Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography
2020; Elsevier BV; Volume: 295; Issue: 50 Linguagem: Inglês
10.1074/jbc.ac120.016154
ISSN1083-351X
AutoresDaniel W. Kneller, G.N. Phillips, Kevin L. Weiss, Swati Pant, Qiu Zhang, Hugh O’Neill, Leighton Coates, Andrey Kovalevsky,
Tópico(s)Computational Drug Discovery Methods
ResumoThe main protease (3CL Mpro) from SARS–CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL Mpro possesses an unusual catalytic dyad composed of Cys145 and His41 residues. A critical question in the field has been what the protonation states of the ionizable residues in the substrate-binding active-site cavity are; resolving this point would help understand the catalytic details of the enzyme and inform rational drug development against this pernicious virus. Here, we present the room-temperature neutron structure of 3CL Mpro, which allowed direct determination of hydrogen atom positions and, hence, protonation states in the protease. We observe that the catalytic site natively adopts a zwitterionic reactive form in which Cys145 is in the negatively charged thiolate state and His41 is doubly protonated and positively charged, instead of the neutral unreactive state usually envisaged. The neutron structure also identified the protonation states, and thus electrical charges, of all other amino acid residues and revealed intricate hydrogen-bonding networks in the active-site cavity and at the dimer interface. The fine atomic details present in this structure were made possible by the unique scattering properties of the neutron, which is an ideal probe for locating hydrogen positions and experimentally determining protonation states at near-physiological temperature. Our observations provide critical information for structure-assisted and computational drug design, allowing precise tailoring of inhibitors to the enzyme's electrostatic environment. The main protease (3CL Mpro) from SARS–CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL Mpro possesses an unusual catalytic dyad composed of Cys145 and His41 residues. A critical question in the field has been what the protonation states of the ionizable residues in the substrate-binding active-site cavity are; resolving this point would help understand the catalytic details of the enzyme and inform rational drug development against this pernicious virus. Here, we present the room-temperature neutron structure of 3CL Mpro, which allowed direct determination of hydrogen atom positions and, hence, protonation states in the protease. We observe that the catalytic site natively adopts a zwitterionic reactive form in which Cys145 is in the negatively charged thiolate state and His41 is doubly protonated and positively charged, instead of the neutral unreactive state usually envisaged. The neutron structure also identified the protonation states, and thus electrical charges, of all other amino acid residues and revealed intricate hydrogen-bonding networks in the active-site cavity and at the dimer interface. The fine atomic details present in this structure were made possible by the unique scattering properties of the neutron, which is an ideal probe for locating hydrogen positions and experimentally determining protonation states at near-physiological temperature. Our observations provide critical information for structure-assisted and computational drug design, allowing precise tailoring of inhibitors to the enzyme's electrostatic environment. COVID-19, a deadly disease caused by the novel coronavirus SARS–CoV-2 (severe acute respiratory syndrome–coronavirus 2), is a pandemic of extraordinary proportions, disrupting social life, travel, and the global economy. The development of vaccines and therapeutic intervention measures promises to mitigate the spread of the virus and to alleviate the burdens COVID-19 has caused in many communities in recent months (1Liu C. Zhou Q. Li Y. Garner L.V. Watkins S.P. Carter L.J. Smoot J. Gregg A.C. Daniels A.D. Jervey S. Albaiu D. 