The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and X-ray diffraction at room temperature

Abstract

The NSP3 macrodomain of SARS CoV 2 (Mac1) removes ADP-ribosylation post-translational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the COVID-19 pandemic. Here, we determined neutron and X-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site, and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a re-evaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

Fig. 1.

The NSP3 macrodomain (Mac1) reverses mono-ADP-ribosylation. (A) Chemical structure of ADPr showing the C1” covalent attachment point with a red arrow. (B) Cartoon of the multi-domain NSP3 showing Mac1 with ADPr bound in the active site (PDB code 7KQP). (C) Structure of ADPr bound in the macrodomain active site (PDB code 7KQP) with the changes in protein structure upon ADPr binding indicated with black arrows. (D) Summary of the crystal structures reported in this work.

Fig. 2.

Crystal structures of Mac1 determined using neutron diffraction. (A) Neutron quality Mac1 crystals grown in the P4₃, P2₁ and C2 space groups. The P4₃ crystal is shown in the quartz capillary used for data collection, while the P2₁ and C2 crystals are shown prior to mounting. (B) Molecular surface showing Mac1 neutron structures. D₂O molecules within 5 Å of the protein surface are shown with sticks/spheres. The adenosine and catalytic sites are shaded green and yellow respectively. (C) Plot showing the occupancy of backbone amide deuterium atoms in the three Mac1 crystal structures. (D) Backbone amide deuterium occupancy mapped onto the P2₁ and C2 Mac1 structures. Backbone nitrogens are shown with blue spheres, colored by backbone D occupancy (blue=0%, white=100%). The helices composed of residues 50–70 are shaded red. The average backbone D occupancy for these helices was 82% in the P2₁ structure and 52% in the C2 structure.

Fig. 3.

Protonation states of Mac1 histidine residues assigned by neutron diffraction. (A) The location of histidine (teal spheres) and cysteine (salmon spheres) residues mapped onto the Mac1 structure (PDB code 7KQO). (B) Chemical structures showing the three possible protonation states of histidine. (C) Histidine protonation states assigned based on NSL density maps (maps are shown in Fig. S5). The tautomers assigned to the high resolution P4₃ X-ray structure (PDB code 7KQO) by the program Reduce are also shown. (D) NSL density maps reveal the hydrogen bond network connecting His45 and ADPr in the C2 structure (PDB code 7TX5). The protein is shown with a white cartoon/stick representation and the 2mF_O-DF_C NSL density map is shown with blue mesh (contoured at 2.5 σ). (E) An extensive hydrogen bond network connects the Asn37 side chain with a surface histidine (His86). The P4₃ structure (protomer A, PDB code 7TX3) and the corresponding 2mF_O-DF_C NSL density map is shown with blue mesh (contoured at 2.5 σ). (F) An aromatic-thiol bond was observed between Tyr68 and Cys81 in protomer A of the P4₃ structure (PDB code 7TX3). The 2mF_O-DF_C NSL density map is shown with blue mesh (contoured at 1 σ).

Fig. 4.

Protein flexibility and hydrogen bond networks in the Mac1 active site. (A) Alignment of P4₃, P2₁ and C2 Mac1 neutron and X-ray crystal structures determined at 100/293/310 K (PDB codes 7KQO, 7KQP, 7KR0, 7KR1, 7TWH, 7TX3, 7TX4, 7TX5). (B) Cα RMSF calculated from structures shown in (A) mapped onto the Mac1 structure. (C) Top: plot showing Cα RMSF of Mac1 structures determined at 100 K (black line) and those at 293/310 K (blue line). Bottom: plot showing Cα B-factors from Mac1 structure. B-factors were normalized by Z-score as described in the Methods section. (D,E,F) Active site hydrogen bond networks assigned based on NSL density maps for the P4₃ neutron structure (protomer A, PDB code 7TX3). The protein is shown with a white stick/cartoon representation and the 2mF_O-DF_C NSL density map is shown with a blue mesh (contoured at 2 σ around the asparagine/glutamine side chains). Hydrogen bonds (<3.5 Å) are shown with dashed black lines. (G, H, I) Same as (D,E,F), but showing the hydrogen bond networks in the C2 ADPr co-crystal structure (PDB code 7TX5). The NSL density maps are shown with a blue mesh (contoured at 3, 2 and 2.5 σ in G, H and I).

