Determination of protein-ligand binding modes using fast multi-dimensional NMR with hyperpolarization

Abstract

Elucidation of small molecule-protein interactions provides essential information for understanding biological processes such as cellular signaling, as well as for rational drug development. Here, multi-dimensional NMR with sensitivity enhancement by dissolution dynamic nuclear polarization (D-DNP) is shown to allow the determination of the binding epitope of folic acid when complexed with the target dihydrofolate reductase. Protein signals are selectively enhanced by polarization transfer from the hyperpolarized ligand. A pseudo three-dimensional data acquisition with ligand-side Hadamard encoding results in protein-side [¹³C, ¹H] chemical shift correlations that contain intermolecular nuclear Overhauser effect (NOE) information. A scoring function based on this data is used to select pre-docked ligand poses. The top five poses are within 0.76 Å root-mean-square deviation from a reference structure for the encoded five protons, showing improvements compared with the poses selected by an energy-based scoring function without experimental inputs. The sensitivity enhancement provided by the D-DNP combined with multi-dimensional NMR increases the speed and potentially the selectivity of structure elucidation of ligand binding epitopes.

Fig. 1. (a) 2D SOFAST-HMQC spectra showing the methyl chemical shift region of 0.34 mM DHFR measured after admixing of 5.3 mM hyperpolarized folic acid (blue). A spectrum of the same sample after decay of the hyperpolarization is underlaid in gray. The spectra are recorded with 40 points in the indirect dimension. The 1D traces at the top are positive sum projections of the 2D spectra. (b) 1D slices extracted at several ¹³C chemical shifts, as indicated by the dashed lines, from both the hyperpolarized and non-hyperpolarized 2D spectra.

Fig. 2. (a) Structure of folic acid. (b) Hyperpolarized ¹H spectrum of folic acid with peak assignments labeled. DMSO designates the dimethyl sulfoxide signal from the glassing matrix used for DNP hyperpolarization.

Fig. 3. (a) Hyperpolarized ¹H spectra of folic acid in the presence of preloaded DHFR, measured as the first scan in the DNP experiment with 1° excitation and encoded according to the Hadamard matrix in the text with selective inversion on resonances a–c. (b) The corresponding 2D SOFAST-HMQC spectra of enhanced protein signals in the methyl region.

Fig. 4. Hadamard reconstructed SOFAST-HMQC spectra (blue) of the methyl group chemical shift region of the protein. The ligand protons from which polarization originated are indicated above each spectrum. Underlaid in gray is the conventional HSQC spectrum of the protein. Red dots and methyl group labels indicate all assignments overlapped with the observed signals in the reconstructed DNP spectra. Additional assigned peaks in the conventional spectrum are represented with black dots.

Fig. 5. Evaluation of ligand trial poses ranked by NOE score. (a) Overlay of the five docked poses with the best NOE score (blue) and the ligand in the crystal structure (red; PDB: 1RE7 (ref. 46)). For the overlay, the two protein structures were aligned on all atoms using PyMOL (The PyMOL Molecular Graphics System, Version 2.2, Schrödinger, LLC), and plotted using UCSF Chimera. The five encoded protons of the ligand are shown as spheres in all of the poses. Protein methyl groups from the crystal structure that are within 5 Å of the five selected poses are represented with gray spheres. (b) Correlation plot of NOE score vs. RMSD between the trial pose and the crystal structure. The blue circles represent the five poses displayed in (a). The RMSD values in the three panels are calculated considering the five ligand protons encoded with selective inversion (left), heavy atoms in the ligand structure excluding the glutamate portion (middle), and heavy atoms in the whole ligand structure (right).

Fig. 6. Pulse sequence for the [¹H–¹³C]-SOFAST-HMQC experiment with ligand resonances encoded by a Hadamard scheme. After the injection (t_inj. = 375 ms) and sample stabilization (t_stab. = 385 ms), the NMR experiment was triggered. Repeated 20 ms EBURP π/2 pulses followed by pulsed-field gradients G_x (44.8 G cm^–1), G_y (38.6 G cm^–1), G_z (33.5 G cm^–1) were applied for water suppression. The two π/2 pulses on the ¹³C channel were applied with γB₁/2π = 11.4 kHz. A 12.7 ms dual Gaussian shaped pulse with flip angle of π was applied simultaneously on two ligand ¹H resonances followed by a pulsed field gradient G_z (47.7 G cm^–1). The first ¹H scan was acquired after a hard pulse with a small flip angle (1°) for enhancement determination of hyperpolarized ligand signals. In the following [¹H–¹³C]-SOFAST-HMQC pulse sequence, a 2.8 ms PC9 shaped pulse (flip angle 2π/3, ±2 ppm bandwidth) and a 1.9 ms RSNOB shaped π-pulse were centered at 0 ppm for selective methyl proton excitation and refocusing. The coherence transfer delay was set to 1/(2J_CH) as Δ = 3.5 ms. A ¹³C GARP decoupling sequence (γB₁/2π = 3.1 kHz) was applied during the acquisition. Pulsed field gradients were applied with G_z,1 (7.5 G cm^–1) and G_z,2 (4.8 G cm^–1). A total of 40 × 1200 points were acquired for the ¹³C and ¹H dimensions, with t_1,max = 8.2 ms and t_2,max = 100 ms, respectively.