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

Significance The determination of active site protonation states is critical for a full mechanistic understanding of enzymatic transformations. However, hydrogen atom positions are challenging to extract using the standard tools of structural biology. Here, we make use of a joint solid-state NMR, X-ray crystallography, and first-principles computational approach that enables the investigation of enzyme catalysis at this fine level of chemical detail. For tryptophan synthase, this allows us to peer along the reaction coordinates into and out of the α-aminoacrylate intermediate. Through this process, we are developing a high-resolution probe for structural biology that is keenly sensitive to hydrogen atom positions—complementing diffraction methods yet able to be applied under conditions of active catalysis in microcrystalline and non-crystalline materials.


This PDF file includes:
Data Availability S2 Supplementary text: First-Principles Calculations and Statistical Methods S2 Figures S1-S3 Positional Variances from NMR Crystallography S6 Expanded Materials and Methods S10 Schemes S1-S2 S18 Tables S1-S9 S20 Figures  First principles calculations were performed using a cluster-based model of the active site as described previously (2). The clusters for E(A-A) and E(A-A)(BZI) were constructed from the corresponding crystal structures 4HN4 and 4HPX by selecting all atoms within 7 Å of the cofactor-substrate complex (Fig. S1). This selection was expanded to include complete residues and were modified as follows: (1) residues that were not part of continuous backbone segments and with only two atoms within the initial 7 Å cut were deleted (Q142, V201, I238, D383); (2) residues with two backbone atoms and no side chain residues within 7 Å were converted to alanine (N145, K167, D305, S308, V309, L349, N375, L376, R379); (3) residues with two side chain atoms and no backbone atoms within 7 Å were truncated by removing the backbone atoms and replacing C α with a methyl group (β-site residues F280, H313, K382); (4) N-terminal nitrogen atoms were replaced with a hydrogen atom and C-terminal carbonyls were capped with an -NH 2 group; (5) the Cs + Initial proton orientations in the active site clusters were set using molecular dynamics (MD) simulations of the αβ dimer. These simulations were performed using the Amber package with GPU acceleration (3,4). Coordinates for StTS were obtained from crystal structures 4HN4 for E(A-A) and 4HPX for E(A-A)(BZI) with crystal waters retained (5). Amber force field FF14SB was applied to protein atoms (6). Ligands were parametrized using General Amber Force Field (GAFF) and AM1-bcc charge model (7,8). Only hydrogens were minimized and the MD simulations were carried out using Generalized Born implicit solvent model (GB-Neck2) (9) at constant temperature of 298 K controlled by Langevin thermostat. Two MD trajectories for each system were collected over 50 ns at 1 ps interval with 2 fs time step. The trajectories were further processed with CPPTRAJ software to contain 5000 frames for analysis (10). The systems were visualized and analyzed using Visual Molecular Dynamics software (11). Dihedral data was collected for residues Thr110, Thr190, Ser235, Ser377, Ser351 and Tyr186 in both systems. In E(A-A), the conformation and orientation for the three water molecules WAT583, WAT604, and WAT914 were also analyzed. The initial conformation and the position of the polar hydrogens in the respective residues and water molecules were selected based on the mean value of the normally distributed dihedral data for each residue.
All cluster models were subsequently geometry optimized in Gaussian09 (12) at the DFT B3LYP level of theory using the Grimme D3 empirical dispersion correction (13) and a two-tier, locally-dense basis set (14-16) with 6-31G(d,p) for the PLP/substrate complex and 6-31G for all other atoms. Except as noted below, all atoms on or within 4 Å of the PLP/substrate complex and all hydrogen atoms in the cluster could adjust while the remaining atom coordinates were fixed at their crystallographic values. The only exceptions were the ε-amino group nitrogen of βLys87 and the oxygen atoms of the three waters adjacent to the serine moiety for the αaminoacrylate, which were also held fixed at their crystallographic coordinates during the geometry optimizations.