Fig. 5.

Mac1 active site water networks are robust to changes in crystal packing, temperature and pH. (A) Water network in the Mac1 active site from the 1.1 Å P4₃ X-ray structure obtained at room temperature (PDB code 7TWH). Waters were considered part of the active site network if they were within 3.5 Å of an active site hydrogen bond acceptor/donor. For clarity, only the side chains of selected residues are shown. The protein is shown with white cartoon/sticks, the waters are shown as teal spheres, and hydrogen bonds with dashed black lines. (B) Plot showing the number of contacts for water molecules shown in (A). (C) Plot showing B-factors for the active site H₂O/D₂O molecules in the room temperature P4₃ X-ray structure and the P2₁ and C2 neutron structures. Solvent molecules are numbered according to (A). (D) Real-space correlation coefficients (RSCC) for active site D₂O molecules in the P4₃, P2₁ and C2 neutron structures calculated using the 2mF_O-DF_C NSL density map. A line is drawn at an RSCC=0.8, which has previously been used as a threshold for assessing whether a D₂O is correctly oriented (49). (E) Variation in water orientations across 100 independent rounds of refinement. The plot shows distances between the average deuterium position of protomer A and B of the P4₃ structure. For comparison, the D₂O oxygen distances between the A and B protomers are shown. (F) Selected active site D₂O molecules showing the 2mF_O-DF_C NSL density map (purple mesh contoured at 2 σ) and 2mF_O-DF_C electron density map (blue mesh/surface contoured at 2 σ). Hydrogen bonds are shown with dashed black lines. For the C2 structure, ADPr specific hydrogen bonds are shown with pink dashed lines. (G) Histogram showing water protein-distances in the 0.85 Å P4₃ X-ray structure determined at 100 K (PDB code 7KQO). Waters were grouped based on whether a matching water molecule within 0.5 Å was found in the P4₃ structure determined at 293 K (PDB code 7TWH). The histogram was generated with a bin width of 0.2 Å. (H) Same as (G) but showing B-factors for all water molecules in the 100 K P4₃ structure. The histogram was generated with a bin width of 5 Ų. (I) Scatter plot showing correlation between water B-factor at 100 and 293 K. The red line shows a linear fit of the data using GraphPad Prism. (J) Plot showing distances between H₂O oxygen atoms between protomer A of the P4₃ X-ray structures determined at 293 and 100 K (PDB codes 7TWH and 7KQO). (K) Plot showing the B-factors of active site H₂O molecules in the P4₃ X-ray structures determined at 293 and 100 K. B-factors were normalized by Z-score using the B-factors from all the H₂O molecules in a structure. (L) Plot showing the RMSF of active site water molecules calculated across the seven structures determined from pH 4 to 10 using the P4₃ crystal form at 100 K. Because of pH-dependent binding of buffer components in the active site of protomer A, only the waters in protomer B are shown.

Fig. 6.