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Thus, the enzyme is considered a promising target for the design and development of SARS–CoV-2–specific protease inhibitors and for repurposing existing clinical drugs (9Dai W. Zhang B. Jiang X.-M. Su H. Li J. Zhao Y. Xie X. Jin Z. Peng J. Liu F. Li C. Li Y. Bai F. Wang H. Cheng X. et al.Structure-based design of antiviral drug candidates targeting the SARS–CoV-2 main protease.Science. 2020; 368 (32321856): 1331-133510.1126/science.abb4489Crossref PubMed Scopus (823) Google Scholar, 10Zhang L. Lin D. Sun X. Curth U. Drosten C. Sauerhering L. Becker S. Rox K. Hilgenfeld R. Crystal structure of SARS–CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors.Science. 2020; 368 (32198291): 409-412Crossref PubMed Scopus (1855) Google Scholar, 11Jin Z. Du X. Xu Y. Deng Y. Liu M. Zhao Y. Zhang B. Li X. Zhang L. Peng C. Duan Y. Yu J. Wang L. Yang K. 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Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS–CoV-2 viral replication by targeting the viral main protease.Cell Res. 2020; 30 (32541865): 678-69210.1038/s41422-020-0356-zCrossref PubMed Scopus (427) Google Scholar, 15Riva L. Yuan S. Yin X. Martin-Sancho L. Matsunaga N. Pache L. Burgstaller-Muehlbacher S. De Jesus P.D. Teriete P. Hull M.V. Chang M.W. Chan J.F.-W. Cao J. Poon V.K.-M. Herbert K.M. et al.Discovery of SARS–CoV-2 antiviral drugs through large-scale compound repurposing.Nature. 2020; 586 (32707573): 113-11910.1038/s41586-020-2577-1Crossref PubMed Scopus (463) Google Scholar). SARS–CoV-2 3CL Mpro is a cysteine protease (16Gorbalenya A.E. Snijder E.J. Viral cysteine proteases.Perspect. Drug Discov. Des. 1996; 6 (32288276): 64-8610.1007/BF02174046Crossref PubMed Scopus (94) Google Scholar, 17Tong L. Viral proteases.Chem. Rev. 2002; 102 (12475203): 4609-462610.1021/cr010184fCrossref PubMed Scopus (136) Google Scholar) and is catalytically active as a homodimer (Fig. 1). Its amino acid sequence is 96% homologous to the earlier SARS–CoV 3CL Mpro, and the catalytic efficiencies of the two enzymes are similar (10Zhang L. Lin D. Sun X. Curth U. Drosten C. Sauerhering L. Becker S. Rox K. Hilgenfeld R. Crystal structure of SARS–CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors.Science. 2020; 368 (32198291): 409-412Crossref PubMed Scopus (1855) Google Scholar, 11Jin Z. Du X. Xu Y. Deng Y. Liu M. Zhao Y. Zhang B. Li X. Zhang L. Peng C. Duan Y. Yu J. Wang L. Yang K. Liu F. et al.Structure of Mpro from COVID-19 virus and discovery of its inhibitors.Nature. 2020; 582 (32272481): 289-29310.1038/s41586-020-2223-yCrossref PubMed Scopus (2253) Google Scholar, 18Anand K. Ziebuhr J. Wadhwani P. Mesters J.R. Hilgenfeld R. Coronavirus main protease (3CLpro) structure: basis for design of anti-SARS drugs.Science. 2003; 300 (12746549): 1763-176710.1126/science.1085658Crossref PubMed Scopus (1275) Google Scholar, 19Huang C. Wei P. Fan K. Liu Y. Lai L. 3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism.Biochemistry. 2004; 43 (15078103): 4568-457410.1021/bi036022qCrossref PubMed Scopus (163) Google Scholar, 20Solowiej J. Thomson J.A. Ryan K. Luo C. He M. Lou J. Murray B.W. Steady-state and pre-steady-state kinetic evaluation of severe acute respiratory syndrome coronavirus (SARS–CoV) 3CLpro cysteine protease: development of an ion-pair model for catalysis.Biochemistry. 2008; 47 (18237196): 2617-263010.1021/bi702107vCrossref PubMed Scopus (30) Google Scholar). The ∼34-kDa enzyme has three distinct domains: catalytic domains I (residues 8–101) and II (residues 102–184) and the α-helical domain III (residues 201–303), which is required for protein dimerization (10Zhang L. Lin D. Sun X. Curth U. Drosten C. Sauerhering L. Becker S. Rox K. Hilgenfeld R. Crystal structure of SARS–CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors.Science. 