Table 1.
Data collection and refinement statistics.
Fig 1.
Overall monomer structure of H. pylori Csd2121–308.
(A) Schematic representation of secondary structures of Csd2121–308 and topology diagram of Csd2121–308. Secondary structures have been defined by the STRIDE program [42]. α-Helices, β-strands, 310-helices, and loops are shown as cylinders (colored in red), arrows (skyblue), flat cylinders (yellow), and solid lines (grey), respectively. Loop 1 (β1-β2 loop; cyan), Loop 2 (β4-β5 loop; red), Loop 3 (β8-β9 loop; skyblue), and Loop 4 (β9-β10 loop; purple) form the putative substrate-binding groove of the Csd2 LytM domain. A dotted line indicates the disordered C-terminal region. (B) Ribbon diagram of Csd2121–308 monomer structure (chain A of Csd2-Csd2 dimer), colored as in the topology diagram in Fig 1A. Close-up views on the right represent the surface representation of the putative substrate-binding groove formed by four loops of the LytM domain (top) and the degenerated active site without a metal ion (bottom). Close-up views on the right have different orientations from the monomer ribbon diagram on the left to show the details more clearly. Side chains of Glu165, Asp169, and His248, corresponding to the conserved Zn2+-coordinating residues, are shown in stick models. Asp169 and His248 belong to the HxxxD and HxH motifs of LytM domains, respectively. In Csd2, Glu165 replaces the histidine residue in the HxxxD motif and it is indicated by the modified H/ExxxD motif. No Zn2+ ion is bound to the Csd2 LytM domain.
Fig 2.
H. pylori Csd2121–308 exists as homodimer in the crystal.
(A) Two different views of the H. pylori Csd2121–308 homodimer structure are shown in ribbon diagram. One Csd2121–308 monomer (chain A colored in yellow-green) and the other Csd2121–308 monomer (chain A’ colored in darker green) from the adjacent asymmetric unit form a homodimer around a crystallographic two-fold symmetry axis (indicated by a dotted arrow) in the crystal. The secondary structure elements of the helical domain are labeled in the side view (top), while most of the secondary structures are labeled in the top view (bottom). (B) Close-up view of the dimer interface. Residues at the dimer interface are shown in stick models. Blue dotted lines represent hydrogen bonds. (C) The interface between Csd2121–308 monomers. One Csd2121–308 monomer (chain A) is shown as a ribbon diagram (in yellow-green) and the other Csd2121–308 monomer (chain A’ from the adjacent asymmetric unit) is shown in the electrostatic surface diagram. This view of the dimer is slightly different from the top view in Fig 2A to show the details more clearly. The dimer interface is hydrophobic in the center and is surrounded by negatively charged surfaces.
Fig 3.
SEC-MALS and equilibrium sedimentation experiments to determine the oligomeric state of H. pylori Csd2 in solution.
(A) Two Csd2 constructs (Csd2121–308 and Csd2140–251) used in these experiments are schematically represented with the secondary structure elements colored as in Fig 1A. Csd2140–251 lacks the helical domain. Csd2121–308 was used for structure determination. (B) SEC-MALS data for two Csd2 protein samples. The black solid lines represent the measured molecular masses. The average molecular masses from MALS analyses are compared with the calculated masses in the table below the chromatography profiles. (C) Equilibrium sedimentation data for two Csd2 protein samples. For Csd2121–308 (top), the circles are experimental data measured at a speed of 35,000 rpm and 5.1 μM protein monomer concentration and the solid line is a fitting line for a reversible monomer-dimer equilibrium model. The two dotted lines are fitting lines for ideal homogeneous monomer and dimer models. Distributions of the residuals for monomer (dotted line), dimer (solid line), and reversible monomer-dimer equilibrium (circles) models are shown in the inset panel. For Csd2140–251 (bottom), the circles are experimental data measured at a speed of 35,000 rpm and 14.5 μM protein monomer concentration and the solid line is a fitting line for a reversible monomer-dimer equilibrium model. The two dotted lines are fitting lines for ideal homogeneous monomer and dimer models. Distributions of the residuals for monomer (dotted line), dimer (solid line), and reversible monomer-dimer equilibrium (circles) models are shown in the inset panel. Equilibrium sedimentation data indicate that both Csd2121–308 and Csd2140–251 are in reversible monomer-dimer equilibrium in solution.
Fig 4.
Analysis of the complex formation between H. pylori Csd1 and Csd2.
(A) SDS-PAGE analysis of the Csd1 and Csd2 complex formation. Lane M: pre-stained protein ladder. Lanes 1 and 2 for Csd154-312-Csd2121-308 complex: pooled fractions either unbound (lane 1) or bound (lane 2) to the affinity chromatography. Lane 3 (unbound) and lane 4 (bound) for Csd175-312-Csd2121-308 complex. Lane 5 (unbound) and lane 6 (bound) for Csd191-312-Csd2121-308 complex. Lane 7 (unbound) and lane 8 (bound) for Csd1125-312-Csd2121-308 complex. Lane 9: the peak fraction of the Csd1125-312-Csd2121-308 complex after size exclusion chromatography. This complex was crystallized for structure determination. (B) SEC-MALS data for the Csd154–312-Csd2121–308 complex. The red line represents the size exclusion chromatography profile. The grey line represents the measured molecular mass, whose average value agrees well with the calculated molecular mass of a 1:1 complex, as shown in the table below the chromatography profile.
Fig 5.
Overall monomer structure of H. pylori Csd1125–312.
