for recognition by the LCa ectodomain; the latter lacks a canonical peptidoglycan-docking groove conserved in other PGRPs. The interface, revealed in atomic detail, between TCT and the receptor complex highlights the importance of the anhydro-containing disaccharide in bridging the two ectodomains together and the critical role of diaminopimelic acid as the specificity determinant for PGRP interaction.

The innate immune system in multicellular animals has developed to discriminate infectious nonself from noninfectious self. Central to this first line of host defense against microbial infections are pattern-recognition receptors (PRRs), which recognize unique structures of conserved components found in pathogens but not in the host (1). In Drosophila, several PRRs responsible for detecting bacterial infections belong to the family of peptidoglycan recognition proteins (PGRPs), which contain a conserved PGRP domain structurally similar to the bacteriophage T7 lysozyme (2). Peptidoglycan (PG) is one of the pathogen-associated components found only in bacteria but not in eukaryotes and is exploited by the innate immune system as the signature of microbial nonself. Polymeric PG is composed of repeating units of muropeptide composed of disaccharide N-acetyl glucosaminyl (GlcNAc)-N-acetylmuramic acid (MurNAc) linked to a stem peptide of d- and l- (or meso-) amino acids, where the third amino acid is mostly lysine in Gram-positive bacteria and diaminopimelic acid (DAP) in Gram-negative bacteria.

DAP-type PG induces innate immune responses in Drosophila by activating the immune deficiency pathway (35). A naturally occurring fragment of DAP-type PG, known as tracheal cytotoxin (TCT; GlcNAc-1,6-anhydro-MurNAc-l-Ala-γ-d-Glu-meso-DAP-d-Ala), strongly activates the immune deficiency pathway, and its recognition requires PGRP-LCa and -LCx (3, 5), which are generated by alternative splicing from the PGRP-LC gene (6). PGRP-LCa and -LCx contain identical cytoplasmic and transmembrane domains but distinct PGRP-like ectodomains (6). TCT is found in cells of most Gram-negative bacteria because this cell-wall fragment (muropeptide) is constantly released from PG during the remodeling of the polymer that occurs during cell growth and division and is then recycled and its sugar and peptide components reincorporated back into PG (7). TCT, which is released from growing cells of Bordetella, is a virulence factor responsible for epithelial cytopathology associated with pertussis infection (8). TCT binds directly to the ectodomain of PGRP-LCx but not to PGRP-LCa; however, TCT induces heterodimerization of the LCa and LCx ectodomains (9, 10). TCT requires its unique GlcNAc-MurNAc(anhydro) moiety for optimal immunestimulatory activity (3, 5). Recently, the structure of the LCa ectodomain was shown to explain its lack of TCT-binding ability by revealing an obstructed PG-docking groove, which has led to a model of TCT recognition by the PGRP-LCa/LCx complex (9). In this model, the LCx ectodomain first engages a typical muropeptide-docking interaction, as observed in the structure of PGRP-IαC in complex with a synthetic PG subunit analog (11). Next, PGRP-LCa recognizes the exposed structural features of TCT while the latter is bound to the LCx ectodomain. This model explains how TCT brings PGRP-LCa and -LCx receptors in close vicinity, allowing their cytoplasmic domains to dimerize for receptor activation (12). The confirmation of this model, however, requires structural determination of TCT bound to the PGRP-LCa and -LCx complex.

We expressed the LCa and LCx ectodomains in Hi-5 cells and purified the two fragments as described (13). TCT was purified from Escherichia coli as described previously (5). The three components were mixed at a molar ratio of 1:1:6 (LCa:LCx:TCT) before crystallization screening. Crystals with the symmetry of space group P212121 (a = 41.123 Å, b = 79.695 Å, c = 114.389 Å) were grown from a mother liquor containing 20% polyethylene glycol 1500, 5% ethylene glycol, and 100 mM phosphate–citrate at pH 4.2. The structure of LCa-TCT-LCx complex was determined by molecular replacement with coordinates of the LCa ectodomain and PGRP-SA as search models (9, 14). The final model contains all 64 nonhydrogen atoms of TCT, residues Asp354 to the C-terminal residue Ser520 of PGRP-LCa and residues Met334 to Glu499 of PGRP-LCx, with 198 solvent molecules; as well as N-linked glycans to the LCa residues Asn389 and Asn515 (see table S1 for crystallographic data and refinement statistics).

