key: cord-0985359-3qpq62ac
authors: Alhammad, Yousef M.O.; Kashipathy, Maithri M.; Roy, Anuradha; Gagné, Jean-Philippe; McDonald, Peter; Gao, Philip; Nonfoux, Louis; Battaile, Kevin P.; Johnson, David K.; Holmstrom, Erik D.; Poirier, Guy G.; Lovell, Scott; Fehr, Anthony R.
title: The SARS-CoV-2 conserved macrodomain is a mono-ADP-ribosylhydrolase
date: 2020-10-28
journal: bioRxiv
DOI: 10.1101/2020.05.11.089375
sha: e7f5b1e420bb9b77a9d28ddc9e83900f4f97ca78
doc_id: 985359
cord_uid: 3qpq62ac

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 900 thousand deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of 48 SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and 49 hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These 50 results are an important first step for the design and testing of potential therapies targeting this 51 unique protein domain. 52 53

The recently emerged pandemic outbreak of COVID-19 is caused by a novel coronavirus 55 named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1, 2). As of September 56 16, 2020, this virus has been responsible for ~ 30 million cases of COVID-19 and >900,000 57 deaths worldwide. SARS-CoV-2 is a member of the lineage B β-CoVs with overall high 58 sequence similarity with other SARS-like CoVs, including SARS-CoV. While most of the 59 protein domains likely have similar functions, and will be instrumental in the design and testing 121 of novel therapeutic agents targeting the CoV Mac1 protein domain. 122

Structure of the SARS-CoV-2 Mac1 complexed with ADP-ribose. To create recombinant 125 SARS-CoV-2 Mac1 for structure determination and enzyme assays, nucleotides 3348-3872 of 126 SARS-CoV-2 isolate Wuhan-hu-1 (accession number NC_045512), representing amino acids 127 I1023-K1197 of rep1a, were cloned into a bacterial expression vector containing an N-terminal 128 6X-His tag and TEV cleavage site. We obtained large amounts (>100 mg) of purified 129 recombinant protein (Fig. S1A) . A small amount of this protein was digested by the TEV 130 protease to obtain protein devoid of any extra tags for crystallization and used to obtain crystals 131 from which the structure was determined (Fig. S1B ). Our crystallization experiments resulted in 132 the same crystal form (needle clusters) from several conditions, but only when ADP-ribose was 133 added to the protein. This represents an additional crystal form (P21) amongst the recently 134 determined SARS-CoV-2 macrodomain structures (29-31). 135

The structure of SARS-CoV-2 Mac1 complexed with ADP-ribose was obtained using X-136 ray diffraction data to 2.2 Å resolution and contained four molecules in the asymmetric unit that 137 were nearly identical. The polypeptide chains could be traced from V3-M171 for subunits A/C 138 and V3-K172 for subunits B/D. Superposition of subunits B-D onto subunit A (169 residues 139 aligned) yielded RMSD deviations of 0.17 Å, 0.17 Å and 0.18 Å respectively between Cα atoms. 140

As such, subunit A was used for the majority of the structure analysis described herein. The 141 SARS-CoV-2 Mac1 protein adopted a fold consistent with the MacroD sub-family of 142 macrodomains that contains a core composed of a mixed arrangement of 7 β-sheets (parallel and 143 antiparallel) that are flanked by 6 α-helices ( Fig. 2A-B) . 144

As mentioned above, apo crystals were never observed for our construct, though the apo 145 structure has been solved by researchers at The Center for Structural Genomics of Infectious 146 Diseases (PDB 6WEN) (30) and the University of Wisconsin-Milwaukee (PDB 6WEY) (32) . 147

Further analysis of the amino acid sequences used for expression and purification revealed that 148 our construct had 5 additional residues at the C-terminus (MKSEK) and differs slightly at the N-149 terminus as well (GIE vs GE) relative to 6WEN. In addition, the sequence used to obtain the 150 structure of 6WEY is slightly shorter than SARS-CoV-2 Mac1 at both the N and C-terminal 151 regions (Fig. S2A) . To assess the effect of these additional residues on crystallization, chain B 152 of the SARS-CoV-2 Mac1, which was traced to residue K172, was superimposed onto subunit A 153 of PDB 6W02 (31), a previously determined structure of ADP-ribose bound SARS-CoV-2 Mac1. 154

