Homodimeric PAD4 and residues located at the dimer interface.

<p>Homodimer of human PAD4 (PDB code: 1WDA, <b>Panel A</b>). The active site and calcium-binding site are indicated. Five calcium ions (Ca1–Ca5) are indicated as yellow balls. The substrate analog, benzoyl-L-arginine amide (BAG), is shown as a stick model. Panels B and C display the amino acid residues in the dimer interface of PAD4, which are represented by ball-and-stick models. <b>Panel B</b>: Arg8(A) is ion-paired with Asp547(B), and Asp273(A) is ion-paired with Arg544(B). <b>Panel C</b>: The hydrogen-bonding network formed by Tyr435(B), Tyr237(A) and Glu281(A). The figures were generated with PYMOL (DeLano Scientific LLC, San Carlos, CA, USA).</p

Continuous sedimentation coefficient distributions of the WT PAD4 and dimer interface mutants in the presence of calcium ions.

<p>The enzyme concentrations were 0.1, 0.3 and 1.0 mg/mL in 50 mM Tris-HCl and 250 mM NaCl (pH 7.6) at 20°C. The concentration of the calcium ion was 10 mM. (<b>A</b>) WT; (<b>B</b>) WT with Ca²⁺; (<b>C</b>) R8K mutant; (<b>D</b>) R8K with Ca²⁺; (<b>E</b>) Y435N mutant; (<b>F</b>) Y435N with Ca²⁺; (<b>G</b>) R8E mutant; (<b>H</b>) R8E with Ca²⁺.</p

Multiple sequence alignments of the PAD family.

<p>The amino acid sequences of PADs were identified using the BLAST <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021314#pone.0021314-Altschul1" target="_blank">[48]</a>, and the alignments were generated with ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021314#pone.0021314-Higgins1" target="_blank">[49]</a>. <b>Panel A</b>: Multiple sequence alignments of 27 isoforms of peptidylarginine deiminase. This figure was generated using the BioEdit sequence alignment editor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021314#pone.0021314-Hall1" target="_blank">[50]</a>. <b>Panel B</b>: Sequence conservation with error bars for the dimer interface residues. This figure shows the frequency of conservation for the respective amino acid residues at a given position <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021314#pone.0021314-Crooks1" target="_blank">[51]</a>.</p

Continuous sedimentation coefficient distributions of the PAD4 interface mutants in monomer-dimer equilibriums.

<p>The enzyme concentrations used in the experiments were 0.1, 0.3 and 1 mg/mL in 50 mM Tris-HCl and 250 mM NaCl (pH 7.6) at 20°C. (<b>A</b>) WT; (<b>B</b>) R8A mutant; (<b>C</b>) R8K mutant; (<b>D</b>) R8Q mutant; (<b>E</b>) D547A mutant; (<b>F</b>) D547E mutant; (<b>G</b>) D547N mutant; (<b>H</b>) R8A/D547A double mutant.</p

Continuous sedimentation coefficient distributions of the dimeric PAD4 WT and interface mutants.

<p>The enzyme concentrations used in the experiments were 0.1, 0.3 and 1 mg/mL in 50 mM Tris-HCl and 250 mM NaCl (pH 7.6) at 20°C. (<b>A</b>) WT; (<b>B</b>) Y237A mutant; (<b>C</b>) E281A mutant; (<b>D</b>) Y237A/E281A double mutant; (<b>E</b>) D273A/R544A double mutant; (<b>F</b>) R8H mutant.</p

Continuous sedimentation coefficient distributions of the monomeric PAD4 interface mutants.

<p>The enzyme concentrations used in the experiments were 0.1, 0.3 and 1 mg/mL in 50 mM Tris-HCl and 250 mM NaCl (pH 7.6) at 20°C. (<b>A</b>) WT; (<b>B</b>) R8L mutant; (<b>C</b>) R8E mutant; (<b>D</b>) R8E/D547E double mutant; (<b>E</b>) Y435A mutant; (<b>F</b>) Y435N mutant.</p

Correlation plots for the dissociation constant (<i>K</i>_d), Hill coefficient (<i>h</i>), and catalytic constant (<i>k</i>_cat).

<p>The data points were derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021314#pone-0021314-t001" target="_blank">Table 1</a>. Closed circle: dimeric PAD4 (WT, R8H, Y237A, E281A, Y237A/E281A, and D273A/R544A); open circle: PAD4 in dimer-monomer equlibrium (R8A, R8K, R8Q, D547A, D547E, D547N, and R8A/D547A); closed triangle: monomeric PAD4 (R8E, R8L, R8E/D547E, Y435A, and Y435N). <b>Panel A: </b><i>K</i>_d versus <i>h</i>. <b>Panel B: </b><i>K</i>_d versus <i>k</i>_cat. <b>Panel C: </b><i>k</i>_cat versus <i>h</i>.</p

Kinetic Parameters of the Human WT and Mutant PAD4s.

a<p><i>K</i>_m,BAEE: Michaelis constant for benzoyl-L-arginine ethyl ester (BAEE) as the <i>in vitro</i> substrate for PAD4.</p>b<p><i>K</i>_0.5,Ca: half-saturation constant for Ca²⁺.</p>c<p><i>k</i>_cat: catalytic constant.</p>d<p><i>h</i>: the Hill coefficient.</p>e<p>M: monomer; D: dimer.</p>f<p><i>K</i>_d: dissociation constant between the monomer and dimer without Ca²⁺; <i>K</i>_d,Ca: dissociation constant between the monomer and dimer with 10 mM Ca²⁺.</p>g<p>ND: not determined.</p

Rescue of AZ-mediated inhibition of the activity of ODC by wild-type and mutant AZI.

<p>ODC (20 µg/mL) was preincubated with AZ (30 µg/mL) and then treated with various concentrations of AZI. Open circles: AZI_WT; closed circles: mutant AZI. (<b>A</b>) AZI_K125N. (<b>B</b>) AZI_ K140M. (<b>C</b>) AZI_K125N/K140M. The molar ratio of AZ monomer versus ODC monomer was fixed at 3.5.</p

Sequence alignment and structures of ODC and AZI.

<p>(<b>A</b>) Pairwise sequence alignment between ODC and AZI in the putative AZ-binding element. (<b>B</b>) Structure of human ODC monomer (PDB code: 1D7K). (<b>C</b>) Structure of mouse AZI monomer (PDB code: 3BTN). The putative AZ-binding site in ODC is colored in deep green and that in AZI is colored in hot pink. This figure was generated with PyMOL (DeLano Scientific LLC, San Carlos, CA, USA).</p