2020; 368 (32198291): 409-412Crossref PubMed Scopus (1855) Google Scholar, 11Jin Z. Du X. Xu Y. Deng Y. Liu M. Zhao Y. Zhang B. Li X. Zhang L. Peng C. Duan Y. Yu J. Wang L. Yang K. Liu F. et al.Structure of Mpro from COVID-19 virus and discovery of its inhibitors.Nature. 2020; 582 (32272481): 289-29310.1038/s41586-020-2223-yCrossref PubMed Scopus (2253) Google Scholar). Importantly, the monomeric enzyme shows no catalytic activity, as was demonstrated for SARS–CoV 3CL Mpro (21Fan K. Wei P. Feng Q. Chen S. Huang C. Ma L. Lai B. Pei J. Liu Y. Chen J. Lai L. Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase.J. Biol. Chem. 2004; 279 (14561748): 1637-164210.1074/jbc.M310875200Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 22Hsu W.-C. Chang H.-C. Chou C.-Y. Tsai P.-J. Lin P.-I. Chang G.-G. Critical assessment of important regions in the subunit association and catalytic action of the severe acute respiratory syndrome coronavirus main protease.J. Biol. Chem. 2005; 280 (15831489): 22741-2274810.1074/jbc.M502556200Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 23Lin P.-Y. Chou C.-Y. Chang H.-C. Hsu W.-C. Chang G.-G. Correlation between dissociation and catalysis of SARS–CoV main protease.Arch. Biochem. Biophys. 2008; 472 (18275836): 34-4210.1016/j.abb.2008.01.023Crossref PubMed Scopus (40) Google Scholar, 24Xia B. Kang X. Activation and maturation of SARS–CoV main protease.Protein Cell. 2011; 2 (21533772): 282-29010.1007/s13238-011-1034-1Crossref PubMed Scopus (78) Google Scholar). The catalytic site of 3CL Mpro employs a noncanonical Cys145–His41 dyad thought to be assisted by a water molecule hydrogen-bonded to the catalytic histidine (18Anand K. Ziebuhr J. Wadhwani P. Mesters J.R. Hilgenfeld R. Coronavirus main protease (3CLpro) structure: basis for design of anti-SARS drugs.Science. 2003; 300 (12746549): 1763-176710.1126/science.1085658Crossref PubMed Scopus (1275) Google Scholar, 25Kneller D.W. Phillips G. O'Neill H.M. Jedrzejczak R. Stols L. Langan P. Joachimiak A. Coates L. Kovalevsky A. Structural plasticity of SARS–CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography.Nat. Commun. 2020; 11 (32581217)320210.1038/s41467-020-16954-7Crossref PubMed Scopus (222) Google Scholar). The cysteine thiol group functions as the nucleophile during the first step of the hydrolysis reaction by attacking the carbon atom of the scissile peptide bond. Substrate hydrolysis requires the catalytic dyad to be in the zwitterionic state with deprotonated Cys145 and protonated His41, which either can be generated through a proton transfer from the Cys145 thiol to the His41 imidazole by a general acid-base mechanism (19Huang C. Wei P. Fan K. Liu Y. Lai L. 3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism.Biochemistry. 2004; 43 (15078103): 4568-457410.1021/bi036022qCrossref PubMed Scopus (163) Google Scholar) or may already be present before substrate binding (20Solowiej J. Thomson J.A. Ryan K. Luo C. He M. Lou J. Murray B.W. Steady-state and pre-steady-state kinetic evaluation of severe acute respiratory syndrome coronavirus (SARS–CoV) 3CLpro cysteine protease: development of an ion-pair model for catalysis.Biochemistry. 2008; 47 (18237196): 2617-263010.1021/bi702107vCrossref PubMed Scopus (30) Google Scholar, 26Paasche A. Zipper A. Schäfer S. Ziebuhr J. Schirmeister T. Engels B. Evidence for substrate binding–induced zwitterion formation in the catalytic Cys-His dyad of the SARS-Co-V main protease.Biochemistry. 