S4
For each optimized structure, NMR shieldings were calculated at the DFT B3LYP level of theory, employing a three-tier, locally dense basis set with the PLP/substrate, βLys87, BZI, and proximal water atoms at 6-311+G(2d,p), atoms within 4Å of this subgroup at 6-311G(d,p), and all remaining atoms at 6-31G. NMR shielding values were converted to chemical shifts using the following linear rescaling relationships, derived at the same level of theory and bases (14,15 O (14). RMSE for the chemical shift tensor components were 4.2 ppm and 13.7 ppm for 13 C and 15 N respectively (2). The a priori determination of the global uncertainties for the predicted shifts allows for the quantitative comparison of the predicted and experimental shifts as described below. Tables  S1-S4.

Reduced-χ 2 Analysis
The structural models were ranked using the reduced-χ 2 statistic (17), a quantitative measure of the agreement between their first-principles predicted chemical shifts and the experimental NMR parameters, In this expression, the summation i runs over all N measured NMR shifts, f is the number of adjustable model parameters (0 for the direct ranking of models; 1 for the exchange model with optimized populations),  exp i is the experimental chemical shift,  model i is the corresponding predicted shift for a given model, and 2 i s is the nuclide-specific weighting derived by setting i s to the corresponding root-mean-square error derived from benchmark studies described above.
Importantly, the reduced-χ 2 allows for chemical shift data from multiple nuclides ( 13 C, 15 N, 17 O, etc.) and of different types (isotropic, tensor principal components) to be combined into a single statistic. For a priori assigned weights, the reduced-χ 2 allows for rigorous evaluation of the consistency of a model with the experimental data (17).

Bayesian Probability Analysis
The overall confidence in the identification of the experimental structure was also quantified using Bayesian probability analysis following the approach of Engel et al. (18). In the context of NMR-assisted crystallography, S5 the application of Bayes' Theorem gives the following expression for the posterior probability,   * p M y , that M is the experimental structure given the measured set of chemical shifts y * Here,   * p y M is the probability of observing chemical shifts y * for a given structure M, and   pM is the prior probability of structure M matching the experimental structure. The probabilities   * p y M were determined relative to the probability of the best-fit (BF) model based on the one-sided p-value (z) from the corresponding F-distribution evaluated at the critical value set by the ratio of the reduced-χ 2 for model M and the best fit model (17) The p-value was then converted into a relative probability for model M according to using a fixed relative probability    * 1 BF p y M . Reduced-χ 2 for the 10 best exchange models for E(A-A) and E(A-A)(BZI) are given in Tables S6 and S7, along with their relative probabilities,   * p y M .
Following Engel et al. (18), we also incorporated into our analysis the possibility that the experimental structure was not part of our ensemble through the introduction of a virtual structure, M , whose shifts were taken as the mean of the shifts for the other candidate structures. In this case, the virtual structure was found to have negligible probability of matching the experimental structure for both E(A-A) and E(A-A)(BZI) (Tables S6, S7).
The prior probability   pM was used to introduce consistency with the observed temperature dependence of the Schiff base nitrogen chemical shift. Temperature dependence at this site requires exchange models that include tautomeric exchange at the Schiff base nitrogen; such models were assigned relative probabilities    1 pM while all other models were assigned    0 pM .
Taking into account both the isotropic and anisotropic chemical shift data, along with the observed temperature dependence of the Schiff base nitrogen, the proposed exchange model was found to be the most probable experimental structure with 88.5% confidence for E(A-A), and 99.6% confidence for E(A-A)(BZI). All probabilities are summarized in Tables S6 and S7.