Analysis of the crystal packing of 6W02 indicates that the additional residues at the C-terminus 155 would clash with symmetry related molecules (Fig. S2B ). This suggests that the presence of 156 these extra residues at the C-terminus likely prevented the generation of the more tightly packed 157 crystal forms obtained for 6W02 and 6WEY, which diffracted to high resolution. 158

The ADP-ribose binding pocket contained large regions of positive electron density 159 consistent with the docking of ADP-ribose (Fig. 3A) . The adenine forms two hydrogen bonds 160 with D22-I23, which makes up a small loop between β2 and the N-terminal half of α1. The side 161 chain of D22 interacts with N6, while the backbone nitrogen atom of I23 interacts with N1, in a 162 very similar fashion to the SARS-CoV macrodomain (6). This aspartic acid is known to be 163 critical for ADP-ribose binding for alphavirus macrodomains (26, 27) . A large number of 164 contacts are made in the highly conserved loop between β3 and α2 which includes many highly-165 conserved residues, including a GGG (motif) and N40, which is completely conserved in all 166 enzymatically active macrodomains (33). N40 is positioned to make hydrogen bonds with the 3' 167 OH groups of the distal ribose, as well as a conserved water molecule ( Fig. 3B-C) . K44 and G46 168 also make hydrogen bonds with the 2' OH of the distal ribose, G48 makes contact with the 1' 169 OH and a water that resides near the catalytic site, while the backbone nitrogen atom of V49 170 hydrogen bonds with the α-phosphate. The other major interactions with ADP-ribose occur in 171 another highly conserved region consisting of residues G130, I131, and F132 that are in the loop 172 between β6 and α5 (Fig. 3B) . The α-phosphate accepts a hydrogen bond from the nitrogen atom 173 of I131, while the β-phosphate accepts hydrogen bonds from the backbone nitrogen atom of 174 G130 and F132. The phenyl ring of F132 may make van der Waals interactions with the distal 175 ribose to stabilize it, which may contribute to binding and hydrolysis (34). Loops β3-α2 and β6-176 α5 are connected by an isoleucine bridge that forms a narrow channel around the diphosphate 177 which helps position the terminal ribose for water-mediated catalysis (6). Of all these residues, is 178 not exactly clear which ones are important for ADP-ribose binding, hydrolysis, or both. 179

Additionally, a network of direct contacts of ADP-ribose to solvent along with water mediated 180 contacts to the protein are shown (Fig. 3C) . 181

Comparison of SARS-CoV-2 Mac1 with other CoV macrodomain structures. We 182 next sought to compare the SARS-CoV-2 Mac1 to other deposited structures of this protein. Superposition with Apo (6WEN) and ADP-ribose complexed protein (6W02) yielded RMSD of 184 0.48 Å (168 residues) and 0.37 Å (165 residues), respectively, indicating a high degree of 185 similarity ( Fig. S3A-B) . Comparison of the ADP-ribose binding site of SARS-CoV-2 Mac1 with 186 that of the apo structure (6WEN) revealed minor conformational differences in order to 187 accommodate ADP-ribose binding. The loop between β3 and α2 (H45-V49) undergoes a change 188 in conformation and the sidechain of F132 is moved out of the ADP-ribose binding site (Fig.  189 S3C). Our ADP-ribose bound structure is nearly identical to 6W02, except for slight deviations 190 in the β3-α2 loop and an altered conformation of F156, where the aryl ring of F156 is moved 191 closer to the adenine ring ( Fig. S3 C-D) . However, this is likely a result of crystal packing as 192 F156 adopts this conformation in each subunit and would likely clash with subunit residues 193 related by either crystallographic or non-crystallographic symmetry. 194

We next compared the ADP-ribose bound SARS-CoV-2 Mac1 structure with that of 195 SARS-CoV (PDB 2FAV) (6) and MERS-CoV (PDB 5HOL) (35) Mac1 proteins. Superposition 196 yielded RMSD deviations of 0.71 Å (166 residues) and 1.06 Å (161 residues) for 2FAV and 197 5HOL, respectively. Additionally, the ADP-ribose binding mode in the SARS-CoV and CoV-2 structures almost perfectly superimposed ( Fig. 4A-D) . The conserved aspartic acid 199 residue (D22, SARS-CoV-2) that binds to adenine, is localized in a similar region in all 3 200 proteins although there are slight differences in the rotamers about the Cb-Cg bond. The angles 201 between the mean planes defined by the OD1, CG and OD2 atoms relative to SARS-CoV-2 202 Mac1 is 23.1 o and 46.5 o for the SARS-CoV and MERS-CoV Mac1 structures, respectively. 203

