Comparison of the catalytic site in open and closed structures of prolyl oligopeptidase family enzymes.

<p>A: Detailed view of the active site of TbOPB in the open and closed state. Open and closed states are superposed to clearly show the localised structural changes that occur upon substrate binding/domain closure. Open state residues are coloured grey, closed state residues are coloured green, and the bound Antipain is coloured magenta. In the open state, the propeller domain Glu172 is a long distance from the active site residues. Arg650 is in a catalytically incompetent conformation in which it binds to the catalytic triad Asp648, displacing the catalytic triad His683 and the His loop from the active site, rupturing the catalytic triad and inactivating the enzyme. Upon domain closure, Glu172 is brought into the active site, where it pulls Arg650 into the catalytically competent position, away from the catalytic triad Asp648, which allows His683 and the His loop to swing into the active site, where His683 can complete the catalytic triad and activate the enzyme. B: ApPREP open (PDB code 3IUN) and closed (PDB code 3IVM) structures. C: ApAAP open (PDB code 3O4J) and closed (PDB code 2HU8) structures. Open and closed states are coloured, superposed, and shown in the same orientation as (A) to facilitate comparison between the different PREP family enzymes. Very similar global and local conformational changes are apparent in these structures, supporting a conserved mechanism of catalytic regulation in different PREP family enzymes. Residues in the open and closed structures are labeled in italic and bold type, respectively.</p

The TbOPB structure.

<p>A: Cartoon of the TbOPB open state dimer. The large distance between the α/β-hydrolase and propeller domains is apparent. B: Cartoon of the TbOPB-Antipain closed state dimer. The α/β-hydrolase and β-propeller domains are in much closer contact, effectively packed against each other. The α/β-hydrolase domains are in the same positions in (A) and (B) to facilitate easy visualization of the structural changes. The protein chains are coloured blue to red from the N to C termini. C, D: Comparison of the open (C) and closed (D) states of the enzyme. A single protein chain of each dimer is shown for clarity. Functionally important residues and loops that undergo localised structural changes between the open and closed states are coloured green, and the bound Antipain is coloured magenta.</p

PISA analysis of the peptidase and propeller domain interface in the open and closed states of TbOPB, ApPREP and ApAAP.

<p>PDB codes for the analyses are shown. These are the same as those used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079349#pone-0079349-g003" target="_blank">Fig. 3</a>.</p

Data collection, phasing and refinement statistics.

<p>Numbers in parentheses refer to values in the highest resolution shell.</p>a<p><i>R</i>_sym = Σ_jΣ_h|<i>I</i>_h,j−<<i>I</i>_h>|/Σ_jΣ_h<<i>I</i>_h> where <i>I</i>_h,j is the jth observation of reflection h, and <<i>I</i>_h> is the mean intensity of that reflection.</p>b<p><i>R</i>_cryst = Σ||<i>F</i>_obs|−|<i>F</i>_calc||/Σ|<i>F</i>_obs| where <i>F</i>_obs and <i>F</i>_calc are the observed and calculated structure factor amplitudes, respectively.</p>c<p><i>R</i>_free is equivalent to <i>R</i>_cryst for a 4% subset of reflections not used in the refinement.</p

The catalytic site of TbOPB.

<p>A: Electron density of the active site of the TbOPB-Antipain closed state complex, contoured at the 0.8 σ level, where σ represents the RMS electron density for the unit cell. Contours more than 1.4 Å from any of the displayed atoms have been removed for clarity. TbOPB carbon atoms are coloured green, and Antipain carbon atoms are coloured magenta. B: Detailed view of the active site of the TbOPB-Antipain closed state complex. The catalytic triad Asp648, His683, and Ser563 form a charge relay. Ser563 forms a covalent hemiacetal transition state analogue complex with Antipain. The oxyanion is stabilized by Ala564 and Tyr482. The P1 Arg is bound to Glu607, Glu655, Arg650, and has a π-stacking interaction with Phe589. The P2 carbonyl is hydrogen bonded to Arg650. The LmOPB-antipain structure (PDB code 2XE4) determined previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079349#pone.0079349-McLuskey1" target="_blank">[19]</a> is superimposed and is shown in line representation. The P3 Arg interacts with Ser523 and Leu617, whilst in TbOPB interactions are with Asp214 and Lys208 of the propeller domain. The P4 Phe does not make any specific interactions with enzyme residues in either TbOPB or LmOPB. TbOPB and LmOPB residues are labeled in bold and italic type, respectively.</p

Expression of the TGF-β signaling machinery in lungs of neonatal and adult rats.