Positional Variances from NMR Crystallography:
The positional uncertainties in the NMR-refined crystal structures were estimated following the method of Hofstetter and Emsley (19), adapted for NMR crystallography within an enzyme active site. Our protocol is summarized in the following steps: 1. An ensemble of slightly perturbed structures was generated using low-temperature molecular dynamics Low-T MD for E(A-A) and E(A-A)(BZI) were performed on the TS αβ dimer using the Amber package with GPU acceleration (3,4). Coordinates for StTS were obtained from crystal structures 4HN4 for E(A-A) and 4HPX for E(A-A)(BZI) with crystal waters retained (5) along with the refined active site and protonation states determined via NMR crystallography. Amber force field FF14SB was applied to protein atoms (6). Ligands were parametrized using the General Amber Force Field (GAFF) and AM1-bcc charge model (7,8).
Both the E(A-A) and E(A-A)(BZI) systems were minimized in a three-step process: hydrogens, side chains, and full protein, with the refined region within 6Å of the β subunit ligand "0JO" (the PLP cofactor + covalently attached ligands) restrained. Explicit TIP3P solvent was added to obtain a rectangular water box with edges at least 12 Å from any protein atom. Particle Mesh Ewald and SHAKE algorithms were applied for long range interactions and fixed heavy-atom-hydrogen bonds, respectively (20,21). Positive ions (Na + ) were added to neutralize the overall charge of the proteins. The solvated systems were minimized again in two steps: water molecules only, and then the whole system, with the same region within 6 Å restrained.
Next, the systems were gradually heated at constant pressure from 5K to 150K at 5K intervals. 300 ps simulations were carried out at each temperature with a time step of 2 fs using the thermal fluctuation configuration ensemble. Trajectories were processed to contain 300 frames (1ps per frame) for each interval. The final ensemble of perturbed crystal structures was composed of 1200 structures, 300 structures each at 5, 10, 25 and 45 K.
2. First principles chemical shifts and reduced-χ 2 were calculated for the active site region of each structure For each of the 1200 structures in the ensemble, the active site region consisting of the PLP-substrate covalent complex, the 3 adjacent waters or BZI, and residues βSer377(C α ), βHis86(C β ), βLys87(C γ ), βGlu109, βThr110, βGly111, βAla112, βGly232, βGly233, βGly234, βGly235, βAsn236, βGln114(bb), and βHis115(bb) were extracted for NMR chemical shift calculations. Only the side chain atoms of residues βSer377, βHis86, and βLys87 were included, truncated at the indicated atom and capped with a hydrogen. Only the backbone atoms and side chains through C β were included for βGln114 and βHis115. Terminal residues in contiguous chains were capped with hydrogen atoms. E(A-A) structures contained 192 atoms and E(A-A)(BZI) structures contained 199.
For each structure, first principles chemical shifts were calculated at the DFT level of theory (B3LYP/6-31G). These shifts were used to calculate a reduced-χ 2 relative to a reference structure using Eq. S4; only atoms for which experimental chemical shifts were measured were included in the reduced-χ 2 . The reference structure was chosen from the ensemble using the selection criterion that the sum of the reduced-χ 2 for all other structures was minimized. A separate reference structure was chosen at each temperature.
3. Anisotropic displacement parameters were defined at each atom by the correlation of positional deviations with reduced-χ 2 The positional deviations of each atom relative to its temperature-specific reference structure were used to shows the 95% confidence cutoff in the reduced-χ 2 . The black and gray lines indicate ±1, 2, and 3 times the width of the Gaussian distribution at a given reduced-χ 2 .
define a covariance matrix of displacements and an atom-specific principal axis system (19). The correlation of positional deviations along a PAS axes (r i ) and the corresponding reduced-χ 2 was used to fit a 2D probability density at each atom (l)  The NMR-derived anisotropic displacement parameters (ADP) were defined as the Gaussian widths in the principal axis system positional distributions drawn at the 95% confidence limit of the chemical shift reduced-χ 2 These were transformed back to the coordinate frame of the crystal structure using standard tensor methods (22,23), and were incorporated into an edited version of the protein crystal structure file along with the refined active site coordinates from NMR-assisted crystallography. These are included as part of the Supplementary Materials as files 4HN4.active_site_refined_NMRX.PDB and 4HPX.active_site_refined_NMRX.PDB. The active sites with positional variances drawn at the 95% probability level are shown in Fig. S3.

