Structural Basis of the Heterodimer Formation between Cell Shape-Determining Proteins Csd1 and Csd2 from Helicobacter pylori

Abstract

Colonization of the human gastric mucosa by Helicobacter pylori requires its high motility, which depends on the helical cell shape. In H. pylori, several 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. H. pylori Csd1, Csd2, and Csd3/HdpA are M23B metallopeptidase family members and may act as d,d-endopeptidases to cleave the d-Ala4-mDAP3 peptide bond of cross-linked dimer muropeptides. Csd3 functions also as the d,d-carboxypeptidase to cleave the d-Ala4-d-Ala5 bond of the muramyl pentapeptide. To provide a basis for understanding molecular functions of Csd1 and Csd2, we have carried out their structural characterizations. We have discovered that (i) Csd2 exists in monomer-dimer equilibrium and (ii) Csd1 and Csd2 form a heterodimer. We have determined crystal structures of the Csd2121–308 homodimer and the heterodimer between Csd1125–312 and Csd2121–308. 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. 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. Structural knowledge of these proteins sheds light on the events that regulate the cell wall in H. pylori.

A Genome-Wide Helicobacter pylori Morphology Screen Uncovers a Membrane-Spanning Helical Cell Shape Complex

“…5A and C). The Csd1/Csd2 results further support published structural findings by An et al, who reported that Csd2 exists as both a monomer and dimer and that Csd1 and Csd2 form a heterodimer (17). An interaction signal was not detected between Csd7 and CcmA by BACTH ( Fig.…”

“…Wild-type (no tag) and RecA-FLAG extracts were used as negative controls (see Table S5). In accordance with published findings and our interaction data, mass spectrometry identified both Csd1 and Csd2 as top hits for both the Csd1-FLAG and Csd2-FLAG IP samples (Table 3; see Table S6 to S8) (17). For Csd7-FLAG, Csd1, Csd2, and CcmA were among the top scoring protein hits (Table 3).…”

“…As previously reported, Csd1 and Csd2 are predicted M23 family endopeptidases, possessing characteristic LytM domains with active site motifs HXXXD and HXH (9,17). In Csd2, the first two histidines of these motifs are not conserved and are replaced with Glu165 and Lys246, respectively, suggesting that Csd2 may not possess PG hydrolase activity (9,17). In further agreement with this observation, when we mutated the Csd2 aspartic acid residue in the HXXXD motif to either a cysteine (Csd2 D169C ) or alanine (Csd2 D169A ), cells were motile and displayed wild-type helical morphology ( Fig.…”

“…As previously reported, Csd1 and Csd2 are predicted M23 family endopeptidases, possessing characteristic LytM domains with active site motifs HXXXD and HXH (9,17). In Csd2, the first two histidines of these motifs are not conserved and are replaced with Glu165 and Lys246, respectively, suggesting that Csd2 may not possess PG hydrolase activity (9,17).…”

“…Csd7 is conserved within the Helicobacter genus, and topology modeling predicts that it is a polytopic inner membrane protein. We show that Csd7 is required for the stability of Csd1 and Csd2, two previously characterized Csd factors (9,17), and directly interacts with these two periplasmic proteins as part of a larger helical cell shape-promoting complex linking periplasmic and cytosolic factors.…”