Both PGRP-LCx and PGRP-LCa are type II membrane proteins with N-terminal cytoplasmic and C-terminal ectodomains. Both the LCx and LCa ectodomains contain a central five-stranded β sheet flanked by five major helices (Fig. 1, A and B). However, unlike PGRP-LC, whose surface groove is obstructed by two LCa-specific helical insertions and cannot bind TCT (9), the LCx ectodomain possesses a typical PG-docking groove found in other PGRPs (11, 1417). In the complex, TCT binds into the docking groove on LCx and makes intimate contacts with the groove-lining residues (Fig. 1C), so that one side of TCT is buried within the docking groove. The TCT-loaded LCx ectodomain buries a total surface of 1326 Å2, consistent with their nanomolar affinity (18). The GlcNAc-MurNAc(anhydro) moiety of bound TCT, the B1-H2 loop and the H2 helix of the docking groove, as well as the H3-B4 loop of LCx together form the recognition surface of 685 Å2 for PGRP-LCa and interact with LCa residues from the H1 helix, the H1-B1 loop, and the H2 helix (Fig. 1, B and C). The groove region and the N-linked glycans of the LCa ectodomain are not involved in interaction with TCT and PGRP-LCx. No conformational change is induced in the LCa ectodomain upon binding; the root mean square deviation (RMSD) of the Cα positions of the complexed and uncomplexed structures after superposition is 0.42 Å. Also, no major conformational change is expected in the LCx ectodomain upon TCT binding; the overall structure of TCT-docked LCx is comparable to that of PGRP-SA, with an RMSD of 0.84 Å.

Fig. 1.

The structure of TCT bound to the ectodomains of PGRP-LCa and PGRP-LCx. (A) Ribbon diagram showing the front view of the LCx ectodomain and bound TCT in stick representation. (B) The LCa-TCT-LCx complex viewed parallel to the membrane surface. The LCa and LCx ectodomains are in blue and green ribbon models, respectively. The membrane linkers have been modeled as dashed coils. (C) The ternary complex as viewed from extracellular space toward the cell membrane. The surface complementarity between the LCx ectodomain (in green molecular surface) and TCT is shown in a space-filling representation. The complexed LCa ectodomain is shown as the blue ribbon model.

The entire TCT is well ordered in the complex crystal (Fig. 2A). The PG-docking groove of LCx exhibits a high degree of shape complementarity to TCT and accommodates the ligand in its low-energy extended conformation. TCT makes van der Waals contacts throughout its elongated stem peptide chain with the docking-groove residues. The TCT-contacting residues of LCx are concentrated in the two sequence regions where insertions occur in LCa (Fig. 2B) (9). The TCT-LCx interaction is further stabilized by 25 strategic hydrogen bonds (Fig. 3 and table S2). In the structure, the pyranose ring of MurNAc, adopting an envelope conformation due to an anhydro bond (Fig. 2A), is cradled by the LCx groove base formed by Thr366/x and Ala367/x. The carbonyl group of 2-acetamide and the equatorial 3-OH of GlcNAc, in the chair form, contact the imidazole ring of His365/x and the backbone amide of Glu480/x, respectively (Fig. 3). GlcNAc appears to dock loosely to the LCx groove with its pyranose ring partially protruding into the solvent space. However, the GlcNAc ring is tethered to the lactyl group of MurNAc and the carbonyl group of d-Glu in TCT, as well as to Ser477/x, Ala478/x, and Thr479/x, through an extensive network of hydrogen bonds involving three water molecules (Fig. 3).

Fig. 2.

TCT in the crystal and its recognition residues in the LCa and LCx ectodomains. (A) FoFc difference-Fourier omit map of TCT at 2.1 Å resolution displayed in green mesh at 2.0σ level. (B) Aligned sequences of the LCx (green) and LCa (blue) ectodomains highlight the TCT-interacting residues in red. The LCx residues contacting the LCa ectodomain are shaded in blue; the LCa residues that interact with the LCx ectodomain are shaded in green. LCa-specific insertion residues are underlined. Secondary-structure elements in the LCx ectodomain are indicated above the alignment. The disulfide-linked Cys residues are boxed in yellow. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 3.

Interaction of TCT with the PG-docking groove in the LCx ectodomain. TCT is shown as pink sticks, and the LCx ectodomain is in ribbon representation with side chains of the TCT-interacting residues shown as green sticks. Hydrogen-bonding interactions are shown in yellow and listed in table S2. Water molecules are shown as red spheres.

All PGs from bacteria contain the same carbohydrate backbone; however, dependent upon bacterial species, the stem peptides that linked to MurNAc vary in the composition and modification of the amino acid in the third position. In most Gram-positive bacteria, the third amino acid is Lys; in most Gram-negative bacteria, the third residue is meso-DAP. It was suggested that the third amino acid of the PG stem peptide is the specificity determinant for recognition by PGRPs (4). meso-DAP differs from Lys only by the presence of a carboxyl group on the Cϵ with d-chirality. In the structure, the carboxyl group of meso-DAP is the only residue recognized by an electrostatic interaction; it forms a bidentate salt bridge with the guanidinium group of Arg413/x and hydrogen bonds to the main-chain amide of Asp395/x (Fig. 3); these two residues and Trp394/x form a binding pocket for the DAP group. Furthermore, because the groove that accommodates DAP is guarded by three solvent-exposed basic residues Arg349/x, Lys423/x, and Arg427/x (not shown), a lysine in a monomeric PG ligand would strongly destabilize docking interaction. However, it should be noted that the ϵ-amino group of Lys in polymeric PG will not retain a basic charge if a cross bridge linking this amino group to the carboxyl group of d-Ala of another stem peptide via the pentaglycine linker is present. Mellroth and colleagues have observed binding of PGRP-LCx toward both polymeric Lys- and DAP-type PGs in vitro (10). The residue corresponding to Arg413/x in PGRP-SA is a threonine. The absence of positively charged residues in PGRP-SA in this region is consistent with its preferred binding to Lys-type PG.