S8
In the text, the average positional RMSD for a given atom type is reported as

Reported ADPs:
In this preliminary analysis, ADPs are reported only for atoms with measured chemical shifts and their covalently-or hydrogen-bonded neighbors. This restriction can likely be relaxed, a subject of ongoing work. The current restriction recognizes a fundamental difference in the application of NMR crystallography to an active site compared to its application in organic molecular crystals (19,24): active sites are typically modeled using a cluster-based approach in which the measured chemical shifts are for atoms concentrated at the center of the model, while molecular crystals are typically modeled in a unit-cell based approach and have measured chemical shifts distributed throughout. Displacements of atoms at the periphery of cluster models are expected to have little influence on the measured chemical shifts at the center of the cluster, and therefore are expected to be unconstrained. To better quantify this, the sensitivity of the reduced-χ 2 to atomic displacements was assessed by forming its gradient with respect to single atom displacements using finite differences. It is known that single atom displacements lead to unphysical models and unrealistically large changes in chemical shifts within a covalent network (19), but this is expected to be a less severe problem for displacements outside of the shift region. These simulations confirm that atoms whose ADP have been reported show the largest gradients -more than 3 orders of magnitude larger than for atoms at the edge of the model.

Fig. S3: Anisotropic displacement parameters for (A) E(A-A) and (B) E(A-A)(BZI) displayed for atoms
with experimental chemical shifts and their neighboring atoms. ADP ellipsoids are drawn at the 95% probability level (scale factor 2.8). The CPK scheme is used to designate atoms and their ellipsoids (C grey; H white; O red; P orange). Images rendered in UCSF Chimera (1).

Materials and Methods: Protein Preparation:
Tryptophan synthase was prepared by overexpression of StTS in E. coli BL21 cells as previously described (25,26). Samples were prepared with the following isotopic labeling schemes for the cofactor and protein components: 1. Protein and cofactor unlabeled/natural abundance isotopomer concentration; 2. Protein 15 Nlabeled at lysine ε-nitrogen sites (ε-15 N-Lys TS); and 3. Protein with the PLP cofactor selectively 13 C enriched at carbon sites C2, C2', and C3 and 15 N enriched at the pyridine ring nitrogen (2,2',3-13 C 3 , 15 N-PLP; TS). Isotopically labeled PLP was prepared as described below and exchanged into the β-subunit active site as detailed in (25).
Observation of NH 4 + bound in the monovalent cation binding site was unexpected. The Na + form of E(A-A) produces ammonium pyruvate at an accelerated rate relative to the Cs + form, and we hypothesize that the NH 4 + displaces the Na + , which binds more weakly than Cs + (33)(34)(35).

Microcrystalline Protein Samples for Solid-State NMR:
Microcrystalline samples of TS were prepared by diluting enzyme solution 1:1 with 50 mM Cs + -bicine buffer, pH 7.8, containing 14% PEG-8000 and 3.0 mM spermine (25). Microcrystals were collected and washed with 50 mM Cs + -bicine, pH 7.8, containing 8% PEG-8000, 1.8 mM spermine, and 3 mM N-(4'trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9; a high affinity alpha site ligand and analogue of the natural α-site substrate 3-idole-D-glycerol-3'-phosphate (IGP) (28) Chemical shift tensor principal axis components were determined by a fit of the sideband intensities in the slow MAS spectra using Herzfeld-Berger analysis (42), implemented within Bruker BioSpin's Topspin 3.6 processing software. Standard errors were determined by a numerical calculation of the covariance matrix (43) in Mathematica, using spectra that were zero-filled once and processed without line broadening; this ensures that extrapolated data points (from zero-filling more than once) were not included in the calculation of the covariance matrix and that an accurate estimate of uncorrelated RMS noise was obtained.

N DNP Experiments
MAS-DNP NMR spectra were collected at 600 MHz (14.1 T) using a Bruker AVIII DNP NMR system (Bruker Biospin) equipped with a Bruker 3.2 mm, 1 H/ 13 C/ 15 N MAS-DNP probe cooled to ~95 K. Microwaves were delivered using a gyrotron source operating at 395 GHz and output optimized for ~12 W at the probe base (46). Microwaves on/off spectra were collected with a MAS frequency of 10 kHz and a 13 C CP-echo pulse sequence with a 1 H CP ramp from 40 to 70 kHz, a 13 C RF field of 50 kHz, 85 to 100 kHz Spinal64 1 H decoupling (36) and a recycle delay of 5s. An AsymPolPOK (47,48) concentration of ~5 mM was used, resulting in enhancements >60 and buildup times on the order of 3 s.

