Structural insights into the bactericidal mechanism of human peptidoglycan recognition proteins

Abstract

Peptidoglycan recognition proteins (PGRPs) are highly conserved pattern-recognition molecules of the innate immune system that bind bacterial peptidoglycans (PGNs), which are polymers of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) cross-linked by short peptide stems. Human PRGPs are bactericidal against pathogenic and nonpathogenic Gram-positive bacteria, but not normal flora bacteria. Like certain glycopeptide antibiotics (e.g., vancomycin), PGRPs kill bacteria by directly interacting with their cell wall PGN, thereby interfering with PGN maturation. To better understand the bactericidal mechanism of PGRPs, we determined the crystal structure of the C-terminal PGN-binding domain of human PGRP-I beta in complex with NAG-NAM-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala, a synthetic glycopeptide comprising a complete PGN repeat. This structure, in conjunction with the previously reported NMR structure of a dimeric PGN fragment, permitted identification of major conformational differences between free and PGRP-bound PGN with respect to the relative orientation of saccharide and peptide moieties. These differences provided structural insights into the bactericidal mechanism of human PGRPs. On the basis of molecular modeling, we propose that these proteins disrupt cell wall maturation not only by sterically encumbering access of biosynthetic enzymes to the nascent PGN chains, but also by locking PGN into a conformation that prevents formation of cross-links between peptide stems in the growing cell wall.

Fig. 1.

Structure of PGN and PGN derivatives. (A) Schematic representation of Lys-type PGNs. Lys-type PGN peptides are usually cross-linked through a peptide bridge composed of one to five glycines. The fragment shown in red corresponds to GMPP. (B) Chemical structure of GMPP. (C) GMPP₂. (D) MPP (R¹, H) and MPP-Dap (R¹, COOH).

Fig. 2.

Structure of the PGRP-IβC–GMPP complex. (A) Overall structure. Disulfide bonds are shown in purple. Labeling of secondary structure elements follows the numbering for unbound PGRP-IαC in ref. . The N and C termini are indicated. The bound GMPP is shown in ball-and-stick representation, with carbon atoms in cyan, nitrogen atoms in blue, and oxygen atoms in red. The final σ_A-weighted 2F_o–F_c electron density map for the GMPP ligand at 1.5 σ is contoured in blue. (B) Stereoview of interactions between PGRP-IβC and GMPP at the PGN-binding site. The bound GMPP is shown in stick representation, with carbon atoms in yellow, nitrogen atoms in blue, and oxygen atoms in red. Hydrogen bonds are drawn as dotted lines. Bound waters (W1–5) mediating hydrogen bonds between PGRP-IβC and GMPP are shown as cyan balls. (C) Schematic representation of interactions between PGRP-IβC and GMPP. GMPP is shown in blue; hydrogen bonds are shown as dotted lines. Residues making van der Waals contacts with GMPP are indicated by arcs with spokes radiating toward the ligand moieties they contact. Water-mediated interactions between GMPP and PGRP-IβC are omitted for clarity.

Fig. 3.

Structural comparison between PGRP-bound PGN analogs in crystal structures and unbound GMPP₂ in solution. (A) Conformational comparison of GMPP, MPP, TCT, and GMPP₂. GMPP, MPP, and TCT are from crystal structures of complexes with human PGRP-IβC, human PGRP-IαC (16), and Drosophila PGRP-LE (27), respectively; GMPP₂ is from the unliganded NMR structure (17). The structures are superposed through the pyranose ring of NAM (for MPP, GMPP, and GMPP₂) or NAM(1,6-anhydro) (for TCT). (B) Superposition of unbound GMPP₂ onto GMPP in the PGRP-IβC–GMPP complex. GMPP and GMPP₂ are shown in ball-and-stick representations, with carbon atoms in yellow and green, respectively, nitrogen atoms in blue, and oxygen atoms in red. Of the two GMPP units in GMPP₂, the first unit, comprising the NAG₁-NAM₁ disaccharide, is superposed onto GMPP in the complex. The peptide stem of GMPP₂ attached to NAM₁ is buried within PGRP-IβC and is shown in pale green. (C) Alternative superposition of unliganded GMPP₂ onto GMPP bound to PGRP-IβC. In this case, the second GMPP unit of GMPP₂, containing NAG₂-NAM₂, is superposed onto GMPP in the PGRP-IβC–GMPP structure. The peptide stem of GMPP₂ attached to NAM₂, shown in pale green, is buried inside PGRP-IβC. (D) Modeled PGRP-IβC–GMPP₂ structure.

Fig. 4.

Possible interaction of PGRPs with the bacterial cell wall. (A) Top view of a structural model of a PGRP-IβC molecule bound to cell wall PGN. PGRP-IβC and the cell wall are shown in molecular surface representation with the glycan strands of PGN in red, the peptide stems in yellow, PGRP-IβC in purple, and the PGRP-bound peptide stem in cyan. This model was constructed by docking PGRP-IβC onto a GMPP unit of a perpendicular model of the cell wall (17) in which the PGN strands are orthogonal to the cell membrane. The strands form a honeycomb pattern, with pore sizes determined by the extent of cross-linking. Small pores are formed by cross-linking each PGN strand to three neighboring strands. The PGRP-IβC molecule is situated in an incompletely cross-linked region of the growing cell well where a missing PGN strand creates a larger pore. (B) Side view of the model in A, in which the cell wall has been cut away to expose the bound PGRP protein. (C) Comparison of cross-linked and PGRP-bound peptide stems. A cross-linked peptide stem in the model of cell wall PGN is shown in blue. The same peptide stem, but in its PGRP-bound conformation, is shown in cyan.