Helical shape of Helicobacter pylori requires an atypical glutamine as a zinc ligand in the carboxypeptidase Csd4

Abstract

Peptidoglycan modifying carboxypeptidases (CPs) are important determinants of bacterial cell shape. Here, we report crystal structures of Csd4, a three-domain protein from the human gastric pathogen Helicobacter pylori. The catalytic zinc in Csd4 is coordinated by a rare His-Glu-Gln configuration that is conserved among most Csd4 homologs, which form a distinct subfamily of CPs. Substitution of the glutamine to histidine, the residue found in prototypical zinc carboxypeptidases, resulted in decreased enzyme activity and inhibition by phosphate. Expression of the histidine variant at the native locus in a H. pylori csd4 deletion strain did not restore the wild-type helical morphology. Biochemical assays show that Csd4 can cleave a tripeptide peptidoglycan substrate analog to release m-DAP. Structures of Csd4 with this substrate analog or product bound at the active site reveal determinants of peptidoglycan specificity and the mechanism to cleave an isopeptide bond to release m-DAP. Our data suggest that Csd4 is the archetype of a new CP subfamily with a domain scheme that differs from this large family of peptide-cleaving enzymes.

Keywords: Carboxypeptidase; Cell Shape; Cell Wall; Helicobacter pylori; Meso-diaminopimelic Acid; Metalloenzyme; Peptidoglycan; Tripeptide; Zinc.

FIGURE 1.

The synthetic scheme for the tripeptide substrate.

FIGURE 2.

The crystal structure of Csd4 (PDB code 4WCL). A, the overall monomeric structure of Csd4 with Zn²⁺ and m-DAP bound. The catalytic domain and domains 2 and 3 are colored blue, gray, and green, respectively. B and D, overall (B) and active site (D) magnified electrostatic surface potential of Csd4 contoured at ±3 k_bT/e_c. Electropositive regions are colored blue; electronegative regions are colored red; position of buried Zn²⁺ indicated with a star. C, distribution of conserved residues mapped onto the surface of Csd4. Most conserved regions are colored blue; the least conserved is colored red. E, two-dimensional interaction map between Csd4 and the product m-DAP (light gray). The predicted catalytic water highlighted in bold type. F, corresponding Zn-Csd4 active site with key ligands and an omit F_o − F_c difference density map for the density of the bound m-DAP product contoured at 3 σ.

FIGURE 3.

Complementation and Western blot analysis of Csd4–3x-Flag control and Csd4 C-terminal truncations. The strains used were LSH18 (Δcsd4), LSH100 WT (no 3x-Flag tag), KBH19 (WT-3x-Flag), KBH35 (T1–3x-Flag), and KBH37 (T2–3x-Flag). A, 1000× phase contrast images of wild-type and mutant H. pylori. B, smooth histograms displaying population cell curvature (x axis) as a density function (y axis). C, Western blot analysis of Csd4 (detected by anti-Flag M2 antibody); LSH100 WT (−); KBH19 (+) with a predicted molecular mass of 48 kDa; KBH35 (T1) with a predicted molecular mass of 39 kDa; KBH37 (T2) with a predicted molecular mass of 28 kDa; and Cag3 with a predicted molecular mass of 55 kDa. Equivalent amounts of cell extract based on optical density of the culture were loaded for each strain.

FIGURE 4.

Tripeptide substrate binding site (PDB code 4WCN). A, key zinc and substrate interactions with Csd4 are shown. Zinc ligands are colored white; tripeptide ligands are colored cyan; the predicted catalytic water is red, and its ligands are orange; zinc is gray; and key interactions are shown as dotted lines. Hydrophobic residues and other waters are not shown. B, structural alignment between substrate- and product-bound Csd4. The view is of the active site in A with a 30° rotation about the x axis. C, tripeptide omit F_o − F_c difference map contoured at 2 σ generated prior to the addition of the tripeptide to the model (purple) and from the final model (green) showing well defined omit density for the deeply buried portions of the tripeptide substrate. Increased tripeptide flexibility in relation to the degree of surface exposure are indicated by a B-factor based coloring scheme (blue = ∼20 Ų and green = ∼60 Ų).

FIGURE 5.

Two-dimensional tripeptide-Csd4 interaction map (PDB code 4WCN). Tripeptide substrate is highlighted in gray; waters are colored cyan; iodide is in green; red bristled arcs depict nearby hydrophobic interactions; and zinc cofactor and its ligands are not shown. The figure was drawn using LigPlot⁺ (43).

FIGURE 6.

