Fast Quantitative Validation of 3D Models of Low-Affinity Protein-Ligand Complexes by STD NMR Spectroscopy

Abstract

Low-affinity protein-ligand interactions are important for many biological processes, including cell communication, signal transduction, and immune responses. Structural characterization of these complexes is also critical for the development of new drugs through fragment-based drug discovery (FBDD), but it is challenging due to the low affinity of fragments for the binding site. Saturation transfer difference (STD) NMR spectroscopy has revolutionized the study of low-affinity receptor-ligand interactions enabling binding detection and structural characterization. Comparison of relaxation and exchange matrix calculations with ¹H STD NMR experimental data is essential for the validation of 3D structures of protein-ligand complexes. In this work, we present a new approach based on the calculation of a reduced relaxation matrix, in combination with funnel metadynamics MD simulations, that allows a very fast generation of experimentally STD-NMR-validated 3D structures of low-affinity protein-ligand complexes.

Scheme 1. M Matrix Definition

Figure 1

RedMat analysis of 2,7-anhydro-Neu5Ac binding to RgNanH-GH33 (PDB code 4X4A). (a) Two-dimensional sketch of the structure of the 2,7-anhydro-Neu5Ac ligand. The labels associated with the ligand proton are shown next to it. (b) Comparison between the calculated (from the X-ray structure; red bars) and experimental (blue bars) relative STD₀ factors (binding epitope mapping) of the nonexchangeable protons of 2,7-anhydro-Neu5Ac binding to RgNanH-GH33. A NOE R-factor of 0.13 was obtained. (c) Two-dimensional plot representing the NOE R-factor as a function of the ligand RMSD for the docking poses obtained for 2,7-anhydro-Neu5Ac binding to RgNanH-GH33. The docking score of each pose is indicated by the color code shown in the legend. The data point corresponding to the X-ray structure is highlighted with a dashed circle. (d) Superposition of three frames of the funnel-MD simulation (protein in light gray cartoon, and ligand in magenta sticks) and the X-ray structure (protein in gray colored cartoon, and ligand in yellow sticks) of the complex. The protein residues within 12 Å from the ligand, shown as wheat-colored lines, were included in the calculation of theoretical STD₀ values. (e) Evolution of the NOE R-factor of 2,7-anhydro-Neu5Ac over 400 ns of funnel-MD simulation. The fragment of the trajectory where the ligand adopts an X-ray-type of orientation is highlighted with a dashed circle and corresponds with the lower ligand RMSD region shown in (f). Unconnected data points indicate MD simulation frames when the ligand is dissociated from the protein and, hence, no NOE R-factor is calculated. (f) Evolution of the root-mean-square deviation (RMSD) for the 2,7-anhydro-Neu5Ac ligand (all atoms except the protons considered) with respect to the protein-binding site (residues within 6 Å from the ligand considered). The fragment of the trajectory where the ligand adopts an X-ray-type of orientation is highlighted with a dashed circle.

Figure 2

RedMat analysis of two triazolopyridazine ligands binding to BRD4 (PDB codes 5M3A (a–c) and 5M39 (d–f)). (a,d) 2D sketch of the structure of 3-methyl-6-(1-methyl-5-phenoxy-1H-pyrazol-4-yl)[1,2,4]triazolo[4,3-b]pyridazine (a, Ligand 1) and 6-(3,4-dimethoxyphenyl)-3-methyl[1,2,4]triazolo[4,3-b]pyridazine (d, Ligand 2). Ligand proton numberings are shown. (b,e) Comparison between the calculated (from the X-ray structure; blue bars) and experimental (red bars) relative STD₀ factors (binding epitope mapping) of the non-exchangeable protons of Ligand 1 (b) and Ligand 2 (e) binding to BRD4. NOE R-factors of 0.16 and 0.12 were obtained (using a cutoff of 10 Å), respectively, showing a very good agreement between the crystal and solution-state structures of the complexes. (c–f) Evolution of the NOE R-factor over the funnel-MD simulation of Ligand 1 (c, cutoff of 12 Å) and Ligand 2 (f, cutoff of 10 Å) binding to BRD4. The segments of the trajectories where both ligands adopt an X-ray-type of orientation are highlighted with a dashed oval and correlate well with the lower ligand RMSD regions along the simulations (ESI, Figure S5).