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

An DR, Kim HS, Kim J, Im HN, Yoon HJ, Yoon JY, Jang JY, Hesek D, Lee M, Mobashery S, Kim SJ, Lee BI, Suh SW - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biophysics and Chemical Biology, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea.

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.

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Overall monomer structure and topology of H. pylori Csd3Δ41. (a) Ribbon diagram of the Csd3Δ41 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 Csd3Δ41 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 ▶).
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fig1: Overall monomer structure and topology of H. pylori Csd3Δ41. (a) Ribbon diagram of the Csd3Δ41 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 Csd3Δ41 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 ▶).

Mentions: We have determined the structure of Csd3Δ41 (Fig. 1 ▶) using SAD data from a Pt-derivatized form 2 crystal. The model of the form 2 crystal was refined at 1.98 Å resolution to an Rwork and Rfree of 20.8 and 23.9%, respectively (Table 1 ▶). The form 2 crystal contains one monomer of Csd3Δ41 in the asymmetric unit. This model of Csd3Δ41 excludes six residues (Pro251–Arg256) in a disordered loop near the metal-binding site as well as four residues Gly333–Thr336 and two histidines at the end of the C-terminal affinity tag. The model of the form 1 crystal was refined at 2.00 Å resolution to an Rwork and Rfree of 20.3 and 25.6%, respectively (Table 1 ▶). The form 1 crystal contains two monomers (chains A and B) of Csd3Δ41 in the asymmetric unit. Chains A and B are related by non­crystallographic twofold symmetry. In both chains A and B, four residues Gly333–Thr336 and the C-terminal affinity tag (LEHHHHHH) are disordered. Chains A and B are highly similar to each other, with an r.m.s. deviation of 0.67 Å for 359 Cα atoms. However, they show larger structural deviations from the chain of the form 2 crystal, with r.m.s. deviations of 1.61 and 1.85 Å for 353 Cα atoms in chains A and B, respectively. The largest deviations occur in the η1 and α6 helices, with deviations of 6.65 and 5.58 Å at the Cα atoms of Pro167 and Gly366, respectively (Supplementary Fig. S1). The observed structural variation is likely to be owing to the inherent flexibility of these regions and also owing to different crystal contacts.


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

An DR, Kim HS, Kim J, Im HN, Yoon HJ, Yoon JY, Jang JY, Hesek D, Lee M, Mobashery S, Kim SJ, Lee BI, Suh SW - Acta Crystallogr. D Biol. Crystallogr. (2015)

Overall monomer structure and topology of H. pylori Csd3Δ41. (a) Ribbon diagram of the Csd3Δ41 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 Csd3Δ41 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 ▶).
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fig1: Overall monomer structure and topology of H. pylori Csd3Δ41. (a) Ribbon diagram of the Csd3Δ41 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 Csd3Δ41 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 ▶).
Mentions: We have determined the structure of Csd3Δ41 (Fig. 1 ▶) using SAD data from a Pt-derivatized form 2 crystal. The model of the form 2 crystal was refined at 1.98 Å resolution to an Rwork and Rfree of 20.8 and 23.9%, respectively (Table 1 ▶). The form 2 crystal contains one monomer of Csd3Δ41 in the asymmetric unit. This model of Csd3Δ41 excludes six residues (Pro251–Arg256) in a disordered loop near the metal-binding site as well as four residues Gly333–Thr336 and two histidines at the end of the C-terminal affinity tag. The model of the form 1 crystal was refined at 2.00 Å resolution to an Rwork and Rfree of 20.3 and 25.6%, respectively (Table 1 ▶). The form 1 crystal contains two monomers (chains A and B) of Csd3Δ41 in the asymmetric unit. Chains A and B are related by non­crystallographic twofold symmetry. In both chains A and B, four residues Gly333–Thr336 and the C-terminal affinity tag (LEHHHHHH) are disordered. Chains A and B are highly similar to each other, with an r.m.s. deviation of 0.67 Å for 359 Cα atoms. However, they show larger structural deviations from the chain of the form 2 crystal, with r.m.s. deviations of 1.61 and 1.85 Å for 353 Cα atoms in chains A and B, respectively. The largest deviations occur in the η1 and α6 helices, with deviations of 6.65 and 5.58 Å at the Cα atoms of Pro167 and Gly366, respectively (Supplementary Fig. S1). The observed structural variation is likely to be owing to the inherent flexibility of these regions and also owing to different crystal contacts.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biophysics and Chemical Biology, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea.

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.

Show MeSH
Related in: MedlinePlus