Calcium binding in <i>S. pyogenes</i> Csn2 structure.

<p>The CA1 (<b>A</b>) and CA2 (<b>B</b>) sites are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033401#pone-0033401-g002" target="_blank">Figure 2B</a>, and coordinating oxygen atoms are shown in red. Among the two CA1 sites, the one adjacent to monomer B, which has lower B factors, is shown. The missing water molecule in the CA1 site, represented as a blue sphere, is modeled based on a comparison with the other CA1 site. Asp122 is shown in its calcium-binding conformation. The difference electron density map for the calcium ions was contoured at 10<i>σ</i>. The distances between the calcium ions and the coordinating atoms are also indicated.</p

Comparison of <i>S. pyogenes</i> Csn2 structure with the metal identified as calcium, sodium, potassium or water.

<p>Values in parentheses are average distances observed in medium-resolution (2.0 to 2.5 Å) structures in the Protein Data Bank <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033401#pone.0033401-Zheng1" target="_blank">[9]</a>.</p

Crystal Structure of <em>Streptococcus pyogenes</em> Csn2 Reveals Calcium-Dependent Conformational Changes in Its Tertiary and Quaternary Structure

<div><p>Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute a microbial immune system against invading genetic elements, such as plasmids and phages. Csn2 is an Nmeni subtype-specific Cas protein, and was suggested to function in the adaptation process, during which parts of foreign nucleic acids are integrated into the host microbial genome to enable immunity against future invasion. Here, we report a 2.2 Å crystal structure of <em>Streptococcus pyogenes</em> Csn2. The structure revealed previously unseen calcium-dependent conformational changes in its tertiary and quaternary structure. This supports the proposed double-stranded DNA-binding function of <em>S. pyogenes</em> Csn2.</p> </div

Tetrameric structure of <i>S. pyogenes</i> Csn2.

<p><b>A</b>: Tetrameric arrangement of <i>S. pyogenes</i> Csn2. Monomer A is colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033401#pone-0033401-g002" target="_blank">Figure 2B</a>, and monomer B is shown in pink. The tetramer is viewed along the two-fold symmetry axis. The two <i>S. pyogenes</i> Csn2 monomers found in the asymmetric unit are enclosed by the black dashed line. <b>B</b>: Analytical size-exclusion chromatography of <i>S. pyogenes</i> Csn2. Elution profiles with different buffer conditions are represented by different colors. Elution volumes for molecular weight standards are also indicated. <b>C</b>: Electrostatic potential surface (red = −25 kT, blue = +25 kT) of the <i>S. pyogenes</i> Csn2 tetramer. Pymol (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>) was used to calculate APBS electrostatics including the bound calcium ions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033401#pone.0033401-Baker1" target="_blank">[27]</a>.</p

Double-stranded DNA binding of <i>S. pyogenes</i> Csn2.

<p><b>A</b>: Sequences of <i>S. pyogenes</i> CRISPR and control DNA fragments used for an electrophoretic mobility shift assay. The repeat and the first spacer of <i>S. pyogenes</i> CRISPR are shown in red and green, respectively. The control DNA fragment contains the promoter site of the <i>Early Responsive to Dehydration Stress 1</i> gene from <i>A. thaliana</i>. <b>B</b>: An electrophoretic mobility shift assay was performed with 150 ng of dsDNA (90 bp) and increasing concentrations of <i>S. pyogenes</i> Csn2. The molar ratio of DNA to <i>S. pyogenes</i> Csn2 tetramer is indicated for each lane.</p

Sequence alignment and monomeric structure of <i>S. pyogenes</i> Csn2.

<p><b>A</b>: Sequence alignment of Csn2 homologues from <i>S. pyogenes</i>, <i>E. faecalis</i> and <i>S. thermophilus</i>. Secondary structure elements are indicated based on the <i>S. pyogenes</i> Csn2 structure. The calcium coordinating residues within the CA1 and CA2 sites are marked with orange and purple triangles, respectively. <b>B</b>: Structure of <i>S. pyogenes</i> Csn2 monomer A. The globular α/β domain, the extended α-helical domain and the hinge regions are shown in green, cyan and yellow, respectively. Secondary structure elements are also indicated. Bound calcium ions in the CA1 and CA2 sites are represented as orange and purple spheres, respectively.</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

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

TPI stabilization patches (TSPs).

<p>(A) Structure-based sequence alignment of TaTPI with other TPIs from <i>Pyrococcus woesei</i> (PwTPI), <i>Methanocaldococcus jannaschii</i> (MjTPI), <i>Thermoproteus tenax</i> (TtTPI), <i>Homo sapiens</i> (HsTPI), <i>Gallus gallus</i> (GgTPI), <i>Thermotoga maritima</i> (TmTPI), and <i>Geobacillus stearothermophilus</i> (GsTPI). Strictly conserved amino acid residues are highlighted in red shaded boxes and moderately conserved amino acid residues are colored in red. Conserved residues are enclosed in blue boxes and TSP regions are enclosed in red boxes. The alignment figure was prepared using <i>ESPript</i> program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145331#pone.0145331.ref032" target="_blank">32</a>]. (B) Structural comparison of TaTPI and HsTPI. The red dotted and solid circles represent TSP regions in TaTPI and HsTPI, respectively.</p

Dimeric conformation of TaTPI.

<p>(A) Structure-based sequence alignment of helix 5 region among TPIs from <i>Thermoplasma acidophilum</i> (TaTPI), <i>Pyrococcus woesei</i> (PwTPI), <i>Methanocaldococcus jannaschii</i> (MjTPI), <i>Thermoproteus tenax</i> (TtTPI), <i>Homo sapiens</i> (HsTPI), <i>Gallus gallus</i> (GgTPI), <i>Thermotoga maritima</i> (TmTPI), and <i>Geobacillus stearothermophilus</i> (GsTPI). Strictly conserved amino acid residues are highlighted in red shaded boxes and moderately conserved amino acid residues are colored in red. Helix 5 regions of TPIs from <i>Thermoplasma acidophilum</i> (PDB ID: 5CSR) and <i>Pyrococcus woesei</i> (PDB ID: 1HG3) are shown as cartoon representation with transparent surfaces, where amino acid residues are colored according to normalized consensus hydrophobicity scale [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145331#pone.0145331.ref031" target="_blank">31</a>]. (B) and (C) Structural comparison of helix 4 and helix 6 in TaTPI (gray) and PwTPI (green and magenta, each from different monomers). The important amino acid residues in tetrameric interface are shown as stick model. (D) Analytical ultracentrifugation experiment of TaTPI. Sedimentation equilibrium distribution (circle) of TaTPI at 26,000 rpm and 20°C is plotted as circle. Concentration of TaTPI was 16.8 u<i>M</i> (0.41 mg ml^-1) in 20 m<i>M</i> Tris-HCl, pH 7.5, and 0.2 <i>M</i> NaCl. Solid line is a fitting line for a homogeneous dimer (2x) model and dotted lines are fitting lines for homogeneous monomer (1x) and tetramer (4x) models. Calculated molecular weight for TaTPI monomer from its amino acid compositions is 24,671 daltons. Inset graph shows distributions of the residuals for homogeneous 1x, 4x (solid lines), and 2x (circle) models, respectively. The random distribution of the residuals for the 2x model indicates that TaTPI exists as homogeneous dimers in solution.</p