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

<div><p>Colonization of the human gastric mucosa by <i>Helicobacter pylori</i> requires its high motility, which depends on the helical cell shape. In <i>H</i>. <i>pylori</i>, several genes (<i>csd1</i>, <i>csd2</i>, <i>csd3</i>/<i>hdpA</i>, <i>ccmA</i>, <i>csd4</i>, <i>csd5</i>, and <i>csd6</i>) play key roles in determining the cell shape by alteration of cross-linking or by trimming of peptidoglycan stem peptides. <i>H</i>. <i>pylori</i> Csd1, Csd2, and Csd3/HdpA are M23B metallopeptidase family members and may act as d,d-endopeptidases to cleave the d-Ala⁴-<i>m</i>DAP³ 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 Csd2_121–308 homodimer and the heterodimer between Csd1_125–312 and Csd2_121–308. Overall structures of Csd1_125–312 and Csd2_121–308 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 <i>H</i>. <i>pylori</i>.</p></div

SEC-MALS and equilibrium sedimentation experiments to determine the oligomeric state of <i>H</i>. <i>pylori</i> Csd2 in solution.

<p>(A) Two Csd2 constructs (Csd2_121–308 and Csd2_140–251) used in these experiments are schematically represented with the secondary structure elements colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164243#pone.0164243.g001" target="_blank">Fig 1A</a>. Csd2_140–251 lacks the helical domain. Csd2_121–308 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 Csd2_121–308 (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 Csd2_140–251 (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 Csd2_121–308 and Csd2_140–251 are in reversible monomer-dimer equilibrium in solution.</p

Overall structures of apo- and G3P-bound TaTPI.

<p>(A) Homodimer of apo-TaTPI is shown in carton representation, where α-helix, β-strand, and loop are colored in red, yellow, and green, respectively. (B) G3P-bound TaTPI. Only monomer of homodimeric G3P-bound TaTPI is demonstrated to emphasize a conformational change compared with apo-TaTPI. Extra positive electron density in <i>F</i>_<i>o</i><i>-F</i>_<i>c</i> omit map contoured at 3.0 δ is shown as a blue mesh, which is modelled as G3P later. Amino acid residues interacting with G3P in catalytic site and G3P are shown as stick model; carbon, oxygen, phosphorus, and nitrogen atoms are colored in green, red, orange, and, blue, respectively. Loop 6 and loop 7 regions, which show a distinctive conformational change upon binding of G3P, are represented in orange and magnified for clarity.</p

Comparison of Csd2_121–308 homodimer and Csd1_125–312-Csd2_121–308 complex (heterodimer I).

<p>Csd2_121–308 homodimer (top left) and the Csd1_125–312-Csd2_121–308 complex (heterodimer I) (bottom left) are shown in ribbon diagrams; they are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164243#pone.0164243.g002" target="_blank">Fig 2A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164243#pone.0164243.s001" target="_blank">S1 Fig</a>, respectively. Electrostatic surface diagrams of Csd2_121–308 chain A’ of Csd2 homodimer and Csd1_125–312 chain C of the Csd1_125–312-Csd2_121–308 heterodimer are shown on the right. Highly negatively-charged surfaces surround the hydrophobic interface of the Csd2_121–308 homodimer, whereas largely positively-charged surfaces surround the hydrophobic interface of Csd1_125–312 of the Csd1_125–312-Csd2_121–308 heterodimer.</p

Overall monomer structure of <i>H</i>. <i>pylori</i> Csd1_125–312.

<p>(A) Schematic representation of secondary structures of Csd1_125–312 and topology diagram of Csd1_125–312. Secondary structures have been defined by the STRIDE program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164243#pone.0164243.ref042" target="_blank">42</a>]. 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 Csd1_125–312 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 2mF_o − DF_c map (grey colored mesh) are shown at the 1.0 <i>σ</i> level.</p

Structure and Stability of the Dimeric Triosephosphate Isomerase from the Thermophilic Archaeon <i>Thermoplasma acidophilum</i>

<div><p><i>Thermoplasma acidophilum</i> is a thermophilic archaeon that uses both non-phosphorylative Entner-Doudoroff (ED) pathway and Embden-Meyerhof-Parnas (EMP) pathway for glucose degradation. While triosephosphate isomerase (TPI), a well-known glycolytic enzyme, is not involved in the ED pathway in <i>T</i>. <i>acidophilum</i>, it has been considered to play an important role in the EMP pathway. Here, we report crystal structures of apo- and glycerol-3-phosphate-bound TPI from <i>T</i>. <i>acidophilum</i> (TaTPI). TaTPI adopts the canonical TIM-barrel fold with eight α-helices and parallel eight β-strands. Although TaTPI shares ~30% sequence identity to other TPIs from thermophilic species that adopt tetrameric conformation for enzymatic activity in their harsh physiological environments, TaTPI exists as a dimer in solution. We confirmed the dimeric conformation of TaTPI by analytical ultracentrifugation and size-exclusion chromatography. Helix 5 as well as helix 4 of thermostable tetrameric TPIs have been known to play crucial roles in oligomerization, forming a hydrophobic interface. However, TaTPI contains unique charged-amino acid residues in the helix 5 and adopts dimer conformation. TaTPI exhibits the apparent T_d value of 74.6°C and maintains its overall structure with some changes in the secondary structure contents at extremely acidic conditions (pH 1–2). Based on our structural and biophysical analyses of TaTPI, more compact structure of the protomer with reduced length of loops and certain patches on the surface could account for the robust nature of <i>Thermoplasma acidophilum</i> TPI.</p></div

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.