2014; 53 (25196915): 5930-594610.1021/bi400604tCrossref PubMed Scopus (60) Google Scholar). However, the protonation states of the 3CL Mpro catalytic site have not been experimentally determined. The enzyme recognizes a general amino acid sequence of Leu-Gln↓Ser-Ala-Gly, where ↓ marks the cleavage site, but displays some substrate sequence promiscuity. The active-site cavity is located on the surface of the protease and can bind substrate residues in positions P1´ through P5 in the substrate-binding subsites S1ʹ–S5, respectively (Fig. 2). Subsites S1, S2, and S4 are shaped into well-formed binding pockets, whereas S1ʹ, S3, and S5 are located on the protein surface with no defined shape. The peptide bond cleavage occurs between the substrate residues at the C-terminal position P1ʹ and N-terminal position P1.Figure 2SARS–CoV-2 3CL Mpro active site architecture indicating the positions of substrate-binding subsites S1′–S5 and the oxyanion hole.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Current structure-assisted drug design efforts are mainly directed toward reversible and irreversible covalent inhibitors that mimic the protease substrate binding to subsites S1ʹ–S5 in the active-site cavity (9Dai W. Zhang B. Jiang X.-M. Su H. Li J. Zhao Y. Xie X. Jin Z. Peng J. Liu F. Li C. Li Y. Bai F. Wang H. Cheng X. et al.Structure-based design of antiviral drug candidates targeting the SARS–CoV-2 main protease.Science. 2020; 368 (32321856): 1331-133510.1126/science.abb4489Crossref PubMed Scopus (823) Google Scholar, 10Zhang L. Lin D. Sun X. Curth U. Drosten C. Sauerhering L. Becker S. Rox K. Hilgenfeld R. Crystal structure of SARS–CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors.Science. 2020; 368 (32198291): 409-412Crossref PubMed Scopus (1855) Google Scholar, 11Jin Z. Du X. Xu Y. Deng Y. Liu M. Zhao Y. Zhang B. Li X. Zhang L. Peng C. Duan Y. Yu J. Wang L. Yang K. Liu F. et al.Structure of Mpro from COVID-19 virus and discovery of its inhibitors.Nature. 2020; 582 (32272481): 289-29310.1038/s41586-020-2223-yCrossref PubMed Scopus (2253) Google Scholar, 12Rathnayake A.D. Zheng J. Kim Y. Perera K.D. Mackin S. Meyerholz D.K. Kashipathy M.M. Battaile K.P. Lovell S. Perlman S. Groutas W.C. Chang K.-O. 3C-like protease inhibitors block coronavirus replication in vitro and improve survival in MERS-CoV-infected mice.Sci. Transl. Med. 2020; 12 (32747425)eabc533210.1126/scitranslmed.abc5332Crossref PubMed Google Scholar, 14Ma C. Sacco M.D. Hurst B. Townsend J.A. Hu Y. Szeto T. Zhang X. Tarbet B. Marty M.T. Chen Y. Wang J. Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS–CoV-2 viral replication by targeting the viral main protease.Cell Res. 2020; 30 (32541865): 678-69210.1038/s41422-020-0356-zCrossref PubMed Scopus (427) Google Scholar), whereas the dimer interface can also be explored for the design of dimerization inhibitors (27Barrila J. Bacha U. Freire E. Long-range cooperative interactions modulate dimerization in SARS 3CLpro.Biochemistry. 2006; 45 (17154528): 14908-1491610.1021/bi0616302Crossref PubMed Scopus (62) Google Scholar, 28Goyal B. Goyal D. Targeting the dimerization of the main protease of coronaviruses: a potential broad-spectrum therapeutic strategy.ACS Comb. Sci. 2020; 22 (32402186): 297-30510.1021/acscombsci.0c00058Crossref PubMed Scopus (172) Google Scholar). Knowledge of the SARS–CoV-2 3CL Mpro active-site cavity structure at an atomic level of detail, including the actual locations of hydrogen atoms, can provide critical information to improve rational drug design. The presence or absence of hydrogen atoms at specific sites on amino acid residues determines their protonation states and, thus, their electrical charges, defining the electrostatics and hydrogen-bonding interactions. Of note, half of all atoms in protein and small-molecule drugs are hydrogen. X-ray crystallography is typically the standard experimental method for structure-assisted drug design but cannot reliably locate