Probing the Catalytic Roles of Arg548 and Gln552 in the Carboxyl Transferase Domain of the \u3cem\u3eRhizobium etli\u3c/em\u3e Pyruvate Carboxylase by Site-directed Mutagenesis

The roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase were investigated using site-directed mutagenesis. Mutation of Arg548 to alanine or glutamine resulted in the destabilization of the quaternary structure of the enzyme, suggesting that this residue has a structural role. Mutations R548K, Q552N, and Q552A resulted in a loss of the ability to catalyze pyruvate carboxylation, biotin-dependent decarboxylation of oxaloacetate, and the exchange of protons between pyruvate and water. These mutants retained the ability to catalyze reactions that occur at the active site of the biotin carboxylase domain, i.e., bicarbonate-dependent ATP cleavage and ADP phosphorylation by carbamoyl phosphate. The effects of oxamate on the catalysis in the biotin carboxylase domain by the R548K and Q552N mutants were similar to those on the catalysis of reactions by the wild-type enzyme. However, the presence of oxamate had no effect on the reactions catalyzed by the Q552A mutant. We propose that Arg548 and Gln552 facilitate the binding of pyruvate and the subsequent transfer of protons between pyruvate and biotin in the partial reaction catalyzed in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase

Ligand Recognition by the TPR Domain of the Import Factor Toc64 from <i>Arabidopsis thaliana</i>

<div><p>The specific targeting of protein to organelles is achieved by targeting signals being recognised by their cognate receptors. Cytosolic chaperones, bound to precursor proteins, are recognized by specific receptors of the import machinery enabling transport into the specific organelle. The aim of this study was to gain greater insight into the mode of recognition of the C-termini of Hsp70 and Hsp90 chaperones by the <u>T</u>etratrico<u>p</u>eptide <u>R</u>epeat (TPR) domain of the chloroplast import receptor Toc64 from <i>Arabidopsis thaliana</i> (<i>At</i>). The monomeric TPR domain binds with 1∶1 stoichiometry in similar micromolar affinity to both Hsp70 and Hsp90 as determined by isothermal titration calorimetry (ITC). Mutations of the terminal EEVD motif caused a profound decrease in affinity. Additionally, this study considered the contributions of residues upstream as alanine scanning experiments of these residues showed reduced binding affinity. Molecular dynamics simulations of the TPR domain helices upon peptide binding predicted that two helices within the TPR domain move backwards, exposing the cradle surface for interaction with the peptide. Our findings from ITC and molecular dynamics studies suggest that <i>At</i>Toc64_TPR does not discriminate between C-termini peptides of Hsp70 and Hsp90.</p></div

Biophysical characterization of <i>At</i>Toc64_TPR-H6 using size exclusion chromatography and circular dichroism.

<p><b>A.</b> Elution profile of <i>At</i>Toc64_TPR-H6 from size exclusion chromatography using a Superdex 75 prep grade column. The different shaped stars represent different forms of the protein as described in the figure. The Ve/Vo versus LogMW plot for superdex standards is shown as an inset. Numbers 1–4 represent different standard proteins used as described in the methods. Red triangle represents <i>At</i>Toc64_TPR-H6. <b>B.</b> Schematic representation of secondary structure of <i>At</i>Toc64_TPR. <b>C.</b> CD spectrum profile of the protein obtained after analysis with DICHROWEB.</p

Interactions occurring in the protein-peptide interface generated by Ligplot.

<p><b>A.</b> Interactions between the TPR domain and the C-terminal octapeptide of Hsp70; <b>B.</b> Interactions between the TPR domain and C-terminal octapeptide of Hsp90. The peptide is shown in purple bonds and the protein in brown bonds.</p

The carboxylate clamp.

<p><b>A.</b> Key interacting residues of the TPR domain with the carboxylate of Asp¹²⁴ of Hsp70 (shown in green bonds); <b>B.</b> Key interacting residues of the TPR domain with the carboxylate of Asp¹²⁴ of Hsp90 (shown in magenta bonds). The oxygen atoms of the carboxylate moieties are labeled. The TPR domain is shown with grey bonds.</p

Principal component analysis for the three systems.

<p>Essential dynamics -2D projection of individual trajectories with their first two eigen vectors: vector 1 and vector 2 (<b>A–C</b>) and their corresponding porcupine plots (<b>D–F</b>) for T_Apo (black), T_C70 (green) and T_C90 (magenta). Porcupine plots of the three systems displayed with a cone model. The length and orientation of the cone (red) is positively correlated with the magnitude and direction of motion.</p

Binding isotherms for the interaction of <i>At</i>Toc64_TPR-H6 with Hsp70 and Hsp90.

<p>The ITC isotherms obtained for the Hsp70 octapeptide binding to <i>At</i>Toc64_TPR-H6 (in green) and the Hsp90 octapeptide binding to <i>At</i>Toc64_TPR-H6 (in magenta). The bottom panels show the curves obtained for titration of the octapeptides of Hsp70/90 into TPR (triangles); the C-terminal pentapeptides of Hsp70/90 into TPR (diamonds) and random peptides into TPR (crosses).</p

Key intrapeptide interactions.

<p><b>A.</b> Intrapeptide interaction in the T_C70 system (coloured green) <b>B.</b> Intrapeptide interaction in the T_C90 system (coloured magenta). The TPR domain is shown as a ribbon diagram in grey.</p

The ITC binding data from alanine scanning mutagenesis of the peptide interaction with <i>At</i>Toc64_TPR-H6.

<p><b>A.</b> Data using the C-terminal octapeptide from Hsp70. <b>B.</b> Data using the C-terminal octapeptide of Hsp90. Asterik (*) suggests that the heat change during binding event is quite low and the signal to noise ratio is high. Hence the K_d is considered as no binding.</p

Computational Alanine Scanning for both ligand bound systems.

<p>Point mutations to alanine were performed computationally for each residue (in bold) of the Hsp70 and Hsp90 octapeptides. Changes in binding free energy (ΔΔG) in kcal/mol is calculated using MMGBSA.</p