Scheme1.The structure of peptidoglycan showing both crosslinkeda nd non-crosslinkedpeptide side chains.T he inset shows the structure of meso-diaminopimelica cid (meso-Dap).Scheme2.Catalytic activities of the enzymesC sd6 and Pgp2. A) Trimming of the non-crosslinkedPGt etrapeptide to give non-crosslinkedPGtripeptide. B) Trimmingo ft he crosslinkedP Gtetrapeptide to give crosslinkedPGt ripeptide.

The syntheses of peptidoglycan (PG)-derived peptides containing meso-diaminopimelic acid (meso-Dap) are typically quite lengthy due to the need to prepare orthogonally protected meso-Dap. In this work, the preparation of the PG pentapeptide containing the isosteric analog meso-oxa-Dap is described. The synthesis relies on the ring opening of a peptide embedded aziridine via the attack of a serine residue. The pentapeptide was attached to a GlcNAc-anhydro-MurNAc disaccharide, to produce a putative substrate for the AmpG pore protein.

The helical morphology of Campylobacter jejuni , a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δ pgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.

Every year, over 600 million people worldwide contract campylobacteriosis, a bacterial food-borne gastroenteritis primarily caused by Campylobacter jejuni. The helical cell shape of C. jejuni, a key colonization factor, is determined by the structure of the peptidoglycan (PG) layer. The helical structure of PG is determined by Pgp2, a LD-carboxypeptidase that cleaves the terminal D-Ala residue from both monomeric and cross-linked PG tetrapeptides. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. Here, we report a 1.6 Å resolution crystal structure that contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues that is located approximately 40 Å from the LD-carboxypeptidase active site. Sitedirected mutagenesis combined with NMR-monitored titration studies were used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LDcarboxypeptidase and the NTF2 domains to guide catalytic activity to induce a helical cell shape.