<p>Changes in the expression of genes encoding components of the TGF-β signaling machinery as assessed by real-time RT-PCR at days P1 and P70; n = 8–15 for each bar. The significance for each bar is indicated by p values, IUGR vs. CO; two-tailed Mann-Whitney test. A: Expression of genes encoding the TGF-β receptors <i>tgfbr1</i>, <i>tgfbr2</i> and <i>tgfbr3</i> at P1 (white bar) and P70 (striped bar). B: Expression of genes encoding the regulatory <i>smad2</i>, <i>smad3</i> and <i>smad4</i> at days P1 (white bar) and P70 (striped bar). C: Expression of genes encoding the inhibitory <i>smad7</i>, <i>smurf2</i> and smad anchor for receptor activation (<i>sara</i>) at days P1 (white bar) and P70 (striped bar).</p

Body weight, respiration and physiological lung parameters of IUGR rats.

<p>A: Body weight (g) and relative lung weight (lung weight/body weight) of rats after IUGR (white bars) and control rats (black bars) at days P1 and P70; n = 8–15 for each bar. B: Architectural changes in lung structure were evident in hematoxylin and eosin-stained lung sections from IUGR rats and control rats at day P70. Measurement of septal thickness (µm) and mean linear intercept (MLI; µm) in IUGR rats and control rats (CO) at day P70; n = 6–10 for each bar. C: Assessment of respiratory system compliance by whole body plethysmography in IUGR rats (white bars) and in the control group (CO; black bars) at P70. n = 15–17 for each bar. D: Expression pattern of genes encoding surfactant protein A (SP-A), SP-C, and SPD. IUGR rats (white bars) and control group (black bars). n = 6–15 for each bar. The significance for each bar is indicated by p values, IUGR vs. Co, n.s. = not significant; two-tailed Mann-Whitney test.</p

Effect of IUGR on the expression of extracellular matrix (ECM) proteins and modulators of the ECM in neonatal rat lungs.

<p>Expression of TGF-β-regulated genes encoding elastin (Eln), tenascin N (Ten), collagens (Coll) I, Coll III, and Fibrillin (A), and genes of ECM modulators including matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 (B) in lungs extracted at day P1 from neonatal rats after IUGR and control rats. The mRNA expression, illustrated as relative fold induction, was assessed by real-time PCR. The control group was normalized to 1 as indicated by a scattered line; n = 15 for each group. The significance for each bar is indicated by p values, IUGR vs. Co; two-tailed Mann-Whitney test.</p

Effect of IUGR on the expression of TGF-β1 in lungs of neonatal and adult rats.

<p>A: Expression of genes encoding TGF-β1 and TGF-β-inducible plasminogen activator inhibitor-1 (PAI-1) during late lung development (day P1). The mRNA expression was assessed by quantitative real-time PCR. The control group was normalized to 1 as indicated by a scattered line; n = 15 for each group. The significance for each bar is indicated by p values, IUGR vs. Co; two-tailed Mann-Whitney test. B: representative immunoblots illustrating expression of TGF-β1 in lungs extracted at day P1 and P70 from rats with and without IUGR. β-actin served as loading control. Immunoblot data were quantified for both day P1 and P70; n = 6 for each bar. The significance for each bar is indicated by p values, IUGR vs. Co; two-tailed Mann-Whitney test.</p

Primer pairs and TaqMan probes used.

<p>*TIMP-1, TIMP-2 and MMP-2 were detected by TaqMan realtime-PCR analysis.</p