The docking energy between Al-CPI and various enzymes.

a)<p>E_RDock, the RDOCK score is defined as: E_elec2+beta×E_sol. E_elec2: the electrostatic energy of the protein complex after the first and second CHARMm minimizations. E_sol: the desolvation energy of the protein complex calculated by the ACE method.</p

Structure of Al-CPI and structural comparison of Al-CPI with CEW cystatin and human cystatin C.

<p>(A) Monomer structure of Al-CPI. (B) Structural comparison of Al-CPI (green), CEW cystatin (cyan), and cystatin C (magenta) around the α-helix core. (C) Structural comparison of Al-CPI (green), CEW cystatin (cyan) and cystatin C (magenta) around the active site segment. (D) Sequence alignment of five type-2 cystatins from <i>A. lumbricoides</i>, chicken egg white (CEW) and soft tick salivary gland (T.S. cystatin) and human cystatin C and D. The amino acid residues partially conserved among the five cystatins are highlighted in blue, and fully conserved are highlighted in red. The amino acid residues indexed with red arrow were selected for distance measurement as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096069#pone-0096069-t002" target="_blank">Table 2</a>.</p

Comparison of Al-CPI with other monomeric type-2 cystatins^a).

a)<p>The Cα of conserved amino acid residues were chosen for all the distance measurements.</p

Structural basis of differential inhibition activity of Al-CPI to cathepsin L (Cat. L) and cathepsin B (Cat. B).

<p>(A) Molecular docking was performed for Al-CPI and Cat. L. (B) Docking of AL-CPI against Cat. B suggested the insert segment of Cat. B would crash with the L2 of Al-CPI. (C) The sequence alignment of four cathepsins showing the unique insert segment of Cat. B.</p

Docking analysis of Al-CPI with cathepsin L.

<p>(A) Docking between Al-CPI and cathepsin L. (B) 90° rotation around the x-axis from the view of (A). (C–E) The detailed analysis of interactions between cathepsin L and Al-CPI N-terminal (C), loop 1 (D) and loop 2 (E). Inhibitor (Al-CPI) residues are indicated with an “i” after the sequence number to distinguish them from those of the enzyme (cathepsin).</p

Data collection and refinement statistics.

<p>The values in parentheses refer to statistics in the highest bin.</p>a)<p>R_merge = ∑_hkl∑_i|I_i(hkl)- | / ∑_hkl∑_iI_i(hkl), where I_i(hkl) is the intensity of an observation and is the mean value for its unique reflection; summations are over all reflections.</p>b)<p>R-factor  = ∑_h|Fo(h)-Fc(h)|/∑_hFo(h), where Fo and Fc are the observed and calculated structure-factor amplitudes, respectively.</p>c)<p>R_free was calculated with 5% of the data excluded from the refinement.</p>d)<p>Root-mean square-deviation from ideal values.</p>e)<p>Categories were defined by MolProbity.</p

Protease inhibition of wild-type Al-CPI and the mutants.

<p>IC₅₀ values of wild type Al-CPI and five mutated forms to cathepsin B, C, L and S were shown. The inhibition activities of Al-CPI and its mutants were analyzed as described in Materials and.</p

Inhibition activities against cathepsin B, C, L and S by recombinant cysteine protease inhibitor <i>Ascaris lumbricoides</i> (Al-CPI).

<p>Cathepsins were incubated with the fluorogenic substrates in the absence or presence of serially diluted Al-CPI in appropriate buffer for 15 min. and reaction was stopped with stopping buffer. The amount of product was measured fluorometrically with excitation at 360 nm and emission at 460 nm. The inhibitory activity of Al-CPI was expressed as a percentage of the total activity detected in reactions without Al-CPI. The half maximal inhibitory concentration (IC₅₀) values of Al-CPI based on initial reaction velocities were determined by nonlinear regression analysis and are shown on each plot. Data shown are from one of three experiments.</p