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UBC Theses and Dissertations

Peptidoglycan binding by Pgp2 and Ape1 determines Campylobacter jejuni helical cell shape Lin, Chang Sheng-Huei

Abstract

The helical morphology of enteropathogenic Campylobacter jejuni is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by cleavages by the LD-carboxypeptidase Pgp2 and the O-acetyl esterase Ape1 within the periplasm. In this thesis, I used X-ray crystallography, biochemical and genetic methods to investigate the interaction interfaces between these two enzymes and PG to select sites for remodeling to generate a helical cell shape. Pgp2 uses two PG binding sites for enzyme activity. The Pgp2 structure consists of LD-carboxypeptidase (LD-CPase) and NTF2 domains, each contains a pocket formed by conserved residues. The LD-CPase pocket also contains the catalytic triad. The nucleophile Cys174 is confirmed to be essential for Pgp2 activity and helical cell shape. The NTF2 pocket, ~40 Å away from the triad, is lined with charged and hydrophobic residues important for full Pgp2 activity and helical shape. Site-directed mutagenesis demonstrates that residues in both binding sites are required for generating helical cell shape. NMR spectroscopy and PG pull-down assays unequivocally demonstrate that both pockets are PG binding sites. Since Pgp2 is likely to form a dimer in C. jejuni, I expect up to four PG binding sites in the dimer. I propose Pgp2 recognizes the tertiary structure of PG involving both the LD-CPase domain and the accessory NTF2 domain to induce a helical cell shape. The Ape1 crystal structure is composed of a SGNH hydrolase domain, a CBM35 domain, and bound acetate located next to the predicted oxyanion hole. Deacetylase activity by Ape1 is assisted by residues derived from loops on the adjacent CBM35 domain. Residues Gln105, Asn121 and Arg123 of the CBM35 domain are hydrogen bonded to the loop forming the oxyanion hole. A model of the an Ape1-hexasaccharide complex suggests an orientation of PG in the active site that diverges from other members of the SGNH superfamily. I propose Ape1 activity is dependent on the length of the PG glycan strand to modulate cell wall homeostasis. Collectively, my thesis contributes to the knowledge about selective PG binding by PG hydrolases as one mechanism to control cell wall remodeling for generating helical shape.

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