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Variable Macro X Domain of SARS-Cov-2 Retains the Ability to Bind ADP-Ribose David N

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Variable Macro X Domain of SARS-Cov-2 Retains the Ability to Bind ADP-Ribose David N bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.014639; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Variable Macro X Domain of SARS-CoV-2 Retains the Ability to Bind ADP-ribose David N. Frick,1* Rajdeep S. Virdi,1 Nemanja Vuksanovic,1 Narayan Dahal,2 & Nicholas R Silvaggi1 Departments of Chemistry & Biochemistry,1 and Physics,2 The University of Wisconsin- Milwaukee, Milwaukee, WI 53217 KEYWORDS: COVID-19, Antiviral Drug target, Severe Acute Respiratory Syndrome, Coronavirus. Supporting Information Placeholder ABSTRACT: The virus that causes COVID-19, SARS- to bind ADP-ribose, that will be referred to here as the CoV-2, has a large RNA genome that encodes numerous “macro X” domain, to differentiate it from the two proteins that might be targets for antiviral drugs. Some downstream “SARS unique macrodomains (SUDs),” 3 of these proteins, such as the RNA-dependent RNA pol- which do not bind ADP-ribose. The macro X domain of ymers, helicase and main protease, are well conserved SARS-CoV-1 is also able to catalyze the hydrolysis of 4 between SARS-CoV-2 and the original SARS virus, but ADP-ribose 1’’ phosphate, albeit at a slow rate. several others are not. This study examines one of the All the above differences preclude the use of the most novel proteins encoded by SARS-CoV-2, a SARS-CoV-1 macro X domain structures as scaffolds to macrodomain of nonstructural protein 3 (nsp3). Alt- design compounds that might target this nsp3 region in hough 26% of the amino acids in this SARS-CoV-2 SARS CoV-2, especially in light of the observation that macrodomain differ from those seen in other corona- the same nsp3 domain from gamma coronaviruses folds viruses, the protein retains the ability to bind ADP- with a closed binding site and does not bind ADP-ribose ribose, which is an important characteristic of beta coro- in vitro.5 Because ADP-ribose binding is a property that naviruses, and potential therapeutic target. could be used to identify possible antiviral agents, the ability of the SARS-CoV-2 macro X domain to bind ADP-ribose was examined using a recombinant purified protein and isothermal titration calorimetry (ITC). The development of antivirals targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), RESULTS AND DISCUSSION the causative agent of the present COVID-19 pandemic,1 will most likely focus on viral proteins and enzymes Hypervariability in the nsp3 Macro X domain— The needed for replication.2 Like other coronaviruses, structures of most of the soluble portions of the SARS- SARS-CoV-2 has a large positive sense (+)RNA genome CoV-1 nsp proteins have been examined at atomic reso- over 30,000 nucleotides long with several open reading lution to help understand coronavirus replication and frames. Most of the proteins that form the viral replicase facilitate antiviral drug discovery. The amino acid se- are encoded by the “rep 1ab” reading frame, which quences of each of these proteins were compared with codes for a 7,096 amino acid-long polyprotein that is the homologous regions of the rep 1ab protein encoded ultimately processed into at least 15 functional peptides, by SARS-CoV-2 (GenPept Accession YP_009724389). five of which are only produced by a translational The most similar proteins were the RNA helicases frameshift event occurring after nsp10 (Fig. 1). Parts of (nsp13) which are identical in all but one of their 603 the SARS-CoV-2 rep 1ab polyprotein are very similar to amino acids: a conservative Val to Ile substitution near the rep 1ab protein of the coronavirus that caused the their C-termini. The RNA-dependent RNA polymerases SARS epidemic in 2003 (which will be referred to here (nsp12) are also well-conserved, sharing all but 34 of as SARS-CoV-1), suggesting the that drugs targeting the 955 amino acids. The primary protease that cleaves the SARS-CoV-1 nsp5-14 might be effective against SARS- polyprotein (nsp5) is also similar in SARS-CoV-1 and CoV-2. However, some portions of the SARS rep 1ab SARS-CoV-2, with only 13 amino acids that differ polyproteins are quite different. among 306 (4.2% different) (Fig. 1). In contrast to the well-conserved SARS nsp5 protease, At the other end of the spectrum are the nsp3 proteins, nsp12 polymerase and nsp13 helicase enzymes, signifi- which are notably more different in the two SARS vi- ruses. Nsp3 is a large multidomain membrane-bound cantly more differences exist between the