Another notable difference is that SARS-CoV and SARS-CoV-2 macrodomains have an 204 isoleucine (I23) following this aspartic acid while MERS-CoV has an alanine (A22). Conversely, 205 SARS-CoV-2 and SARS-CoV Mac1 have a valine instead of an isoleucine immediately 206 following the GGG motif (V49/I48). From these structures it appears that having two isoleucines 207 in this location would clash, and that lineage B and lineage C β-CoVs has evolved in unique 208 ways to create space in this pocket ( Fig. 4D and data not shown). Despite these small differences 209 in local structure, the overall structure of CoV Mac1 domains remain remarkably conserved, and 210 indicates they likely have similar biochemical activities and biological functions. 211

affinities. To determine if the CoV macrodomains had any noticeable differences in their ability 213 to bind ADP-ribose, we performed isothermal titration calorimetry (ITC), which measures the 214 energy released or absorbed during a binding reaction. Macrodomain proteins from human 215 (Mdo2), SARS-CoV, MERS-CoV, and SARS-CoV-2 were purified (Fig. S1A ) and tested for 216 their affinity to ADP-ribose. All CoV Mac1 proteins bound to ADP-ribose with low micromolar 217 affinity (7-16 μM), while human Mdo2 bound with an affinity about 10-times stronger (~220 218 nM) (Fig. 5A-B) . As a control we tested the ability of the MERS-CoV macrodomain to bind to 219 ATP, and only observed minimal binding with mM affinity (data not shown). At higher 220 concentrations, the SARS-CoV-2 macrodomain caused a slightly endothermic reaction, 221 potentially the result of protein aggregation or a change in conformation (Fig. 5A) . The MERS-222

CoV Mac1 had a greater affinity for ADP-ribose than SARS-CoV or SARS-CoV-2 Mac1 in the 223 ITC assay (Fig. 5A-B) , however, our results found the differences between these macrodomain 224 proteins to be much closer than previously reported (9). As an alternate method to confirm ADP-225 ribose binding, we conducted a thermal shift assay. All 4 macrodomains tested denatured at 226 higher temperatures with the addition of ADP-ribose (Fig. S4 ). We conclude that lineage B and 227 lineage C β-CoV Mac1 proteins bind to ADP-ribose with similar affinities. 228

CoV macrodomains are MAR-hydrolases. To examine the MAR-hydrolase activity of 229

CoV Mac1, we first tested the viability of using ADP-ribose binding reagents to detect 230 MARylated protein. Previously, radiolabeled NAD + has been the primary method used to label 231 MARylated protein (16, 17) . To create a MARylated substrate, the catalytic domain of the 232 PARP10 (GST-PARP10 CD) protein was incubated with NAD + , leading to its automodification. 233 PARP10 CD is a standard substrate that has been used extensively in the field to analyze the 234 activity of macrodomains (16, 18, 26, 27) . PARP10 is highly upregulated upon CoV infection 235 (23, 36) and is known to primarily auto-MARylate itself on acidic residues, which are the targets 236 of the MacroD2 class of macrodomains (27). We then tested a panel of anti-MAR, anti-PAR, or 237 both anti-MAR and anti-PAR binding reagents/antibodies for the ability to detect MARylated 238 PARP10 by immunoblot. The anti-MAR and anti-MAR/PAR binding reagents, but not anti-PAR 239 antibody, bound to MARylated PARP10 (Fig. S5) . Therefore, in this work we utilized the anti-240 MAR binding reagent to detect MARylated PARP10. 241

We next tested the ability of SARS-CoV-2 Mac1 to remove ADP-ribose from 242 We conclude that macrodomain proteins are unable to remove PAR from an automodified 272 PARP1 protein under these conditions. 273 ELISA assays can be used to measure ADP-ribosylhydrolase activity of 274 macrodomains. Gel based assays as described above suffer from significant limitations in the 275 number of samples that can be done at once. A higher throughput assay will be needed to more 276 thoroughly investigate the activity of these enzymes and to screen for inhibitor compounds. 277

