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

Jae-Hong Lim,Han-Eol Kim,William E. Goldman,Byung-Ha Oh,Yoshiteru Oshima,Kamna Aggarwal,Shoichiro Kurata,Neal Silverman,Tamaki Yano,Min-Sung Kim

doi:10.1074/jbc.m513030200

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.

Highlights

We suspected that the affinity of peptidoglycan recognition protein (PGRP)-LE for PGN is quite high, such that heterogeneously sized PGN fragments were co-purifying with the protein
Biochemical, and biophysical studies on the interaction between PGRP-LE and tracheal cytotoxin (TCT), we addressed the important question of how PGRPs can discriminate Gram-positive versus Gram-negative bacterial PGN
Arg254 is the key determinant that allows the preferential recognition of DAPtype PGN over lysine-type PGN by PGRP-LE

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction and Preparation of PGRP-LE—The gene segment coding for the PGRP domain (residues 173–345) of Drosophila PGRP-LE was cloned into the pPROEx HTa vector (Invitrogen) by a standard polymerase chain reaction method, and the resulting recombinant protein was expressed in Escherichia coli BL21 (DE3) cells. Bacterial lysate was prepared by sonication in buffer A consisting of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 5 mM ␤-mercaptoethanol, and the supernatant was applied to a Ni2ϩ-nitrilotriacetic acid column (Qiagen). The column was washed with buffer A containing 20 mM imidazole and subsequently washed with a 1:1 mixture of buffer A and 6 M guanidine hydrochloride (pH 8.0). This step for mild protein denaturation was essential to yield a homogeneous form of the protein in subsequent purification steps. The column was washed with buffer A for on-resin protein renaturation.

Generously allowed region

RESULTS

DISCUSSION