Backbone assignments and conformational dynamics in the S. typhimurium tryptophan synthase α-subunit from solution-state NMR

Abstract

Backbone assignments for the isolated α-subunit of Salmonella typhimurium tryptophan synthase (TS) are reported based on triple resonance solution-state NMR experiments on a uniformly ²H,¹³C,¹⁵N-labeled sample. From the backbone chemical shifts, secondary structure and random coil index order parameters (RCI-S²) are predicted. Titration with the 3-indole-D-glycerol 3'-phosphate analog, N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), leads to chemical shift perturbations indicative of conformational changes from which an estimate of the dissociation constant is obtained. Comparisons of the backbone chemical-shifts, RCI-S² values, and site-specific relaxation times with and without F9 reveal allosteric changes including modulation in secondary structures and loop rigidity induced upon ligand binding. A comparison is made to the X-ray crystal structure of the α-subunit in the full TS αββα bi-enzyme complex and to two new X-ray crystal structures of the isolated TS α-subunit reported in this work.

Keywords: Chemical shift assignments; Ligand titration; Protein NMR; Protein dynamics; Relaxation; Tryptophan synthase.

Fig. 1

(a) Ribbon model of the TS α-subunit from the full α₂β₂ bi-enzyme complex (PDB ID: 4HT3). The β-strands forming the TIM barrel are shown in blue. Loops α-L6 and α-L2 are shown in red and display the catalytically active “closed” confirmation. The N-(4’-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9) ligand bound in the active site is shown in green. (b) Close-up of the active site highlighting sidechains that interact with F9; hydrogen bonds are represented by blue lines. Figure prepared using Chimera (Pettersen et al. 2004)

Fig. 2

Two-dimensional ¹H-¹⁵N HSQC spectrum of U-²H-¹³C-¹⁵N αTS at 700 MHz, 298K, pH 6.5, and a protein concentration of 750 μM. The 191 labels indicate backbone amide ¹H and ¹⁵N resonances that could be sequentially assigned with probability > 99%. Peaks connected by thin lines (upper right) correspond to the asparagine and glutamine side chain -NH₂ groups; peaks marked with an asterisk (bottom right) are folded arginine side chain H^ε-N^ε peaks. The resonance for L127 (δ_1H = 8.66 ppm, δ_15N = 134.7 ppm) is not shown

Fig. 3

Assignment probabilities for each residue of the ligand-free αTS obtained using APES (Shin et al. 2006) and the I-PINE webserver (Bahrami et al. 2009; Lee et al. 2019). Color codes: green bars (> 99%); cyan bars (85% - 99%); yellow bars (50% - 85%); red bars (< 50%); gray bars (no assignment). Loop regions α-L2 and α-L6 are indicated

Fig. 4

(a) Difference between the Cα and Cβ secondary chemical shifts for αTS. (b) Secondary structure predictions for αTS from TALOS-N. The propensity of occurrence for α-helix is shown in red and that for β-sheet is shown in blue and inverted. The presence of loops is indicated by the simultaneous presence of helix and strand probabilities. The secondary structure elements from the X-ray crystal structure are indicated across the top (bars α-helices, arrows β-strands) and by the faint red (α-helix) and blue (β-strand) color shades

Fig. 5

RCI-S² values of ligand-free αTS (orange circles) compared to the S² values calculated from the MD trajectory (blue triangles). The secondary structure elements from the X-ray crystal structure are indicated across the top (bars α-helices, arrows β-strands).

Fig. 6

(a) Average chemical shift changes (∆δ_NH) calculated for residues of the isolated αTS at a protein concentration of 750 μM and [ligand]/[protein] ratio of 3. Asterisks have been placed over residues E49, A59, L100, L127, A129, I153, and Y175 and also residues F212, G234, and S235 that are seen to interact with F9 in the active site of αTS in the full TS complex and display themselves (or in their sequential neighborhood) relatively large CSPs upon addition of F9. The secondary structure elements from the X-ray crystal structure are indicated across the top (bars α-helices, arrows β-strands). (b) Mapping of the αTS ligand binding site onto the isolated αTS structure 6OUY from CSPs. The coordinates for the missing loop α-L6 and the F9 molecule (shown in green) have been extracted from the αTS structure of PDB entry 4HT3 after alignment of both PDB entries. Red indicates residues that show relatively large CSPs, white indicates residues with moderate CSPs, while blue indicates residues with low CSPs

Fig. 7

(a) Difference between RCI-S² values for the protein with and without ligand (ΔRCI-S²) for residues of the isolated αTS at a protein concentration of 750 μM and [ligand]/[protein] ratio of 3. Blue colored upright bars signify increased rigidity and red colored inverted bars decreased rigidity. The secondary structure elements from the X-ray crystal structure are indicated across the top (bars α-helices, arrows β-strands). (b) Mapping of the ΔRCI-S² values onto the isolated αTS structure 6OUY. The coordinates for the missing loop α-L6 and the F9 molecule (shown in green) have been extracted from the αTS structure of PDB entry 4HT3 after alignment of both PDB entries. Blue indicates increased rigidify while red indicates increased dynamic upon addition of ligand

Fig. 8

Experimental (a) ¹⁵N-T₁ and (b) ¹⁵N-T₂ relaxation times for residues of αTS with (orange diamonds) and without (blue squares) F9 at 700 MHz, 298K, a protein concentration of 750 μM, and [ligand]/[protein] ratio of 3. The secondary structure elements from the X-ray crystal structure are indicated across the top (bars α-helices, arrows β-strands)

Fig. 9

Average chemical shift differences (Equation 2) for select residues in αTS displaying fast exchange (a-f) and deviations from fast exchange (g-h) at a protein concentration of 750μM and [ligand]/[protein] ratios of 0, 0.5, 1, 1.5, 2, 3 and 5.5. (i) Mapping of the residues showing fast (red) and intermediate (blue) exchange onto the isolated αTS (rose; PDBID: 6OSO) aligned with the α-subunit of the αβ dimer (α-subunit, tan; β-subunit, sky blue; PDBID: 4HT3)