Based on the success of our antibody-based detection of MAR, we developed an ELISA assay 278 that has a similar ability to detect de-MARylation as our gel-based assay, but with the ability to 279 do so in a higher throughput manner (Fig. 8A ). First, MARylated PARP10 was added to ELISA 280 plates. Next, the wells were washed and then incubated with different concentrations of the 281 SARS-CoV-2 Mac1 protein for 60 min. After incubation, the wells were washed and treated with 282 anti-MAR binding reagent, followed by HRP-conjugated secondary antibody and the detection 283 reagent. As controls, we detected MARylated and non-MARylated PARP10 proteins bound to 284 glutathione plates with anti-GST antibody and anti-MAR binding reagents and their 285 corresponding secondary antibodies (Fig. 8B ). SARS-CoV-2 Mac1 was able to remove MAR 286 signal in a dose-dependent manner and plotted to a linear non-regression fitted line (Fig. 8C) . 287

Based on these results, we believe that this ELISA assay will be a useful tool for screening 288 potential inhibitors of macrodomain proteins. 289

Here we report the crystal structure of SARS-CoV-2 Mac1 and its enzyme activity in 291 vitro. Structurally, it has a conserved three-layered α/β/α fold typical of the MacroD family of 292 macrodomains, and is extremely similar to other CoV Mac1 proteins ( Fig. 2-4) . The conserved 293

CoV macrodomain (Mac1) was initially described as an ADP-ribose-1"-phosphatase (ADRP), as 294 it was shown to be structurally similar to yeast enzymes that have this enzymatic activity (37). 295

Early biochemical studies confirmed this activity for CoV Mac1, though its phosphatase activity 296 for ADP-ribose-1"-phosphate was rather modest (6-8). Later, it was shown that mammalian 297 macrodomain proteins could remove ADP-ribose from protein substrates, indicating protein de-298 ADP-ribosylation as a more likely function for the viral macrodomains (33, 38, 39 In this study, we show that the Mac1 proteins from SARS-CoV, MERS-CoV and SARS-303

CoV-2 hydrolyze MAR from a protein substrate (Fig. 6 ). Their enzymatic activities were similar 304 despite sequence divergence of almost 60% between SARS-CoV-2 and MERS-CoV. However, 305 the initial rate associated with the loss of substrate was largest for the SARS-CoV-2 Mac1 306 protein, particularly under multiple-turnover conditions. It is unclear what structural or sequence 307 differences may account for the increased activity of the SARS-CoV-2 Mac1 protein under these 308 conditions, especially considering the pronounced structurally similarities between these 309 proteins, specifically the SARS-CoV Mac1 (0.71 Å RMSD). It is also unclear if these differences 310 would matter in the context of the virus infection, as the relative concentrations of Mac1 and its 311 substrate during infection is not known. We also compared these activities to the human Mdo2 312 macrodomain. Mdo2 had a greater affinity for ADP-ribose than the viral enzymes, but had 313 significantly reduced enzyme activity in our experiments. Due to its high affinity for ADP-314 ribose, it is possible that the Mdo2 protein was partially inhibited by rebinding to the MAR 315 product in these assays. Regardless, these results suggest that the human and viral proteins likely 316 have structural differences that alter their biochemical activities in vitro, indicating that it may be 317 possible to create viral macrodomain inhibitors that don't impact the human macrodomains. We 318 also compared the ability of these macrodomain proteins to hydrolyze PAR. None of the 319 macrodomains were able to hydrolyze either partially or heavily modified PARP1, further 320 demonstrating that the primary enzymatic activity of these proteins is to hydrolyze MAR (Fig.  321   7) . 322

When analyzing viral macrodomain sequences, it is clear that they have at least 3 highly 323 conserved regions (Fig. 1B) (24). The first region includes the NAAN (37-40) and GGG (residues 324 46-48) motifs in the loop between β3 and α2. The second domain includes a GIF (residues 130-325 132) motif in the loop between β6 and α5. The final conserved region is a VGP (residues 96-98) 326 motif at the end of β5 and extends into the loop between β5 and α4. Both of the first two 327 domains have well defined interactions with ADP-ribose (Fig. 3) . However, no one has 328 addressed the role of the VGP residues, though our structure indicates that the glycine may 329 interact with a water molecule that makes contact with the β-phosphate. Identifying residues that 330 directly contribute to ADP-ribose binding, hydrolysis, or both by CoV Mac1 proteins will be 331 critical to determining the specific roles of ADP-ribose binding and hydrolysis in CoV 332 replication and pathogenesis. 333

