Structural Basis for Preferential Recognition of Diaminopimelic Acid-type Peptidoglycan by a Subset of Peptidoglycan Recognition Proteins

Abstract

Drosophila peptidoglycan recognition protein (PGRP)-LCx and -LCa are receptors that preferentially recognize meso-diaminopimelic acid (DAP)-type peptidoglycan (PGN) present in Gram-negative bacteria over lysine-type PGN of Gram-positive bacteria and initiate the IMD signaling pathway, whereas PGRP-LE plays a synergistic role in this process of innate immune defense. How these receptors can distinguish the two types of PGN remains unclear. Here the structure of the PGRP domain of Drosophila PGRP-LE in complex with tracheal cytotoxin (TCT), the monomeric DAP-type PGN, reveals a buried ionic interaction between the unique carboxyl group of DAP and a previously unrecognized arginine residue. This arginine is conserved in the known DAP-type PGN-interacting PGRPs and contributes significantly to the affinity of the protein for the ligand. Unexpectedly, TCT induces infinite head-to-tail dimerization of PGRP-LE, in which the disaccharide moiety, but not the peptide stem, of TCT is positioned at the dimer interface. A sequence comparison suggests that TCT induces heterodimerization of the ectodomains of PGRP-LCx and -LCa in a closely analogous manner to prime the IMD signaling pathway, except that the heterodimer formation is nonperpetuating.Innate immune defenses against pathogens are initiated by pattern recognition receptors that bind conserved stereotypical, rather than particular, molecular structures present in a wide spectrum of microorganisms but absent in the host (1). A representative example of such structures is peptidoglycan (PGN), 3 the major constituent of the cell wall of both Gram-positive and -negative bacteria. The peptidoglycan recognition protein (PGRP) family is a class of pattern recognition receptors that bind, and sometimes cleave, PGN. A total of 13 and four PGRP family members have been identified in Drosophila and humans, respectively (2-5). PGRPs are often characterized based on their polypeptide length. Short form PGRPs, such as PGRP-SA and -SD, contain a single PGRP domain (ϳ180 amino acids) and, in most cases, a signal sequence, leading to the secretion of the proteins. Long form PGRPs, such as PGRP-LC and -LE, contain other domain(s) in addition to the PGRP domain, often including a transmembrane region (6). The PGRP domain is similar in structure to N-acetylmuramoyl-L-alanine amidases, such as T7 lysozyme, and some PGRPs are similarly catalytic, whereas others lack a critical cysteine residue in the catalytic triad (7). Thus, these PGRPs lack catalytic activity but function instead as pattern recognition receptors and/or as antimicrobials. For example, murine PGRP-S is directly antimicrobial and contributes to the neutrophil-mediated killing of bacteria (8, 9), whereas PGRP-SA, -SD, -LC, and -LE are key pattern recognition receptors involved in activation of the Drosophila immune response through the Toll or IMD (immune deficiency) signaling pathway (10 -16).In particular, genetic studies revealed that circulating PGRP-SA and PGRP-SD detect Gram-positive bacteria and activate the Toll pa...

Structural insights into the bactericidal mechanism of human peptidoglycan recognition proteins

“…Crystal structures of PGN fragments bound to human or insect PGRPs (15,16,27,28), including the PGRP-I␤C-GMPP complex, have shown that the fragments adopt a similar overall conformation and interaction mode, with some variations around the saccharide and C terminus of the peptide stem (Fig. 3A).…”

“…The PGN-binding site of PGRP-I␤C, whose general topology is maintained in other PGRPs (15,(21)(22)(23)(24)(25)(26)(27)(28), resides in a long cleft whose walls are formed by helix ␣1 and five loops (␤3-␣1, 1␣-␤4, ␤5-␤6, ␤6-␣2, ␤7-␣3) that project above the central ␤-sheet platform ( Fig. 2 A and B).…”

“…Structure-based sequence alignments with Drosophila PGRP-LE, which preferentially recognizes Dap-type PGNs, revealed that PGRP-I␤C lacks a critical Arg at position 288 (Val in both PGRP-I␤C and -I␣C), but that PGRP-S retains an Arg at this position. In the structure of PGRP-LE bound to a Dap-type PGN ligand (27), Arg-254 makes a bidentate salt bridge with the sidechain carboxylate of Dap. The corresponding Val-288 of PGRP-I␤C, unlike Arg-88 of PGRP-S, could not form this presumably stabilizing interaction.…”

Peptidoglycan recognition protein-SD provides versatility of receptor formation in Drosophila immunity

“…At the third amino acid, L-Lysine is found in the majority of Gram-positive bacteria, whereas meso-diaminopimelic acid (mDAP) is present in Gram-negative bacteria and Gram-positive bacilli (5). Indeed, it has been shown that Lys-type PG is a potent activator of Toll, and DAP-type PG induces primarily the Imd pathway (4,(6)(7)(8)(9)(10).…”

Innate immune response in insects: recognition of bacterial peptidoglycan and amplification of its recognition signal

“…Two crystal structures were recently reported: the PGRP domain of Dm-PGRP-LE in complex with tracheal cytotoxin (TCT), a naturally occurring monomer unit of DAP-type PG (32); and the ectodomains of Dm-PGRP-LCa and Dm-PGRP-LCx in complex with TCT (33). These two reports provided the first evidence that protein-protein interactions induced by the monomeric DAP-type PG from Gram-negative bacteria is likely to be essential to activation of the Imd pathway.…”