Wild-type Csd4 exhibits higher catalytic activity on the tripeptide substrate than its active site variants. A, Csd4 activity was continuously monitored via the activity of meso-diaminopimelate dehydrogenase, which consumes the Csd4 product m-DAP to produce NADPH. Buffer-based activity rate differences of Csd4 and its variants are shown. B, the pH-based activity differences between wild-type Csd4 and the Q46H variant was examined by examining the amount of product produced after 20 min. Mean values are shown with error bars representing the standard deviation based on at least three experimental replicates. The p value for all variants is <0.0005 as compared with wild type in their respective buffers utilizing the t test in A. p values between wild type and Q46H are <0.0006 for all pH values except pH 4.8 in B.

FIGURE 7.

Active site of the zinc-bound Q46H variant (PDB code 4WCM). A, zinc ligands are colored white; m-DAP is in yellow; phosphate is in orange; other residues interacting with the phosphate are colored blue; and phosphate interactions are shown as dotted lines. B, omit F_o − F_c difference density map contoured at 3 σ showing density for a bound phosphate.

FIGURE 8.

Complementation and overexpression analysis of Csd4 active site variants. Strain labels indicate the copy number (1 or 2) and amino acid residue at position 46 (Q, A, or H) of csd4–3x-Flag. Strains with two copies have one copy at the native locus and the second copy at the rdxA locus. The strains used were (−) LSH100 WT (no 3x-Flag tag), KBH54 (Δcsd4), KBH19 (1Q), KBH33 (1H), KBH42 (1A), KBH60 (2H), KBH65 (2Q), and KBH66 (1Q, 1H). A–C, 1000× phase contrast images of wild-type (A) and csd4 mutant H. pylori (B and C) smooth histograms displaying population cell curvature (x axis, B) and population axis length (x axis, C) as a density function (y axis). D, Western blot analysis of Csd4 (detected by anti-Flag M2 antibody, predicted molecular mass of 48 kDa) and Cag3 (periplasmic protein loading control, predicted molecular mass is 55 kDa). Equivalent amounts of cell extract based on optical density of the culture were loaded for each strain. ImageJ software was used for densitometry analysis of Csd4 variant expression relative to Cag3 and is indicated below each lane. M.w, molecular mass; rel. exp., relative expression.

FIGURE 9.

Conservation of zinc and substrate binding residues for select homologs of Csd4. Shown are H. pylori G27 (H.py) YP_002265985.1, Helicobacter canis (H.ca) WP_023929364.1, H. hepaticus ATCC 51449 (H.he) NP_860063.1, Helicobacter pullorum (H.pu) WP_005022665.1, C. jejuni 81–176 (C.je) YP_001001002.1, Desulfovibrio vulgaris str. Miyazaki F (D.vu) YP_002436677.1, Sulfurimonas gotlandica GD1 (S.go 1, SMGD1_0946, WP_008338748.1; S.go 2, SMGD1_2299, WP_008339471.1), Sulfurimonas denitrificans DSM 1251 (S.de) YP_393892.1, Wolinella succinogenes DSM 1740 (W.su 1, WS0230, NP_906487.1; W.su 2, WS0783, NP_906997.1), Arcobacter nitrofigilis DSM 7299 (A.ni) YP_003657196.1, Arcobacter butzleri RM4018 (A.bu) YP_001489842.1, Persephonella marina EX-H1 (P.ma) YP_002731141.1, Caminibacter mediatlanticus TB-2 (C.me 1, CMTB2_05747, WP_007475257.1; C.me 2, CMTB2_06881, WP_007473913.1), and Sulfurospirillum barnesii SES-3 (S.ba) YP_006404626.1. Only the regions containing the Csd4 carboxypeptidase domain are shown. Highlighted are key amino acid ligands in the Csd4 crystal structures: zinc-binding ligands (*), substrate/product binding residues ( ), hydrophobic binding pocket residues (+), and catalytically important glutamate (#).

FIGURE 10.

A bootstrapped tree of the Csd4 family of CPs. Homologs of Csd4 were identified from the nonredundant database at the National Center for Biotechnology Information utilizing BLASTP and an E value cutoff of 1 × 10⁻⁷. Identical protein sequences derived from different strains of the same species and proteins with short alignment coverage were removed. The sequences were aligned with Clustal Omega (28) and a bootstrapped tree (with 500 replicates, subtree pruning and regrafting and five random starts) was generated using PhyML (29) within Seaview (30). *, H. hepaticus and H. cinaedi form their own subbranch and contain a His at the equivalent position of Gln-46.