Structural basis for interaction of O-acetylserine sulfhydrylase and serine acetyltransferase in the Arabidopsis cysteine synthase complex

Abstract

In plants, association of O-acetylserine sulfhydrylase (OASS) and Ser acetyltransferase (SAT) into the Cys synthase complex plays a regulatory role in sulfur assimilation and Cys biosynthesis. We determined the crystal structure of Arabidopsis thaliana OASS (At-OASS) bound with a peptide corresponding to the C-terminal 10 residues of Arabidopsis SAT (C10 peptide) at 2.9-A resolution. Hydrogen bonding interactions with key active site residues (Thr-74, Ser-75, and Gln-147) lock the C10 peptide in the binding site. C10 peptide binding blocks access to OASS catalytic residues, explaining how complex formation downregulates OASS activity. Comparison with bacterial OASS suggests that structural plasticity in the active site allows binding of SAT C termini with dissimilar sequences at structurally similar OASS active sites. Calorimetric analysis of the effect of active site mutations (T74S, S75A, S75T, and Q147A) demonstrates that these residues are important for C10 peptide binding and that changes at these positions disrupt communication between active sites in the homodimeric enzyme. We also demonstrate that the C-terminal Ile of the C10 peptide is required for molecular recognition by At-OASS. These results provide new insights into the molecular mechanism underlying formation of the Cys synthase complex and provide a structural basis for the biochemical regulation of Cys biosynthesis in plants.

Figure 1.

Regulation of Cys Synthesis by Formation of the Cys Synthase Complex. Active forms of OASS and SAT are indicated by red text; inactive (or less active) forms of either enzyme are indicated by black text. Modified from Hell and Hillebrand (2001). CS, Cys synthase.

Figure 2.

Overview of the At-OASS·C10 Peptide Structure. (A) Comparison of the C-terminal sequences of SAT from Arabidopsis (At-SAT), Glycine max (soybean) (Gm-SAT), Escherichia coli (Ec-SAT), and H. influenzae (Hi-SAT). Portions of the At-SAT and Hi-SAT peptides observed crystallographically in structures of At-OASS and Hi-OASS, respectively, are highlighted in red. (B) Ribbon diagram of the At-OASS dimer. The monomers of the dimer are colored in light purple and white. The location of the active site is defined by the Schiff base formed between PLP and Lys-46 (yellow). C10 peptides (colored green or rose in each monomer) are bound in the two active sites of the At-OASS homodimer. (C) Electron density of the C10 peptide is shown as a 2F_o-F_c omit map contoured at 1.2σ. Eight (TEWSDYVI) of 10 residues in the peptide were observable. (D) Stereo view of the molecular surface of At-OASS forming the peptide binding site. The C10 peptide (rose) is shown as a stick drawing. The surface corresponding to the active site PLP is colored yellow. The surface of residues previously implicated in interaction between At-OASS and At-SAT are shown in green.

Figure 3.

The C10 Peptide Binding Site. (A) Stereo view of interactions in the C10 peptide binding site. All modeled residues of the C10 peptide are shown and labeled (rose). Sidechains of At-OASS residues that interact with the C10 peptide are colored yellow. Residues providing peptide backbone contacts are drawn in white without sidechains. Water molecules are shown as red spheres. Dotted lines indicate hydrogen bonds. (B) Schematic drawing of interactions between the C10 peptide (blue) and At-OASS (black). Only the five C-terminal residues of the peptide are shown in the scheme. Hydrogen bonds are shown as dotted lines with distances in angstroms. W, water molecule (red).

Figure 4.

Comparisons of the At-OASS·C10 Peptide Complex. (A) Stereo view of the structural overlay of the At-OASS·C10 peptide complex (rose) and the At-OASS K46A mutant structure (green). The last six residues of the C10 peptide and three key active site residues are drawn as sticks. In the K46A mutant structure, Met is covalently linked to the PLP to form an external aldimine in the active site. (B) Stereo view of the structural overlay of the At-OASS·C10 peptide complex (rose) and the Hi-OASS·peptide complex (green). Amino acid sidechains and residues of each peptide are shown in ball-and-stick representation.

Figure 5.

Calorimetric Titrations of At-OASS with C10 Peptide. (A) Titration of At-OASS with C10 peptide. ITC data are plotted as heat signal (μcal/s) versus time (min). The experiment consisted of 20 injections of 12 μL each of C10 peptide (23.7 μM) into a solution containing At-OASS (1.5 μM) at 25°C. (B) Integrated heat responses per injection from (A), plotted as normalized heat per mole of injectant. The solid line represents the best fit to a two-site binding model. (C) Titration of At-OASS mutants with C10 peptide. The integrated heat responses are plotted as normalized heat per mole of injectant for the T74S (open circles), S75A (closed squares), S75T (open squares), and Q147A (closed circles) At-OASS mutants. The solid line for each mutant represents the best fit to a one-site binding model.

Figure 6.

Comparison of C10 Peptide and Mutant Peptide Interactions with At-OASS. The fluorescence emission signals of At-OASS (1 μM) without peptide, At-OASS + 3 μM C10 peptide, and At-OASS + 3 μM mutant peptide are shown as solid black, dashed black, and solid gray lines, respectively. a.u., arbitrary units.