A Drosophila Pattern Recognition Receptor Contains a Peptidoglycan Docking Groove and Unusual L,D-Carboxypeptidase Activity

Abstract

The Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes. We have determined the crystal structure of PGRP-SA to 2.2-Å resolution and analyzed its peptidoglycan (PG) recognition and signaling activities. We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs. Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation. Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity. As L,D-configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG.

Peptidoglycan recognition proteins (PGRPs)

“…The crystal structures of PGRPs reveal a general design similar to type 2 bacteriophage amidases: they all have three peripheral ␣ helices and several central ␤-sheet strands ( Figure 3) [7,[10][11][12][13]. The front face of the molecule has a cleft that forms a peptidoglycan-binding groove ( Figure 3), and the back of the molecule has a PGRP-specific segment (not present in bacteriophage amidases), which is often hydrophobic and is also …”

“…Bactericidal effect in PMNs (d) [25], although both types of peptidoglycan bind to PGRP-SA [12]. The probable reason for the weak Toll-activating capacity of DAP-type peptidoglycan is that this peptidoglycan, but not Lys-type peptidoglycan, is the substrate for the carboxypeptidase activity of PGRP-SA [12] (Figure 4d).…”

“…On the basis of the conserved structure of the active site of the amidase, several other insect PGRPs are predicted to have amidase activity, whereas several others are not [9,14,15] (Figure 1 and Table 1). One PGRP that is not an amidase, Drosophila PGRP-SA, has an L,D-carboxypeptidase activity with specificity for the bond between DAP and D-Ala of the stem peptide present in peptidoglycan of Gramnegative bacteria and Gram-positive rod bacteria [12] ( Figure 4). The biological significance of this carboxypeptidase activity is not certain.…”

“…Crystallographic analysis of human PGLYRP-1 and the carboxy-terminal PGRP domain of PGLYRP-3, as well as insect PGRP-LB, -SA, -LC and -LE, show that all these PGRPs have a ligand-binding groove that binds peptidoglycan and is specific for MurNAc bound to three peptide-bonded amino acids (muramyl-tripeptide), which is the minimum peptidoglycan fragment hydrolyzed by PGLYRP-2 [7,9,[10][11][12][13][52][53][54][55]. It can accommodate a larger structure, such as GlcNAcMurNAc-tetrapeptide or MurNAc-pentapeptide (Figure 3), but it does not bind muramyl-dipeptide or a peptide without MurNAc [56][57][58].…”

Molecular cloning and functional characterization of peptidoglycan recognition protein 6 in grass carp Ctenopharyngodon idella

“…In addition, some insect PGRPs such as Drosophila PGRP-SC1 and PGRP-LB are N-acetylmuramoyl-L-alanine amidases (Kim et al, 2003;Mellroth et al, 2003), which can hydrolyze proinflammatory peptidoglycans. One insect PGRP, Drosophila PGRP-SA, which is not an amidase, has an L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide peptidoglycan fragments (Chang et al, 2004). Mammals have four PGRPs, namely PGLYRP1, 2, 3, 4.…”