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

ABA and related ABA analogs.

<p>Compounds are labeled accordingly, with (+)-PBI686 representing the photoactive, bioactive ABA-mimetic biotinylated probe used to pull-out putative ABA-binding proteins.</p

Predicted ABA binding to the spinach Rubisco active site.

<p>A) The Rubisco structure was previously solved in the presence of its two 3PGA product molecules (tan sticks with red oxygen atoms and orange phosphorus atoms), which may be used to define the protein’s active site (PDB ID: 1AA1). Small molecule docking of (+)-ABA (cyan sticks with red oxygen atoms) into B) the open, non-activated; C) closed, non-activated (PDB ID: 1RCX); and D) open, activated Rubisco active site, demonstrated the potential for competitive inhibition in a variety of enzyme states. In both non-activated enzyme forms, (+)-ABA was docked to the same location as the 3PGA molecule proximal to Mg2+ (PDB ID: 1AA1, residue 3PG 447) and was stabilized by hydrogen bonding to basic and acid residues (gray sticks) in Rubisco loops. In the activated enzyme form, (+)-ABA was docked to the region between the two 3PGA molecules, making ionic interactions with the Mg2+ cofactor (green spheres).</p

ABA is an inhibitor of spinach Rubisco.

<p>A) ABA is a weak non-competitive inhibitor of Rubisco catalytic (carboxylation) activity. Dixon plot showing effect of various concentrations of (+)-ABA on Rubisco catalytic activity in the presence of 10 μM (squares), 20 μM (diamonds), 50 μM (triangles), 100 μM (+), 200 μM (stars), 800 μM (X), 1200 μM (circle) RuBP substrate. The positioning of the intercept close to the x-axis suggests a non-competitive inhibition, with an estimated K_i of about 2.1 mM. B) ABA is a competitive inhibitor of Rubisco activation (carbamylation). Dixon plot showing effect of various concentrations of (+)-ABA on Rubisco activation in the presence of 1 mM (black diamonds), 7 mM (open squares) and 20 mM (closed triangles) of the activation substrate NaHCO₃. The intercept suggests at least partially competitive inhibition with an approximately K_i of about 130 μM.</p

ABA binds to spinach Rubisco with high affinity.

<p>A) Saturation curve and Scatchard plot representing specific binding of [³H]-(±)-ABA, as a difference between total and non-specific binding signal. B) Competition Binding Assay. Displacement of 25 nM (±) [³H]ABA by non-radiolabeled (+) ABA (diamonds), (-) ABA (circles), PA (squares) and trans-(+) ABA (triangle) at the indicated concentrations.</p

X-ray data collection and structure refinement statistics.

<p>Values in parenthesis are for the highest resolution shell.</p

Co-crystallography of RuBP-bound pea Rubisco with ABA.

<p>A) L-Subunit B showing (+)-ABA (in yellow) bound to a surface cleft adjacent to Y100. While distal from the RuBP (in green) binding site (~ 30 Å away), this site is in closer proximity to the positively charged regulatory latch region comprised of residues R41, R134, K305, H310, R312 and involves residues that immediately precede and follow the 90–97 loop region implicated in binding to Rubisco activase. B) The omit F_o-F_c electron density maps represented by the green hatching at ~ 2 sigma, were calculated without ABA included in the model. (+)-ABA (in yellow with red oxygens) is shown, bound to the A L-subunit. The terminal carboxylate is shown anchored to R139; the ketone forms a short H-bond with Y85; and the hydroxyl group at the chiral center forms a H-bond with K356. The hydroxyl group also has a weak 3.3 Å interaction with a water that is part of an H-bond and ionic matrix involving the side chains of Y100, E88, R358 and Y363.</p

Streptavidin-affinity-enriched PBI686-tagged protein extracts.

<p>Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.</p