High-Confidence Placement of Fragments into Electron Density Using Anomalous Diffraction-A Case Study Using Hits Targeting SARS-CoV-2 Non-Structural Protein 1

Abstract

The identification of multiple simultaneous orientations of small molecule inhibitors binding to a protein target is a common challenge. It has recently been reported that the conformational heterogeneity of ligands is widely underreported in the Protein Data Bank, which is likely to impede optimal exploitation to improve affinity of these ligands. Significantly less is even known about multiple binding orientations for fragments (<300 Da), although this information would be essential for subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here, we use recently reported fragment hits for the SARS-CoV-2 non-structural protein 1 (nsp1) N-terminal domain to propose a general procedure for unambiguously identifying binding orientations of 2-dimensional fragments containing either sulphur or chloro substituents within the wavelength range of most tunable beamlines. By measuring datasets at two energies, using a tunable beamline operating in vacuum and optimised for data collection at very low X-ray energies, we show that the anomalous signal can be used to identify multiple orientations in small fragments containing sulphur and/or chloro substituents or to verify recently reported conformations. Although in this specific case we identified the positions of sulphur and chlorine in fragments bound to their protein target, we are confident that this work can be further expanded to additional atoms or ions which often occur in fragments. Finally, our improvements in the understanding of binding orientations will also serve to improve the rational optimisation of SARS-CoV-2 nsp1 fragment hits.

Keywords: COVID-19; SARS-CoV-2; anomalous difference Fourier map; fragment orientation; non-structural proteins; nsp1; tunable wavelength.

Figure 3

Comparison of the fragment binding site of the previously reported conformation of 10B6 with that obtained using sulphur anomalous difference Fourier maps. (A) The published configuration of 10B6. The 2mF_o-DF_c map of the fragment is shown in cyan. (B) Anomalous difference Fourier maps calculated from data collected at 4.5 keV (orange) and 2.75 keV (marine) with the mF_o-DF_c map (green) in the fragment region. (C) Refined 2mF_o-DF_c map of 10B6 with sulphur atom sitting in the centre of the anomalous peak. The rms deviations for the 2mF_o-DF_c, mF_o-DF_c and anomalous difference Fourier maps are 1.0, 3.0, and 4.0, respectively, for Figure 3, Figure 4, Figure 5 and Figure 6. The electron density mostly accounts for 10B6.

Figure 4

Comparison of the fragment binding site of the previously reported conformation of 11C6 with that obtained using sulphur and chlorine anomalous difference Fourier maps. (A) The published configuration of 11C6. The 2mF_o-DF_c map of the fragment is shown in cyan. (B) Anomalous difference Fourier map calculated from data collected at 4.5 keV (orange) with the mF_o-DF_c map (green) in the fragment binding region. The anomalous peak from 2.75 keV data does not appear in the sulphur location possibly because of the low occupancy of 11C6. (C) Refined 2mF_o-DF_c map of 11C6 with the chlorine and sulphur atoms sitting in the centres of the anomalous peaks. The electron density only partially covers 11C6, but the orientation of the fragment hit could be confirmed by the anomalous signals for sulphur and chlorine.

Figure 5

Comparison of the previously reported single conformation of 5E11 with those obtained using sulphur anomalous difference Fourier maps. (A) The published configuration of 5E11. The 2mF_o-DF_c map of the fragment is shown in cyan. (B) Anomalous difference Fourier maps calculated from datasets collected at 4.5 keV (orange) and 2.75 keV (marine) with the mF_o-DF_c map (green) in the fragment region. (C) Refined 2mF_o-DF_c map of 5E11 in the two orientations with sulphur sitting in the centres of the two anomalous peaks obtained from 4.5 keV data. The electron density mostly covers 5E11 in both distinct orientations, but there is no density around the N-methyl-methanamine substituent, indicating its flexibility and complicating unambiguous placement of the fragment in the absence of sulphur peaks.

Figure 6

Comparison of the fragment binding site of previously reported conformation of 7H2 with that obtained using a chlorine anomalous difference Fourier map. (A) The published configuration of 7H2 with the 2mF_o-DF_c map around the fragment is shown in cyan. (B) Anomalous difference Fourier map calculated from data collected at 4.5 keV (orange) together with the mF_o-DF_c map (green) in the fragment binding region. (C) Refined 2mF_o-DF_c map of 7H2 with the chlorine located in the centre of the anomalous peak. The electron density mostly covers 7H2.

Figure 1

(A) Chemical structures of the four nsp1-targeting fragment hits containing sulphur atoms and chloro substituents. (B) Schematic representation of the four distinct orientations planar fragments can adopt in suboptimal real-life 2mF_o-DF_c maps (rmsd = 1.0, blue mesh), exemplified by 5E11. One hundred and eighty-degree rotations from one orientation to another can occur along horizontal or vertical axes, in particular when substituents are in quasi symmetric positions on one of the ring systems or if these possess some flexibility and are therefore not visible in the electron density.

Figure 1

(A) Chemical structures of the four nsp1-targeting fragment hits containing sulphur atoms and chloro substituents. (B) Schematic representation of the four distinct orientations planar fragments can adopt in suboptimal real-life 2mF_o-DF_c maps (rmsd = 1.0, blue mesh), exemplified by 5E11. One hundred and eighty-degree rotations from one orientation to another can occur along horizontal or vertical axes, in particular when substituents are in quasi symmetric positions on one of the ring systems or if these possess some flexibility and are therefore not visible in the electron density.

Figure 2

Validation of the localisation of anomalous signals of sulphur from (A) Met9, (B) Cys51 and (C) Met85 in the anomalous difference Fourier maps (rmsd = 4.0) of the nsp1-7H2 complex. The anomalous peak at 4.50 keV is coloured in orange, while that at 2.75 keV is shown in marine. Sulphur, nitrogen, and oxygen atoms in amino acids are coloured in yellow, blue and red, respectively for Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6. The two alternative conformations of Met85 are displayed and correlated well with the slightly elongated form of the anomalous signals.