Peptidoglycan binding by a pocket on the accessory NTF2-domain of Pgp2 directs helical cell shape of Campylobacter jejuni

Chang Sheng-Huei Lin,Anson C.K Chan,Jenny Vermeulen,Jacob Brockerman,Arvind S Soni,Martin E Tanner,Erin C Gaynor,Lawrence P Mcintosh,Jean-Pierre Simorre,Michael E.P Murphy

doi:10.1016/j.jbc.2021.100528

Abstract

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 Δpgp2 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.

Highlights

Campylobacter jejuni is a Gram-negative, highly motile and helical-shaped bacterium that is a leading cause of bacterial foodborne gastroenteritis worldwide [1]
The structure of Pgp2 contains an N-terminal helix, the catalytic LD-CPase domain, and the C-terminal NTF2-like fold (NTF2) domain, each connected to the domain by a single loop (Fig. 1A)
An extensive interface with a buried surface area of 1130 Å2 is found between the LD-CPase and NTF2 domains, Figure 1

Summary

RESEARCH ARTICLE

Received for publication, November 27, 2020, and in revised form, March 1, 2021 Published, Papers in Press, March 10, 2021, https://doi.org/10.1016/ j.jbc.2021.100528. McIntosh, Jean-Pierre Simorre , and Michael E. P. Murphy1,* 1Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada; 2Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada; 3Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada; 4University of Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France

Edited by Wolfgang Peti

Results

Experimental procedures

Microscopy and celltool shape analysis

HPLC muropeptide analysis