nsp3 proteins 6 encoded by SARS-CoV-1 and SARS-CoV-2. The most protein, and its clearest role in viral replication is cleav- variation occurs in a domain of Nsp3 domain suspected ing the rep polyprotein. Over 17% of the amino acids in bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.014639; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. the nsp3 protease domain differ between SARS-CoV-1 SARS-CoV-1 structure (PDB 2fav), these contacts likely and SARS-CoV-2. Other parts of nsp3 are even more occur with the two conserved aspartates (residues 40 and variable, like the macrodomains that lie N-terminal to 43 in Fig. 2A). None of the other nucleotides bind with the nsp3 protease domain. Macrodomains consist of four an entropic penalty as seen with ADP-ribose, either, helices that surround a mixed beta sheet. A ligand- suggesting that the ribose moiety becomes structured binding pocket that typically binds ADP-ribose or relat- when bound to the macrodomain. 7 ed compounds lies between the helices and the sheet. The energetics of ADP-ribose binding to the SARS- SARS-CoV-1 has three macrodomains in tandem, but CoV protein are similar to those seen with the same pro- only the first binds ADP ribose. The amino acid se- tein from SARS-CoV-1,9 MERS-CoV.8 Enthalpy and quences of this macro X domain differ by 26% between entropy of binding were also very similar for all three SARS-CoV-1 and SARS-CoV-2 (Fig. 1). protein (Fig. 3D). Unlike what is seen with the macro X Many of the 47 variant residues in the 180 amino acid protein from an alpha coronavirus,5 enthalpy appears to long SARS macro X domain are clustered near its N- drive ADP-ribose binding to the macro X domains of the terminus in a region that is particularly variable in the three beta coronaviruses. three Coronaviridae genera (Fig. 2A). Macro X domains To address the molecular basis for ADP-ribose bind- from coronaviruses that cause the common cold (alpha 5 ing, crystallization experiments were performed using coronaviruses) and the beta coronaviruses like SARS- the purified SARS-CoV-2 protein and the conditions CoV-1 and Middle East respiratory syndrome corona- 8 used to crystallize the homologous protein from SARS- virus (MERS-CoV) all bind ADP ribose. However, the CoV-1.9 Unfortunately, no crystals formed, nor did crys- same domain in gamma coronaviruses does not bind 5 tals form in any of the sparse matrix crystallization ADP-ribose. screens that were tested. Although both proteins possess When atomic models of the SARS-CoV-2 macro X the same N-terminal tag, it is possible that it prevents domain were generated with the MODELLER24 plugin crystallization of the CoV-2 protein, and this idea is for UCSF Chimera (v. 1.14)23 using the structure of the presently being tested by subcloning the protein into SARS-CoV-1 macro X domain (PDB file 2fav)9 as a other expression vectors. template, most of the two protein backbones superim- Conclusion— The significance of the study stems posed, but the N-termini did not (Fig. 2B, C). In fact, the mainly from the demonstration that the SARS-CoV-2 SARS-CoV-2 model with the lowest zDOPE score (- macro X domain binds ADP ribose. This is the first step 0.61) folds with the N-terminus blocking the ADP ribose needed to justify screens for potential antivirals that bind binding site (Fig. 2C), which is reminiscent of what is 5 in place of ADP ribose. More work needs to be done, seen in in the gamma coronaviruses (PDB file 3eke). however, to understand the antiviral potential of such However, in other models, the protein folds in a more compounds because the biological role for ADP-ribose open conformation with a free ADP-ribose-binding cleft binding is still not fully understood. Some work with (Fig. 2B). alpha coronaviruses suggest that ADP-ribose binding by Expression and Purification of the SARS-CoV-2 Mac- the macro X domain is not needed for viral replication.10 ro X domain— An E. coli expression vector for the mac- However, studies with other (+)RNA viruses suggest ro X domain was built to express and N-terminally His- that macrodomains are essential for virulence.11 This tagged protein similar to a SARS-CoV-1 protein studied work is also noteworthy because the synthetic codon- by Egloff et al.9 Upon induction, a one liter culture of optimized plasmid reported here produces up to 100 mg BL21(DE3) cells harboring the vector expresses 50-100 of soluble macro X domain protein per liter of E. coli mg of the macro X domain protein that can be purified culture, and this protein retains a high affinity for ADP in one step to apparent homogeneity using immobilized ribose.
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