Intra-receptor atoms correlation maps: (A) – in ligand-free PYR1 dimer; (B) – in PYR1-HAB1 complex; (C) – in 2ABA-bound PYR1 dimer; (D) – in PYR1-ABA-HAB1 complex.

<p>Strong correlations are represented by low values of the descriptor (green and blue colors), whereas high values indicate a more independent motion (magenta and white colors).</p

Normalized main-chain flexibility profile of PYR1 monomer bound to HAB1 (solid lines) over-imposed on B-factors of the starting crystallographic structure 3QN1 (dashed lines).

<p>Normalized main-chain flexibility profile of PYR1 monomer bound to HAB1 (solid lines) over-imposed on B-factors of the starting crystallographic structure 3QN1 (dashed lines).</p

Abscisic acid binding by the PYR1 ligand pocket induces gate-latch-locking.

<p>(A) – Structure of the apo-PYR1, gate open [PDB ID 3K3K, chain A]. (B) – Structure of ABA-bound PYR1, gate closed [PDB ID 3K3K, chain B]. The lock mechanism involves both direct and water/ions-mediated interactions of residues from gate (residues 85–89) and latch (residues 115–117), as well as hydrophobic interactions and hydrogen bonds throughout the binding pocket's surface. Residues which contribute to hydrogen binding in gate and latch are labeled and shown by orange sticks, while hydrophobic residues in the neighborhood of ABA (colored yellow) are shown by purple sticks. The allosteric rearrangement of gate and latch loops forms a surface for successful PP2C binding. Upon the binding, a conserved PP2C tryptophan 385 (not shown) is inserted between gate and latch and forms water-mediated hydrogen bond with ABA <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003114#pcbi.1003114-Nishimura1" target="_blank">[9]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003114#pcbi.1003114-Melcher1" target="_blank">[11]</a>.</p

Normalized main-chain flexibility profiles of PYR1 monomers

<p> (solid lines) over-imposed on B-factors of the corresponding starting crystallographic structures (chains A and B from PDB entry 3K3K <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003114#pcbi.1003114-Nishimura1" target="_blank">[9]</a>, dashed lines). In the plot, red color represents PYR1 in ABA-bound, closed-lid conformation and blue color represents ABA-free, open lid conformation.</p

Figure 7

<p> atoms correlation maps for PYR1-HAB1 binding areas: (A) – PYR1-ABA-HAB1 complex; (B) – PYR1-HAB1 complex, ABA extracted; (C) – recovered PYR1-ABA-HAB1 complex in which PYR1 was initially shifted against HAB1; (D) – recovered ABA-extracted PYR1-HAB1 complex in which PYR1 was initially shifted against HAB1. In the maps, lower levels of the correlation descriptor represent strong correlations (green and blue regions), and higher levels correspond a relatively uncorrelated motion (white and magenta regions).</p

The PYR1-ABA-HAB1 complex (PDB ID 3QN1) with residues on the binding surface shown by orange (PYR1) and green (HAB1) sticks.

<p>Polar interactions comprise residues H60–E323, K63–S200, K63–E201, I84–G246, S85–G246, G86–R389, S85–E203, P88–Q386, P88–R389, R116–W385, N151–Q384, and L166–E323. Non-polar interactions include residues F61–Y404, I84–G246, R116–Q386, L87–V393, L117–W385, P148–W385, D155–I383, M158–I383, M158–F391, F159–V393, F159–W385, F159–G392, T142–F391, and L166–Y404 <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003114#pcbi.1003114-Dupeux2" target="_blank">[17]</a>. These include both direct and water-mediated interactions. ABA is represented by a translucent surface, which is colored according to the charge distribution: red for positively, blue for negatively, and white for neutrally charged ABA atoms, respectively.</p

PYR1-dimer, 2ABA -bound (modified PDB ID 3NJO after ligand replacement, mutation S88P, minimizations and equilibrations in water) with residues on the binding surface indicated by orange sticks for chain A and green sticks for chain B.

<p>The direct and water-mediated interactions, detected by AccelrysVS employing the same criteria as for the PYR1-ABA-HAB1 complex in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003114#pcbi-1003114-g006" target="_blank">Figure 6</a>, comprise H60–L166, H60–T162, F61–F159, F61–L166, F61–F61, F61–T162, I62–M158, K63–D155, K63–E153, I84–F159, S85–D155, S85–D154, S85–F159, S85–T156, S85–E153, G86–P88, G86–L87, G86–F159, L87–P88, L87–L87, L87–F159, P88–P88, L166–L166 and all reciprocal <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003114#pcbi.1003114-Peterson1" target="_blank">[10]</a>. Ligand molecules in the binding pockets are depicted by surfaces colored according to the charge distribution as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003114#pcbi-1003114-g006" target="_blank">Figure 6</a>.</p

List of 3D PYR1 constructs taken from PDB and modeled <i>in silico</i>, which were used for molecular dynamics simulations and the ECD analysis.

<p>List of 3D PYR1 constructs taken from PDB and modeled <i>in silico</i>, which were used for molecular dynamics simulations and the ECD analysis.</p

Main-chain flexibility profiles of PYR1-ABA-bound closed lid (red line), PYR1 ABA-free closed lid (green line) and PYR1 ABA-free open lid (blue line) monomer constructs with standard deviations indicated by vertical lines.

<p>The simulations were performed at 300 K.</p

Isothermal titration calorimetry analysis of spinach Rubisco-ligand interactions.

<p>Raw ITC data of sequential injections of (A) RuBP (0.75 mM) and (B) (+)-ABA (5 mM) titrated against spinach Rubisco (39.42 μM). The processed fit is shown below each binding isotherm. The binding parameters are shown in the inset. The ligands were titrated until saturation was reached showing a specific binding interaction. The data were fitted using the ‘Independent Binding’ model in the NanoAnalyze software and the best fit is represented by the black line.</p