A peptidoglycan monomer with the glutamine to serine change and basic peptides bind in silico to TLR-2 (403-455)

Bacterial cell wall polysaccharides, such as PGN, bind and activate TLR-2, NOD2 and PGRP on monocytes/macrophages and activate inflammation. We found that the peptides containing basic amino acids (cations) at N -terminus and tyrosine at C-terminus interfered with activating ability of PGN. This finding is significant because the ECD of TLR-2 in vivo encounters a large number of proteins or peptides. Some should bind ECD and "pre-form" TLR-2 to respond or not to its activators, although they cannot activate TLR-2 alone. TLR-2 is receptor for a large number of ligands, including lipopeptides and bacterial cell wall glycoproteins. A binding site for lipopeptides has been identified; however, a binding site for soluble or multimeric PGN has not been proposed. To identify the candidate binding sites of peptides and PGN on TLR-2, we modeled docking of peptides and of the PGN monomer (PGN-S-monomer) to extracellular domain (ECD-TLR-2) of the unbound TLR-2. Quantification, in silico, of free energy of binding (DG) identified 2 close sites for peptides and PGN. The PGN-S-monomer binding site is between amino acids TLR-2, 404-430 or more closely TLR-2, 417-428. The peptide-binding site is between amino acids TLR-2, 434-455. Molecular models show PGN-S-monomer inserts its N -acetyl-glucosamine (NAG) deep in the TLR-2 coil, while its terminal lysine interacts with inside (Glu ⁴⁰³) and outside pocket (Tyr ³⁷⁸). Peptides insert their two N -terminal arginines or their C-terminal tyrosines in the TLR-2 coil. PGN did not bind the lipopeptide-binding site in the TLR-2. It can bind the C-terminus, 572-586 (DG = 0.026 kcal), of "lipopeptide-bound" TLR-2. An additional, low-affinity PGN-binding site is TLR-2 (227-237). MTP, MDP, and lysine-less PGN bind to TLR-2, 87-113. This is the first report identifying candidate binding sites of monomer PGN and peptides on TLR-2. Experimental verification of our findings is needed to create synthetic adjuvant for vaccines. Such synthetic PGN can direct both adjuvant and cancer antigen to TLR-2.