(A) Schematic representation of secondary structures of Csd1125–312 and topology diagram of Csd1125–312. Secondary structures have been defined by the STRIDE program [42]. α-Helices, β-strands, 310-helices, and loops are shown as cylinders (colored in light pink), arrows (blue-green), flat cylinders (yellow), and solid lines (grey), respectively. Loop 1 (β1-β2 loop; cyan), Loop 2 (β4-β5 loop; red), Loop 3 (β8-β9 loop; skyblue), and Loop 4 (β9-β10 loop; purple) form the substrate-binding groove of the Csd1 LytM domain. Dotted lines indicate disordered regions. (B) Ribbon diagram of Csd1125–312 monomer structure (chain C of Csd1-Csd2 dimer I), colored as in the topology diagram in Fig 5A. Close-up views on the right represent the surface representation of the substrate-binding groove formed by four loops of the LytM domain (top) and canonical Zn2+-coordination with three protein ligands and two water molecules (bottom). Dark grey and red spheres represent a Zn2+ ion and water molecules, respectively. Side chains of the Zn2+-coordinating residues (His169, Asp173, and His252) are shown in stick models. Black dotted lines denote penta-coordination of the Zn2+ ion. The electron density for the Zn2+-bound active site in 2mFo − DFc map (grey colored mesh) are shown at the 1.0 σ level.
Fig 6.
Comparison of Csd2121–308 homodimer and Csd1125–312-Csd2121–308 complex (heterodimer I).
Csd2121–308 homodimer (top left) and the Csd1125–312-Csd2121–308 complex (heterodimer I) (bottom left) are shown in ribbon diagrams; they are colored as in Fig 2A and S1 Fig, respectively. Electrostatic surface diagrams of Csd2121–308 chain A’ of Csd2 homodimer and Csd1125–312 chain C of the Csd1125–312-Csd2121–308 heterodimer are shown on the right. Highly negatively-charged surfaces surround the hydrophobic interface of the Csd2121–308 homodimer, whereas largely positively-charged surfaces surround the hydrophobic interface of Csd1125–312 of the Csd1125–312-Csd2121–308 heterodimer.
Fig 7.
The C-terminal tail of Csd2 (chain B’) is bound to the substrate-binding groove in the LytM domain of Csd1 (chain C) in Csd1-Csd2 dimer I.
(A) In the structure of Csd1-Csd2 dimer I, the C-terminal residues (His299−Ala304) of Csd2121–308 from an adjacent asymmetric unit (chain B’ shown in ribbon diagram) occupy the substrate-binding groove of the LytM domain in Csd1125–312 (chain C shown in surface diagram). Four loops of Csd1 LytM domain that form the substrate-binding groove are labeled and colored as in Fig 5. The ribbon diagram is colored as in S1 Fig A close-up view on the right represents the Csd2 C-terminal tail residues located in the substrate-binding groove of Csd1 LytM domain. The Csd2 tail residues (enclosed in the black box) are shown in a stick model, with the electron density shown in mesh. The electron density for the Csd2 tail in the feature-enhance map (FEM) calculated by using PHENIX program [47] (lime colored mesh) and 2mFo − DFc map (magenta colored mesh) are shown at the 1.0 σ level. (B) A detailed view of the interactions between the Csd2 tail residues and the substrate-binding groove of the Csd1 LytM domain (shown in ribbon diagram, colored as in S1 Fig). Both main chains and side chains of the Csd2 tail residues are shown in a stick model, with the candidate peptide bond that might be cleaved by the enzymatic activity of Csd1 is indicated by a red wavy line. Side chains of Csd1125–312 residues interacting with the Csd2 tail residues are shown in a stick model. Grey and red spheres represent the Zn2+ ion and water molecules, respectively. Zn2+-coordination (canonical) and hydrogen bonds with waters are indicated by red and black dotted lines, respectively. (C) Superposition of LytM domains in H. pylori Csd1 (skyblue), H. pylori Csd3 (light green; PDB code, 4RNY), and S. aureus LytM bound with tetraglycine phosphinate (purple; PDB code, 4ZYB) shows that the two water molecules (Wat1 and Wat2) of Csd1125–312 chain C of heterodimer I overlap nicely with side chain oxygen atoms of Glu74 (labeled in light green) from the helix α3 of the inhibitory Domain 1 in Csd3 and also with those of the phosphinate molecule (black). The Csd2 tail is simplified as a poly-alanine model (grey). The bound Zn2+ ions are indicated by grey, purple, and green spheres for H. pylori Csd1, H. pylori Csd3, and S. aureus LytM, respectively. Two dotted lines represent the disordered regions in Loop 1 of Csd1. The metal-coordinating residues in the H(169)xxxD(173) and HxH(252) motifs and the conserved catalytic residues in the H(250)xH motif and an additional catalytic histidine residue H(219) of Csd1, as well as corresponding residues of H. pylori Csd3 and S. aureus LytM, are shown in a stick model. Tyr204 (labeled in red) of S. aureus LytM is shown in a stick model. (D) Sequence alignment of LytM domains in Csd1, Csd2, and Csd3 from H. pylori 26695 strain [Csd1 (HP_Csd1; SWISS-PROT accession code O26068), Csd2 (HP_Csd2; O26069), and Csd3 (HP_Csd3; O25247)], S. aureus LytM (SA_LytM; O33599), and S. simulans lysostaphin (SS_LytM; P10547). Tyr204 of S. aureus LytM is marked by a red star. Conserved residues of the characteristic motifs are colored in blue.