Reorganization of water networks upon ADPr binding. (A) Active site water positions from the structure of ADPr-bound Mac1 determined at 100 K (P4₃ crystal, PDB code 7KQP) compared to the apo structure (PDB code 7KQO). For clarity, only selected side chains of the ADPr-bound structure are shown (white sticks). Hydrogen bonds are shown as dashed black lines. (B) Plot showing distances between the water molecules shown in (A). (C) Plot showing the B-factors for waters shown in (A). (D) Evidence for hydrogen-deuterium exchange in ADPr co-crystallized with Mac1 (C2, PDB code 7TX5). The mF_O-DF_C NSL density map calculated after joint neutron/X-ray refinement but prior to adding deuterium atoms to ADPr is shown with green mesh (contoured at +3 σ). No density was observed for the C2’ and C3’ hydroxyl deuteriums. (E) Alignment of the ADPr-bound Mac1 structures determined using P4₃ and C2 crystals. The two configurations of the terminal ribose are marked (α and β), and the conformational change required to bind the α configuration in the C2 crystal is shown with red arrows. (F) Hydrogen bond networks in the ADPr-bound Mac1 structure determined using neutron diffraction (C2, PDB code 7TX5). The 2mF_O-DF_C NSL density map is shown for bridging water molecules with a purple mesh (contoured at 2.5 σ for W6, W8, W14 and W17, and at 2 σ for W19). The 2mF_O-DF_C electron density map is contoured at 2 σ (blue mesh). Deuterium atoms are colored cyan and hydrogen bonds are shown with dashed black lines. (G) Left: plot showing the RMSF of active site water molecules calculated across the seven ADPr-bound structures determined from pH 4 to 10. Right: B-factors for water molecules in the ADPr-bound structures from pH 4 to 10.

Fig. 7.

Water networks mediate fragment binding in the adenosine site of Mac1. (A) Structure of Mac1 showing density of fragment-protein bridging water molecules, calculated using the 232 previously reported fragment structures (14). The P4₃ 100 K structure (PDB code 7KQO) is shown with a white stick/cartoon representation. Water density was calculated with the GROMAPS tool (56) and is contoured from 5 to 50 σ. (B) Left: chemical structure showing an example of a bridging water molecule. Right: distances from bridging waters to apo waters calculated for all fragment structures. (C) Left: chemical structure showing an example of water displacement. Right: the minimum fragment-water molecule for each fragment structure. (D) W17 acts as a bridge to 64 fragments binding in the adenosine site. The Mac1 structure (PDB code 7KQO) is shown, and W17 from the 64 fragments is shown with yellow spheres. The hydrogen bonds to Leu126 and Ala154 are shown with black dashed lines, and the fragment atoms are shown with spheres colored by atom type. (E,F,G) The W17-Leu126/Ala154 hydrogen bonds are conserved across the 64 bridging fragments, whereas the W17-fragment bonds are variable, based on distance (F) and angle (G). (H) W17 mediates diverse interactions with carboxylic-acid containing fragments and the oxyanion subsite. The fragments are shown with blue sticks. (I,J) W17 is displaced by 2 out of the 178 fragments binding in the adenosine site. Two conformations of Verdiperstat were observed.

Fig. 8.

Mechanism of ADPr-ribose hydrolysis catalyzed by the SARS-CoV-2 NSP3 macrodomain. (A) Composite image showing the ADPr-bound Mac1 structure (white sticks/cartoon/surface, PDB 7KQP). ADPr is shown in the configuration that is compatible with a substrate-assisted mechanism. The terminal ribose adopts the α-configuration and W8 acts as the water nucleophile. For clarity, only the side chains of selected residues are shown. Hydrogen bonds are shown with dashed black lines and the W6-C1” trajectory is shown with a dashed red line. (B) Same as (A), but showing the β-configuration of the terminal ribose that is compatible with His45 acting as a general base to activate W6 as a nucleophile. (C, D) Chemical structures showing the two possible mechanisms for ADPr hydrolysis. (E) Top: structural alignment of the SARS-CoV-2 macrodomain (PDB code 7KQP), Chikungunya virus macrodomain (PDB code 3GPO) and the human macrodomain hMacroD2 (PDB code 4IQY), all in complex with ADPr. Bottom: protein sequence alignment of residues equivalent to residues 40–51 from the SARS-CoV-2 macrodomain. (F) X-ray crystal structure of the NSP3 macrodomain from the Tylonycteris bat coronavirus HKU4 in complex with NAD+ (PDB code 6MEB). The terminal ribose is rotated ~180° relative to ADPr. This configuration matches the model proposed in (B/D).