<p>(A) In the structure of Csd1-Csd2 dimer I, the C-terminal residues (His299<i>−</i>Ala304) of Csd2_121–308 from an adjacent asymmetric unit (chain B’ shown in ribbon diagram) occupy the substrate-binding groove of the LytM domain in Csd1_125–312 (chain C shown in surface diagram). Four loops of Csd1 LytM domain that form the substrate-binding groove are labeled and colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164243#pone.0164243.g005" target="_blank">Fig 5</a>. The ribbon diagram is colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164243#pone.0164243.s001" target="_blank">S1 Fig</a> 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164243#pone.0164243.ref047" target="_blank">47</a>] (lime colored mesh) and 2mF_o − DF_c map (magenta colored mesh) are shown at the 1.0 <i>σ</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164243#pone.0164243.s001" target="_blank">S1 Fig</a>). 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 Csd1_125–312 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 <i>H</i>. <i>pylori</i> Csd1 (skyblue), <i>H</i>. <i>pylori</i> Csd3 (light green; PDB code, 4RNY), and <i>S</i>. <i>aureus</i> LytM bound with tetraglycine phosphinate (purple; PDB code, 4ZYB) shows that the two water molecules (Wat1 and Wat2) of Csd1_125–312 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 <i>H</i>. <i>pylori</i> Csd1, <i>H</i>. <i>pylori</i> Csd3, and <i>S</i>. <i>aureus</i> LytM, respectively. Two dotted lines represent the disordered regions in Loop 1 of Csd1. The metal-coordinating residues in the <b>H(169)</b>xxx<b>D(173)</b> and Hx<b>H(252)</b> motifs and the conserved catalytic residues in the <b>H(250)</b>xH motif and an additional catalytic histidine residue <b>H(219)</b> of Csd1, as well as corresponding residues of <i>H</i>. <i>pylori</i> Csd3 and <i>S</i>. <i>aureus</i> LytM, are shown in a stick model. Tyr204 (labeled in red) of <i>S</i>. <i>aureus</i> LytM is shown in a stick model. (D) Sequence alignment of LytM domains in Csd1, Csd2, and Csd3 from <i>H</i>. <i>pylori</i> 26695 strain [Csd1 (HP_Csd1; SWISS-PROT accession code O26068), Csd2 (HP_Csd2; O26069), and Csd3 (HP_Csd3; O25247)], <i>S</i>. <i>aureus</i> LytM (SA_LytM; O33599), and <i>S</i>. <i>simulans</i> lysostaphin (SS_LytM; P10547). Tyr204 of <i>S</i>. <i>aureus</i> LytM is marked by a red star. Conserved residues of the characteristic motifs are colored in blue.</p

Analysis of the complex formation between <i>H</i>. <i>pylori</i> Csd1 and Csd2.

<p>(A) SDS-PAGE analysis of the Csd1 and Csd2 complex formation. Lane M: pre-stained protein ladder. Lanes 1 and 2 for Csd1_54-312-Csd2_121-308 complex: pooled fractions either unbound (lane 1) or bound (lane 2) to the affinity chromatography. Lane 3 (unbound) and lane 4 (bound) for Csd1_75-312-Csd2_121-308 complex. Lane 5 (unbound) and lane 6 (bound) for Csd1_91-312-Csd2_121-308 complex. Lane 7 (unbound) and lane 8 (bound) for Csd1_125-312-Csd2_121-308 complex. Lane 9: the peak fraction of the Csd1_125-312-Csd2_121-308 complex after size exclusion chromatography. This complex was crystallized for structure determination. (B) SEC-MALS data for the Csd1_54–312-Csd2_121–308 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.</p

<i>H</i>. <i>pylori</i> Csd2_121–308 exists as homodimer in the crystal.

<p>(A) Two different views of the <i>H</i>. <i>pylori</i> Csd2_121–308 homodimer structure are shown in ribbon diagram. One Csd2_121–308 monomer (chain A colored in yellow-green) and the other Csd2_121–308 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 Csd2_121–308 monomers. One Csd2_121–308 monomer (chain A) is shown as a ribbon diagram (in yellow-green) and the other Csd2_121–308 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.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p