It has been shown that the disaccharide moiety and the 1,6-anhydro bond of MurNAc are required for the stimulatory activity (3, 5). In the complex structure, the GlcNAc and MurNAc(anhydro) of TCT bridge together the LCa and LCx ectodomains (Fig. 4A). The formation of 1,6-anhydro bond in MurNAc creates a unique diozolane ring that makes van der Waals contacts with side chains of Leu362/a, Arg401/a, and Thr405/a from LCa, where the folded Arg401 side chain is stabilized by hydrogen bonding to Gln364/a and to ring O5 of MurNAc(anhydro) (Fig. 4A). In addition, the 3-OH of GlcNAc pyranose and 2-acetamide NH make hydrogen bonds to the side chain of Glu409/a, and to Gln364/a and Thr405/a via a water molecule. Several residues from the LCx ectodomain also form part of the LCa interaction surface (Figs. 2B and 4A). Notably, side chains of Phe387/x and Trp392/x from H2 helix make hydrophobic contacts with three leucine residues from LCa (Leu362/a, Leu398/a, and Leu402/a). Moreover, the side chain of Leu362/a is buried within the hydrophobic cleft formed by MurNAc(anhydro) from TCT, and Ala368/x, Val383/x, and Phe387/x (Fig. 4A). Most of the interface residues in LCa show an induced-fit conformational change (Fig. 4B). Likewise, the orientations of many side chains of the LCx interface residues may also be induced as a result of TCT docking. The indole ring of Trp392/x is stacked against the indole ring of Trp394/x, which in turn stacks against the elongated side chain of DAP (Fig. 3); the aromatic ring of Phe387/x is in intimate van der Waals contact with MurNAc. Without bound TCT, both Phe387/x and Trp392/x would not have their side chains oriented properly for LCa interaction. This notion is favored by previous observation that a lactyl-tetrapeptide still retains some stimulatory activity despite the lack of a disaccharide moiety (3, 5); presumably, the stem peptide alone can bind to the LCx docking groove and induce properly oriented Trp392/x through stacking interactions to allow heterodimerization.

Fig. 4.

The LCa ectodomain interface for GlcNAc-MurNAc(anhydro) moiety of TCT and the LCx ectodomain. (A) Interaction of the disaccharide moiety presented by the LCx ectodomain (green) with the LCa ectodomain (blue). (B) Superposition of the interface residues in the complexed (blue) and uncomplexed (gray; PDB code 1Z6I) LCa ectodomains showing induced-fit conformational change of the side chains shown as sticks.

In conclusion, the structure of the LCa-TCT-LCx complex has revealed the molecular interaction between a naturally occurring PG subunit and its PRR complex. The complex structure shows that TCT binds to the docking groove of LCx primarily through its elongated stem peptide and that the bound TCT conformation, which allows its disaccharide GlcNAc-MurNAc(anhydro) to be presented for LCa interaction, is further stabilized by extensive hydrogen bonding. DAP, the specificity determinant of TCT, engages a key electrostatic interaction with Arg413/x. Stacking interactions with the DAP side chain and the MurNAc ring induce proper orientations of side chains of Phe387/x and Trp392/x, which contribute part of the TCT-LCx interface for LCa binding by induced fit. The present complex structure represents the receptor activation step where a monomeric PG ligand brings PGRP-LCa and -LCx receptors together to allow cytoplasmic domains to interact for activation of the immune deficiency pathway. By contrast, activation of this pathway by polymeric PG only requires PGRP-LCx, which can be brought to close proximity by binding to adjacent muropeptide subunits (9, 10). Protein-protein interaction induced by monomeric ligand as visualized by the present work is likely to serve as the general mechanism for pattern recognition for other PGRPs, such as PGRP-SA, that play a double role as both a PG sensor and a signaling molecule.

Note added in proof: During the revision of this paper, the structure of TCT bound to PGRP-LE was reported (19).

Supporting Online Material

www.sciencemag.org/cgi/content/full/311/5768/1761/DC1

Materials and Methods

Tables S1 and S2

References

References and Notes

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