Structural Studies on the Molecular Interactions between Camel Peptidoglycan Recognition Protein, Cpgrp-s and Peptidoglycan Moieties, N-acetylglucosamine and N-acetylmuramic Acid

Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and Nacetylmuramic acid (MurNAc) which are crosslinked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3×10M, 2.6×10M and 1.8×10M respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules A, B, C and D in the asymmetric unit that exist as A-B and C-D units of two neighbouring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α, and IL-6 from human peripheral blood mononuclear cells (PBMCs). The introduction of CPGRP-S to the PGNchallenged cultured PBMCs reduced the expressions of pro-inflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and downregulated macrophage-mediated inflammation indicating its potential applications as an antibacterial agent. INTRODUCTION The first line of defense in the host against invading microorganisms is provided by its innate immune system which contains several proteins including peptidoglycan recognition proteins (PGRPs). PGRPs recognize conserved motifs that are present on the bacterial cell surface and are known as pathogen-associated molecular patterns (PAMPs) (1). Similar patterns are absent in the mammalian systems (2). One of the most commonly occurring PAMPs is peptidoglycan (PGN) molecule which is a polymer of β(1-4)Nacetylglucosamine (GlcNAc) and Nacetylmuramic acid (MurNAc) with all lactyl groups of MurNAc substituted with stem peptides containing alternating Land D-amino acids (3). The peptide chain from one strand is either directly cross-linked to the neighboring chain of polysaccharides or through a short peptide. The polysaccharide strands generally maintain a constant structure in various bacteria. In a contrast, the sequences and conformations of cross-linking peptides may vary considerably. It is well known that in Gram-negative bacteria, the third residue is meso-diaminopimelic acid (m-Dap) while in Gram-positive bacteria, it is L-lysine (L-Lys). In order to characterize the binding properties of CPGRP-S with PGN and various other PAMPs, we have determined the crystal structures of three complexes of CPGRP-S with lipopolysaccharide (LPS, 4), lipoteichoic acid (LTA, 4) and muramyl by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from