While all previous studies of macrodomain de-ADP-ribosylation have primarily used 334 radiolabeled substrate, we obtained highly repeatable and robust data utilizing ADP-ribose 335 binding reagents designed to specifically recognize MAR (40, 41) . The use of these binding 336 reagents should enhance the feasibility of this assay for many labs that are not equipped for 337 radioactive work. Utilizing these binding reagents, we further developed an ELISA assay for de-338 MARylation that has the ability to dramatically increase the number of samples that can be 339 analyzed compared to the gel-based assay. To our knowledge, previously developed ELISA 340 assays were used to measure ADP-ribosyltransferase activities (42) but no ELISA has been 341 established to test the ADP-ribosylhydrolase activity of macrodomain proteins. This ELISA 342 assay should be useful to those in the field to screen compounds for macrodomain inhibitors that 343 could be either valuable research tools or potential therapeutics. 344

The functional importance of the CoV Mac1 domain has been demonstrated in several 345 reports, mostly utilizing the mutation of a highly conserved asparagine that mediates contact with 346 the distal ribose (Fig. 3B) (18, 21, 22) . However, the physiological relevance of Mac1 during 347 SARS-CoV-2 infection has yet to be determined. In addition, the proteins that are targeted by the 348 CoV Mac1 for de-ADP-ribosylation remains unknown. Unfortunately, there are no known 349 compounds that inhibit this domain that could help identify the functions of this protein during 350 infection. The outbreak of COVID-19 has illustrated an urgent need for developing multiple 351 therapeutic drugs targeting conserved coronavirus proteins. Mac1 appears to be an ideal 352 candidate for further drug development based on: i) its highly conserved structure and 353 biochemical activities within CoVs; and ii) its importance for multiple CoVs to cause disease. 354

Targeting Mac1 may also have the benefit of enhancing the innate immune response, as we have 355

shown that Mac1 is required for some CoVs to block IFN production (18, 23). Considering that 356

Mac1 proteins from divergent αCoVs such as 229E and FIPV also have de-ADP-ribosylating 357 activity (16, 17), it is possible that compounds targeting Mac1 could prevent disease caused by 358 of wide variety of CoV, including those of veterinary importance like porcine epidemic diarrhea 359 virus (PEDV). Additionally, compounds that inhibit Mac1 in combination with the structure 360 could help identify the mechanisms it uses to bind to its biologically relevant protein substrates, 361 remove ADP-ribose from these proteins, and potentially define the precise function for Mac1 in 362 SARS-CoV-2 replication and pathogenesis. In conclusion, the results described here will be 363 critical for the design and development of highly-specific Mac1 inhibitors that could be used 364 therapeutically to mitigate COVID-19 or future CoV outbreaks. 365

Plasmids 368

The SARS-CoV macrodomain (Mac1) (residues 1000-1172 of pp1a) was cloned into the 369 pET21a+ expression vector with an N-terminal His tag. The MERS-CoV Mac1 (residues 1110-370 1273 of pp1a) was also cloned into pET21a+ with a C-terminal His tag. SARS-CoV-2 Mac1 371 (residues 1023-1197 of pp1a) was cloned into the pET30a+ expression vector with an N-terminal 372

His tag and a TEV cleavage site (Synbio). The pETM-CN Mdo2 Mac1 (residues 7-243) 373 expression vector with an N-terminal His-TEV-V5 tag and the pGEX4T-PARP10-CD (residues 374 818-1025) expression vector with an N-terminal GST tag were previously described (33). All 375 plasmids were confirmed by restriction digest, PCR, and direct sequencing. 376

A single colony of E. coli cells (C41(DE3)) containing plasmids harboring the constructs 378 of the macrodomain proteins was inoculated into 10 mL LB media and grown overnight at 37°C 379 with shaking at 250 rpm. The overnight culture was transferred to a shaker flask containing 2X 380 1L TB media at 37 o C until the OD600 reached 0.7. The proteins were either induced with 0.4 381 mM IPTG at 37 o C for 3 hours, or 17°C for 20 hours. Cells were pelleted at 3500 × g for 10 min 382 and frozen at -80°C. Frozen cells were thawed at room temperature, resuspended in 50 mM Tris 383 (pH 7.6), 150 mM NaCl, and sonicated using the following cycle parameters: Amplitude: 50%, 384