Evaluation of MAS-DNP samples by X-band EPR spectroscopy:
CW EPR spectra were collected on a Bruker X-Band EMX Nano benchtop spectrometer (Bruker Biospin, Billerica, MA) to verify polarizing agent concentrations and PA dispersion upon freezing as described in Tran et al. (46). DNP sample rotors were placed in 5 mm quartz sample tubes and placed into the cavity with its associated adaptor. Spectra were collected at room temperature (298 K) and at 100 K by using cold nitrogen gas to cool the cavity. 17 17 O spectra at all fields. A total of 2048 or 4096 points were collected and zero-filled to two times the number of real points, spectral widths ranged from 41 kHz to 100 kHz, relaxation delays were between 5 and 10 ms, and acquisition times were between 10 and 25 ms. A 90°-τ -180°-τ -acquire pulse sequence was used to measure the spectra of all intermediates. A 90°-τ-180°-2τ-180°-2τ-180°-τ -acquire (triple-echo, 3π) was additionally used to measure the spectra of the E(A-A) and E(A-A)(BZI) intermediates at 14.1T and higher to improve observation of bound resonances by suppressing signal from the free substrate and reaction product (53). This is based on the selective excitation of the central transition in 17 O QCT NMR which leads to a nutation rate that is three times faster for protein-bound substrates than that of the broadband excitation felt by free substrate in solution (49,54). The power levels for the 180° refocusing pulses were set to those of the 90° pulses for 11.7T, 14,1T, and 16.4T, but were lowered to 10 kHz for 21.1T and 35.2T. In each case the 180° pulse length was set to 1/3 the nominal pulse for water; for 11.7T, 14.1T, and 16.4T, the initial excitation pulse was set to 1/3 the water 90° pulse, while at 21.1T and 35.2T it was set to 1/2 the water 90°. The τ delay times were between 5 and 50 µs. The pulse diagrams and phase cycling lists are provided in our previous publication (53). All spectra were ultimately processed using Bruker's Topspin 3.6 or 4.0 software. Field specific details including hardware are listed below. The spectra at multiple fields were fit simultaneously to extract the isotropic chemical shift (δ iso (ppm)), quadrupole product parameter (P Q (MHz)), and shielding anisotropy product parameter (P SA (ppm)) as described previously (53). The procedure was modified to include additional higher order contributions to the linewidth following Wu and coworkers (50,51). In this analysis, the peak position δ obs and linewidth Δν (S13) These equations apply in the limit of slow isotropic motion and are specific to spin-5/2 nuclei. β is the angle between the principal axis systems of the chemical shift and quadrupolar coupling tensors under the assumption of axial symmetry; here we take β=π/2 (50), which is supported by modeling of the tensors using first principles methods. Following our earlier protocol (53), τ c was set at 232 ns based on QCT experiments at the same 5 fields for the considerably more stable E(Q 3 ) 2AP intermediate.
At each field, the free substrate/product signals were fit and subtracted from the spectrum prior to global fitting of the bound signals. For the SCH E(A-A)(BZI) data only, a reference spectrum for the free substrate/product was first subtracted.
The spectra and fits are shown in Fig. S4. The best-fit parameters are tabulated in Table S8.

Synthesis of 2-Nitro-15 N-aniline:
To a sealed tube was added 1-fluoro-2-nitrobenzene (105 µL, 1.0 mmol), 15 3 (1 mg, 0.5 mol %) and 1 M AcOH (215µL). The reaction was heated to 75 °C for 24 h. The mixture was then cooled to room temperature, filtered through a celite plug, diluted with 3 mL DI water and extracted with CH 2 Cl 2 (2 x 4 mL). The resulting solution was concentrated in vacuo to give a yellow residue. The product was further purified through column chromatography in using 1% MeOH/CH 2 Cl 2 eluent to afford a white solid (10 mg, 42 %

( 15 N)-5-ethoxy-4-methyloxazole
To a vigorously stirred heterogeneous mixture of phosphorous pentoxide (3.6 g, 12.7 mmol) and magnesium oxide (1.2 g, 30 mmol) in 50 mL of dry dichloromethane, under nitrogen atmosphere, was added a solution of ( 15 N)-ethyl 2-formamidopropanoate (600 mg, 4.1 mmol) dissolved in 3 mL of dry dichloromethane. The reaction was refluxed for 24 hrs, then cooled to 0 °C. To the cooled mixture was slowly added 10 mL of water, followed by 50 mL of saturated NaHCO 3 . The biphasic mixture was filtered through celite, then transferred to a separatory funnel, and the organic phase separated.

