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

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

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

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

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

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