Pulse length: 30 seconds, Number of pulses: 12, while incubating on ice for >1min between 385 pulses. The soluble fraction was obtained by centrifuging the cell lysate at 45,450 × g for 30 386 minutes at 4°C. The expressed soluble proteins were purified by affinity chromatography using 387 a 5 ml prepacked HisTrap HP column on an AKTA Pure protein purification system (GE 388

Healthcare). The fractions were further purified by size-exclusion chromatography (SEC) with a 389 Superdex 75 10/300 GL column equilibrated with 20mM Tris (pH 8.0), 150 mM NaCl and the 390 protein sized as a monomer relative to the column calibration standards. To cleave off the His tag 391 from the SARS-CoV-2 Mac1, purified TEV protease was added to purified SARS-CoV-2 Mac1 392 protein at a ratio of 1:10 (w/w), and then passed back through the Ni-NTA HP column. Protein 393 was collected in the flow through and equilibrated with 20 mM Tris (pH 8.0), 150 mM NaCl. 394

The SARS-CoV-2 Mac1, free from the N-terminal 6X-His tag, was used for subsequent 395 crystallization experiments. 396

For the PARP10-CD protein, the cell pellet was resuspended in 50 mM Tris-HCl (pH 397 8.0), 500 mM NaCl, 0.1mM EDTA, 25% glycerol, 1 mM DTT and sonicated as described above. 398

The cell lysate was incubated with 10 ml of Glutathione Sepharose 4B resin from GE Healthcare, 399 equilibrated with the same buffer for 2 hours, then applied to a gravity flow column to allow NaH2PO4/K2HPO4, pH 8.2). Refinement screening was conducted using the additive screen HT 498 (Hampton Research) by supplementing 10% of each additive to the Salt Rx HT E10 condition in 499 a new 96-well UVXPO crystallization plate. The crystals used for data collection were obtained 500 from Salt Rx HT E10 supplemented with 0.1 M NDSB-256 from the additive screen (Fig. S1) . 501

Samples were transferred to a fresh drop composed of 80% crystallization solution and 20% 502

(v/v) PEG 200 and stored in liquid nitrogen. X-ray diffraction data were collected at the 503 Advanced Photon Source, IMCA-CAT beamline 17-ID using a Dectris Eiger 2X 9M pixel array 504 detector. 505

Structure Solution and Refinement: Intensities were integrated using XDS (45, 46) via 506 Autoproc (47) and the Laue class analysis and data scaling were performed with Aimless (48). 507

Notably, a pseudo-translational symmetry peak was observed at (0, 0.31 0.5) that was 44.6% of 508 the origin. Structure solution was conducted by molecular replacement with Phaser (49) using a 509 previously determined structure of ADP-ribose bound SARS-CoV-2 Mac1 (PDB 6W02) as the 510 search model. The top solution was obtained in the space group P21 with four molecules in the 511 asymmetric unit. Structure refinement and manual model building were conducted with Phenix 512 (50) and Coot (51) respectively. Disordered side chains were truncated to the point for which 513 electron density could be observed. Structure validation was conducted with Molprobity (52) and 514 figures were prepared using the CCP4MG package (53). Superposition of the macrodomain 515 structures was conducted with GESAMT (54). 516

All statistical analyses were done using an unpaired two-tailed student's t-test to assess 518 differences in mean values between groups, and graphs are expressed as mean ±SD. Significant p 519 values are denoted with *p≤0.05. 520

The coordinates and structure factors for SARS-CoV-2 Mac1 were deposited to the 522 Worldwide Protein Databank (wwPDB) with the accession code 6WOJ. 523

We'd like to thank Ivan Ahel and Gytis Jankevicius (Oxford University) for providing 525 protein expression plasmids; John Pascal (University of Montreal) and Marie-France Langelier 526 nM) for the indicated times at 37°C. ADP-ribosylated PARP10 CD was detected as described 783 above, and total PARP10 CD and macrodomain protein levels were determine by Coomassie 784 Blue (Fig. S6) . D) Time-dependent substrate concentrations were determined by quantifying 785 band intensity using Image Studio software. The data were then analyzed using Mathematica 12, 786 as described in Methods, to determine the initial rate (k) of substrate decay. Results in C are 787 representative experiments of three independent experiments and data in D represent the 788 combined results of the three independent experiments. 789 