( 15 N)-pyridoxal
To a stirred solution of ( 15 N)-4,5-bis(ethoxycarbonyl)-3-hydroxy-2-methylpyridin-1-ium chloride (100 mg, 0.3 mmol) in 20 mL of dry tetrahydrofuran, under nitrogen atmosphere at 0° C, was added 3.0 mL of LiAlH 4 (1M in THF) and the resulting mixture was then refluxed for 24 hrs. The mixture was cooled, then carefully quenched with 25 mL of water, and filtered through celite. The filtrate was washed with dichloromethane (3 x 10 mL), and the aqueous phase was transferred to a round bottom flask along with manganese dioxide (42 mg, 0.5 mmol). The mixture was acidified with aqueous HCl, and allowed to stir at room temperature for 4 hours. The mixture was then filtered through celite, yielding an aqueous solution of crude ( 15 N)-pyridoxal (21 mL, 15.7 mM) and the solution was used without further purification. UV-Vis showed peaks at 320 and 388 nm. The crude solution was used without further purification for exchange into the apoenzyme as previously described (25). Spectra for the holoenzyme were consistent with literature.
The K229Q mutant ePL kinase was expressed and purified according to (57).                Fig. 3. The time course shows the disappearance of serine and appearance of product signals in the mother liquor. Data collected at -10 C on a 9.4 T Bruker DSX spectrometer equipped with a 4 mm CPMAS probe spinning at a MAS rate of 8 kHz. 3 kHz, and with the bearing gas cooled to -15°C, giving an effective sample temperature of -10°C. The primary signals arise from the isotropic shift and spinning sidebands (with sideband order given above) of the Schiff base nitrogen (286.7 ppm, indicated by the arrow) and natural abundance amide backbone nitrogens (122 ppm, sidebands indicated by asterisks). A fit to the sideband manifold (red) performed in Bruker Topspin 3.6 allows for the extraction of the chemical shift principal axis components (δ11, δ22, δ33) = (525±11, 305±10, 30±14) ppm. There is a second minor peak present, indicated by the double asterisks. Errors were determined by a numerical calculation of the covariance matrix and are reported as ±1 standard error. H-X double resonance 4mm MAS probe, spinning at a MAS rate of 2.5 kHz, and with the bearing gas cooled to -15°C, giving an effective sample temperature of -10°C. The primary signals arise from the isotropic shift and spinning sidebands (with sideband order given above) of the Schiff base nitrogen (292.3 ppm, indicated by the arrow) and natural abundance amide backbone nitrogens (122 ppm, sidebands indicated by asterisks). A fit to the sideband manifold (red) performed in Bruker Topspin 3.6 allows for the extraction of the chemical shift principal axis components (δ11, δ22, δ33) = (544±12, 327±10, 6±15) ppm. Errors were determined by a numerical calculation of the covariance matrix and are reported as ±1 standard error. Both the isotropic and anisotropic chemical shifts indicate that the phosphate group is dianionic. The order of the spinning sidebands is given above each peak. Error bars were determined by a numerical calculation of the covariance matrix and are reported as ±1 standard error.  Both the black and red spectra were acquired with a 25 ms echo period on 15 N before detection, but the black spectrum includes the application of dipolar dephasing pulses on 31 P that attenuate signals from 15 N atoms that are dipolar coupled to 31 P. The dipolar coupling falls off as the inverse cube of the interatomic distance, and the dephasing period used here selects nitrogen atoms within ~3-4 Å of a phosphorous atom. There is resolution of many individual ε-amino group nitrogen sites, but only the signal at 35.6 ppm dephases. Based on proximity to the PLP phosphate group, this resonance is assigned to the ε-amino group of βLys87.