Structure of Csd3 from Helicobacter pylori, a cell shape-determining metallopeptidase

Abstract

Helicobacter pylori is associated with various gastrointestinal diseases such as gastritis, ulcers and gastric cancer. Its colonization of the human gastric mucosa requires high motility, which depends on its helical cell shape. Seven cell shape-determining genes (csd1, csd2, csd3/hdpA, ccmA, csd4, csd5 and csd6) have been identified in H. pylori. Their proteins play key roles in determining the cell shape through modifications of the cell-wall peptidoglycan by the alteration of cross-linking or by the trimming of peptidoglycan muropeptides. Among them, Csd3 (also known as HdpA) is a bifunctional enzyme. Its D,D-endopeptidase activity cleaves the D-Ala(4)-mDAP(3) peptide bond between cross-linked muramyl tetrapeptides and pentapeptides. It is also a D,D-carboxypeptidase that cleaves off the terminal D-Ala(5) from the muramyl pentapeptide. Here, the crystal structure of this protein has been determined, revealing the organization of its three domains in a latent and inactive state. The N-terminal domain 1 and the core of domain 2 share the same fold despite a very low level of sequence identity, and their surface-charge distributions are different. The C-terminal LytM domain contains the catalytic site with a Zn(2+) ion, like the similar domains of other M23 metallopeptidases. Domain 1 occludes the active site of the LytM domain. The core of domain 2 is held against the LytM domain by the C-terminal tail region that protrudes from the LytM domain.

Keywords: HP0506; Helicobacter pylori; LytM; M23B family metallopeptidase; cell-shape determinant; csd3; d,d-carboxypeptidase; d,d-endopeptidase; peptidoglycan hydrolase.

Figure 1

Overall monomer structure and topology of H. pylori Csd3_Δ41. (a) Ribbon diagram of the Csd3_Δ41 monomer (chain A of form 1), with the secondary-structure elements labelled. Domain 1, the core of domain 2 and the LytM domain are shown in bright orange, sky blue and red, respectively. The C-­terminal α-helix (α6) and β-strand (β22) are coloured teal. The green sphere is a Zn²⁺ ion. Side chains of the metal-coordinating residues (Glu74, His259, Asp263 and His341) are shown in stick models (dark grey). The secondary structures were defined by STRIDE (Heinig & Frishman, 2004 ▶). The walls of the active site in the LytM domain are made up of four loops: loop I (the β12–β13 loop), loop II (the β15–β16 loop), loop III (the β19–β20 loop) and loop IV (the β20–β21 loop). (b) Domains of H. pylori Csd3 coloured as in (a). TM, transmembrane helix. Residue numbers for each domain are indicated. (c) Topology diagram of Csd3_Δ41 coloured as in (a). α-Helices, β-strands, 3₁₀-helices and loops are shown as cylinders, arrows, circles and solid lines, respectively. Structure figures were drawn using PyMOL (DeLano, 2002 ▶).

Figure 2

Sequence alignment of four M23B metallopeptidase proteins. Sequence alignment of Csd3 from H. pylori strain 26695 (HP0506; HP_Csd3; SWISS-PROT accession code O25247), NMB0315 from N. meningitidis (NMB_0315; Q9K163), VC0503 from Vibrio cholerae (VC_0503; Q9KUL5) and LytM from S. aureus (SA_LytM; O33599). Red triangles indicate the conserved residues in the HxxxD and HxH motifs (H259-xxx-D263 and H339-x-H341 in H. pylori Csd3) that are important for the metallopeptidase activity. Four loops are indicated by grey boxes.

Figure 3

Metal coordination in Csd3_Δ41 and interactions of the C-terminal α-helix (α6) and β-strand (β22) with the core of domain 2. A ribbon diagram of the Csd3_Δ41 monomer, coloured as in Fig. 1 ▶(a), is shown in the centre. The close-up views on the left represent interactions of the C-terminal strand (β22) with the β6 strand in the core of domain 2 (top) and of the C-terminal helix (α6) with the β-sheet in the core of domain 2 (bottom). Hydrogen-bond interactions are shown as black dotted lines. The close-up views on the right represent the ribbon diagram of the Zn²⁺-binding motif (top) and the surface representation of the substrate-binding groove formed by four loops of the LytM domain (bottom). The electron density for the Zn²⁺ ion in the OMIT mF _o − DF _c map is shown as a light pink mesh (contoured at 10σ). To show the detailed interactions more clearly, the close-up views have slightly different orientations.

Figure 4

LytM domain of Csd3. (a) Two different views of the electrostatic potential surface of the LytM domain of Csd3. The positive and negative electrostatic potentials on the surface are coloured blue and red, respectively. Four loops that form the substrate-binding groove around the Zn²⁺ ion (green sphere) are denoted by loops I–IV. (b) Superposition of LytM domains in four M23B metallopeptidases. The LytM domains of H. pylori Csd3 (red), N. meningitidis NMB0315 (pale green; PDB entry 3slu), V. cholerae VC0503 (sky blue; PDB entry 2gu1) and S. aureus LytM (yellow/orange; PDB entry 1qwy) are superimposed and shown as ribbon diagrams.

Figure 5

Conserved residues in the active site of M23B metallopeptidases. (a) Superposition of LytM domains in two crystal forms of H. pylori Csd3. Chain A of the form 1 crystal (red) and the form 2 crystal (pink) are shown as ribbon diagrams. (b) Superposition of the LytM domains in two M23B family members (NMB0315 and VC0503) coloured as in Fig. 4 ▶(b). Conserved residues are shown as stick models and are labelled (NMB0315 at the top and VC0503 at the bottom). Metal ions and water molecules are shown as green spheres and blue dots, respectively. In NMB0315, the Zn²⁺ ion was replaced by an Ni²⁺ ion during affinity chromatography (Wang et al., 2011 ▶). Wat-1 and Wat-2 are present in NMB0315, where the Ni²⁺ ion is pentacoordinated. Black dotted lines denote hydrogen bonds to Wat-1 in NMB0315. In VC0503, the Zn²⁺ ion is tetracoordinated, with one water molecule (omitted for clarity) located between Wat-1 and Wat-2.

Figure 6

The active site of the Csd3 LytM domain is blocked by domain 1. In this figure, domain 2 is omitted for clarity. (a) Two different views of the interaction between domain 1 (shown as bright orange ribbons) and the LytM domain (shown as a grey surface diagram) of Csd3. (b) Electrostatic surface diagram of domain 1 and a ribbon diagram of the LytM domain (coloured deep teal). Residues of the LytM domain located at the domain interface are shown as stick models and are labelled. (c) Ribbon diagram of domain 1 (bright orange) and the LytM domain (coloured deep teal). Residues at the domain interface are shown as stick models. Hydrogen-bond interactions and salt-bridge interactions are shown as red and black dotted lines, respectively.