Conformational plasticity 599 of the VEEV macro domain is important for binding of ADP-ribose

The crystal structures of Chikungunya and Venezuelan equine 604 encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket

Macrodomains: Structure, Function, Evolution, and 607 Catalytic Activities

Viral Macro 609 Domains Reverse Protein ADP-Ribosylation

The conserved macrodomains of the non-612 structural proteins of Chikungunya virus and other pathogenic positive strand RNA 613 viruses function as mono-ADP-ribosylhydrolases

The Conserved Coronavirus Macrodomain Promotes Virulence 616 and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome 617 Coronavirus Infection

PARPs and ADP-ribosylation in RNA 619 biology: from RNA expression and processing to protein translation and proteostasis

The impact of 622 PARPs and ADP-ribosylation on inflammation and host-pathogen interactions

Mouse hepatitis virus 625 liver pathology is dependent on ADP-ribose-1''-phosphatase, a viral function conserved in 626 the alpha-like supergroup

628 The nsp3 macrodomain promotes virulence in mice with coronavirus-induced 629 encephalitis

The coronavirus macrodomain is required to prevent 632 PARP-mediated inhibition of virus replication and enhancement of IFN expression

The Viral Macrodomain Counters Host Antiviral 635 ADP-Ribosylation

ADP-ribosyl-binding and hydrolase activities of the alphavirus 638 nsP3 macrodomain are critical for initiation of virus replication

Both ADP-Ribosyl-Binding and Hydrolase Activities of the Alphavirus nsP3 642 Macrodomain Affect Neurovirulence in Mice. mBio 11

Chikungunya virus macrodomain is critical for virus replication and virulence

The hepatitis E virus ORF1 'X-domain' residues form a putative 648 macrodomain protein/Appr-1''-pase catalytic-site, critical for viral RNA replication

National Science Foundation (NSF, United States)

High-resolution structure of the SARS-CoV-2 NSP3 Macro X domain

Joachimiak, 654 A

Center for Structural Genomics of Infectious Diseases (CSGID)

Crystal Structure of ADP ribose phosphatase of NSP3 from SARS CoV-2 in the complex 660 with ADP ribose

Molecular Basis for 662 ADP-Ribose Binding to the Mac1 Domain of SARS-CoV-2 nsp3

A family of macrodomain proteins reverses cellular mono-ADP-ribosylation

Macrodomain ADP-ribosylhydrolase and 668 the pathogenesis of infectious diseases

Nsp3 of coronaviruses: Structures and functions of a 670 large multi-domain protein

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A highly specific phosphatase that acts on 676 ADP-ribose 1''-phosphate, a metabolite of tRNA splicing in Saccharomyces cerevisiae

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Deficiency of terminal ADP-ribose protein glycohydrolase 685 TARG1/C6orf130 in neurodegenerative disease

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An ELISA method to estimate the mono ADP-694 ribosyltransferase activities: e.g in pertussis toxin and vaccines

Purification of human PARP-1 697 and PARP-1 domains from Escherichia coli for structural and biochemical analysis. 698

Crystallographic and biochemical analysis 700 of the mouse poly(ADP-ribose) glycohydrolase

Evaluation of Single-Crystal X-Ray-Diffraction Data from a Position-702 Sensitive Detector

Data processing and analysis with the autoPROC toolbox

An introduction to data reduction: space-group determination, scaling 708 and intensity statistics

710 Phaser crystallographic software

PHENIX: a 714 comprehensive Python-based system for macromolecular structure solution

Features and development of Coot

MolProbity: all-atom structure validation for 720 macromolecular crystallography

Developments in the CCP4 molecular-graphics 723 project

Enhanced fold recognition using efficient short fragment clustering

Scaling and assessment of data quality

Improved R-factors for diffraction data analysis in 729 macromolecular crystallography

Global indicators of X-ray data quality

Resolving some old problems in protein crystallography

Linking crystallographic model and data quality

coronavirus macrodomain structures. A) SARS-CoV Mac1 with ADP-ribose (gold) (2FAV) and