Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

Abstract

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C^β and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Keywords: NMR-assisted crystallography; integrated structural biology; pyridoxal-5′-phosphate; solid-state NMR; tryptophan synthase.

Fig. 1.

TS β-site reaction, highlighting the α-aminoacrylate intermediate (41, 82). The PLP cofactor is drawn in black. In stage I of the reaction, L-Ser reacts with the internal aldimine E(Ain) to give the gem-diamine, E(GD₁), and external aldimine, E(Aex₁), intermediates. Subsequent proton abstraction via βLys87 leads to the first carbanionic intermediate, E(C₁), and elimination of the β-hydroxyl group as water gives the α-aminoacrylate intermediate, E(A-A). In stage II, indole makes a nucleophilic attack at C^β of E(A-A) to yield a new C–C bond and the L-Trp carbanionic intermediate, E(C₂), and, upon deprotonation, E(C₃). In the final stages, E(C₃) is reprotonated, leading to the eventual release of L-Trp and the regeneration of E(Ain).

Fig. 2.

The β-subunit active sites for E(A-A) and E(A-A)(BZI) from crystal structures 2J9X and 4HPX, respectively. (A) The E(A-A) intermediate shows three active site waters adjacent to the substrate with the central water forming close contacts to both the substrate C^β and the βLys87 ε-amino group. (B) The E(A-A)(BZI) complex shows BZI displacing the three waters but otherwise inducing only small changes in the active site structure. Images rendered in UCSF Chimera (83).

Fig. 3.

¹⁵N and ¹³C CPMAS SSNMR spectra of microcrystalline TS E(A-A) prepared with the following isotopic labeling: (A) ¹⁵N-labeled on the substrate L-Ser; (B) ¹⁵N-enriched on the PLP cofactor; (C) ¹⁵N-enriched at protein lysine side-chain ε-amino groups; (D) natural abundance isotopomer concentration; (E) ¹³C-labeled at C′ of the L-Ser substrate; (F) ¹³C,¹⁵N-enriched on the PLP cofactor and C^β of the substrate L-Ser; and (G) ¹³C-labeled at C^α of the substrate L-Ser. The top spectra in (E–G) are formed as the difference between the E(A-A) spectra with various cofactor/ligand isotopic labels and the same spectra acquired at natural abundance, emphasizing the resonances for the specific site labels. The large peak at 63.1 ppm is from free serine. Spectra acquired at 9.4 T, −10 °C, and 8 kHz MAS as described in Materials and Methods.

Fig. 4.

Cluster model of the E(A-A) active site. (A) X-ray crystal structure of the TS α₂β₂ heterodimer with the β-subunit active site highlighted in red. (B) Cluster model of the active site for first-principles geometry optimization and chemical shift calculations with protein side chains displayed in wireframe and cofactor and substrate in ball and stick. (C) Protonation sites on or near the cofactor/substrate complex: [a] the βLys87 side chain, [b] the PLP phosphate group, [c] the PLP pyridine ring nitrogen, [d] the PLP phenolic oxygen, [e] the Schiff-base nitrogen, and [f/g] the substrate carboxylate. Shaded nuclei indicate sites for which experimental NMR chemical shifts are reported.

Fig. 5.

Ranking of structural models based on agreement between the experimental and first-principles chemical shifts as quantified by the reduced χ² statistic. (A) The 15 best geometry-optimized active site models for the E(A-A) intermediate; structures and labeling are given in SI Appendix, Schemes S1 and S2. (B) Rankings of the seven best fast-exchange equilibrium models comparing the experimental and first-principles isotropic chemical shifts (red) and Schiff base nitrogen tensor components (blue). 95% confidence limits are shown as the correspondingly colored horizontal bars. (C and D) Model rankings for the E(A-A)(BZI) complex. For both E(A-A) and E(A-A)(BZI), only a single tautomeric exchange model simultaneously satisfies the isotropic and tensor chemical shift restraints.

Fig. 6.

Protonation states, hydrogen bonding interactions, and the placement and orientation of structural waters as revealed by NMR-assisted crystallography in the TS β-subunit active site. (A and B) The E(A-A) intermediate and (C and D) the E(A-A)(BZI) complex. For E(A-A), the position and orientation of the central water molecule highlights the reaction coordinate for the loss of the serine β-hydroxyl and confirms βLys87 as the active site acid–base catalytic residue. The E(A-A)(BZI) complex shows BZI bound in the active site with hydrogen bonding interactions to βGlu109 and the charged ε-amino group of βLys87. BZI is poised for, but unable to initiate, the subsequent bond formation step. Images rendered in UCSF Chimera (83).

Fig. 7.

¹³C SSNMR CPMAS spectra of microcrystalline TS E(A-A) prepared with (A) a 50:50 mixture of singly labeled 2-¹³C-L-Ser and 3-¹³C-L-Ser and (B) doubly labeled 2,3-¹³C₂-L-Ser. For both, the natural abundance ¹³C protein background has been subtracted as in Fig. 3. The C^α and C^β resonances show a minor peak that, along with the major peak, splits into a doublet upon incorporation of doubly labeled L-Ser substrate (deconvolutions shown adjacent to the spectrum). (C) The 2D dipolar driven ¹³C correlation spectrum of E(A-A) formed with U-¹³C₃-L-Ser displays distinct cross-peaks for the major and minor resonances, indicating that they belong to two independent E(A-A) species. One-dimensional spectra acquired as in Fig. 3; 2D Spectra acquired using combined R2-driven (CORD) mixing (73) on a Bruker BioSolids CryoProbe (74) at 14.1 T, −10 °C (sample temp) and 8 kHz MAS as described in Methods and Materials.