Structural Studies of Three Cell Shape Determining Proteins from Helicobacter pylori: Csd1, Csd2, and Csd3

Abstract

Helicobacter pylori is associated with various gastrointestinal diseases, including gastric cancer. Its colonization of the human gastric mucosa requires high motility, which depends on the helical cell shape. In H. pylori, cell shape-determining genes (csd1, csd2, csd3/hdpA, ccmA, csd4, csd5, and csd6) play key roles in determining the cell shape by alteration of cross-linking or by trimming of peptidoglycan stem peptides. Among them, Csd1, Csd2, and Csd3/HdpA belong to the same M23B metallopeptidase family and may act as D,D-endopeptidases to cleave the cross-linking (D-Ala4-mDAP3 bond) of cross-linked dimer muropeptides. Csd3 is a bi-functional enzyme, and it functions also as the D,D-carboxypeptidase to cleave the D-Ala4-D-Ala5 bond of the muramyl pentapeptide. To better understand the role of Csd proteins in H. pylori, I have determined the crystal structures of Csd1 (HP1543 in 26695 strain), Csd2 (HP1544), and Csd3 (HP0506). H. pylori Csd3 exists as monomers in solution. The crystal structure of Csd3 revealed that the Csd3 monomer consists of three domains: domain 1, domain 2, and C-terminal LytM domain. The Csd3 LytM domain contains the catalytic active site with a Zn2+ ion, like similar domains of other M23 metallopeptidases. However, the active site in the Csd3 LytM domain is blocked by domain 1, resulting in a latent and inactive state. H. pylori Csd2 alone exists in monomer-dimer equilibrium and forms a stable heterodimer with H. pylori Csd1 in solution. The crystal structures of Csd2121–308 homodimer and Csd1125–312-Csd2121–308 heterodimer revealed that overall structures of Csd1125–312 and Csd2121–308 monomers are similar to each other, consisting of a helical domain and a LytM domain. The helical domains of both Csd1 and Csd2 play a key role in the formation of homodimers or heterodimers. LytM domains of Csd1 and Csd2 share the same overall fold but a functionally significant difference exists in their active sites. The Csd1 LytM domain contains a catalytic site with a Zn2+ ion, which is coordinated by three conserved ligands and two water molecules, whereas the Csd2 LytM domain has incomplete metal ligands and no metal ion is bound. Although Csd3 and Csd1/Csd2 proteins are LytM homologs in H. pylori, they show distinct features in their domain organization and LytM domains. Structural knowledge of these Csd proteins sheds light on the events that regulate the cell wall in H. pylori.