hydrogen atoms in biological macromolecules, leaving significant gaps in our understanding of biological function and drug binding (29Bax B. Chung C-W. Edge C. Getting the chemistry right: protonation, tautomers and the importance of H atoms in biological chemistry.Acta Crystallogr. D. 2017; 73 (28177309): 131-14010.1107/S2059798316020283Crossref Scopus (39) Google Scholar). Electron clouds scatter X-rays; thus, scattering power is determined by the number of electrons present in an atom, i.e. by its atomic number. Hydrogen, with just a single electron that often participates in highly polarized chemical bonds, is the weakest possible X-ray scatterer and consequently is invisible in X-ray structures with a few exceptions beyond subatomic resolution (30Gardberg A.S. Del Castillo A.R. Weiss K.L. Meilleur F. Blakeley M.P. Myles D.A.A. Unambiguous determination of H-atom positions: comparing results from neutron and high-resolution X-ray crystallography.Acta Crystallogr. D. 2010; 66 (20445231): 558-56710.1107/S0907444910005494Crossref PubMed Scopus (37) Google Scholar, 31Blakeley M.P. Mitschler A. Hazemann I. Meilleur F. Myles D.A.A. Podjarny A. Comparison of hydrogen determination with X-ray and neutron crystallography in a human aldose reductase-inhibitor complex.Eur. Biophys. J. 2006; 35 (16622654): 577-58310.1007/s00249-006-0064-8Crossref PubMed Scopus (25) Google Scholar, 32Lin J. Pozharski E. Wilson M.A. Short carboxylic acid-carboxylate hydrogen bonds can have fully localized protons.Biochemistry. 2017; 56 (27989121): 391-40210.1021/acs.biochem.6b00906Crossref PubMed Scopus (30) Google Scholar). In contrast, atomic nuclei scatter neutrons, where the scattering power of neutrons is independent of the atomic number. Deuterium, a heavy isotope of hydrogen, scatters neutrons just as well as carbon, nitrogen, and oxygen. Neutron crystallography is capable of accurately determining positions of hydrogen and deuterium atoms and visualizing hydrogen-bonding interactions at moderate resolutions (33Niimura N. Podjarny A. Neutron Protein Crystallography. Oxford University Press, Oxford2011Crossref Scopus (40) Google Scholar, 34Golden E.A. Vrielink A. Looking for hydrogen atoms: neutron crystallography provides novel insights into protein structure and function.Aust. J. Chem. 2014; 67: 1751-176210.1071/CH14337Crossref Scopus (10) Google Scholar, 35Oksanen E. Chen J.C.-H. Fisher S.Z. Neutron crystallography for the study of hydrogen bonds in macromolecules.Molecules. 2017; 22 (28387738): 59610.3390/molecules22040596Crossref Scopus (30) Google Scholar, 36Blakeley M.P. Podjarny A.D. Neutron macromolecular crystallography.Emerg. 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D. 2010; 66 (20382986): 339-35110.1107/S0907444910008656Crossref PubMed Scopus (245) Google Scholar), neutrons cause no direct or indirect radiation damage to protein crystals, permitting diffraction data collection at near-physiological (room) temperature, avoiding possible artifacts induced by the use of cryoprotectant chemicals required for X-ray cryo-crystallographic measurements. In neutron crystallographic experiments, protein crystals are usually hydrogen/deuterium–exchanged with the heavy water (D2O) to increase the signal-to-noise ratio of the diffraction pattern because hydrogen has a large incoherent scattering cross-section that increases background. Also, the coherent neutron-scattering length of hydrogen is negative (−3.739 fm) and is therefore observed in the neutron-scattering length (or nuclear) density maps as troughs. At moderate resolutions, the negative neutron-scattering length of hydrogen leads to the density cancelation phenomenon observed for CH, CH2, and CH3 groups as hydrogen atoms attached to carbon atoms cannot exchange with deuterium. Conversely, deuterium has a coherent neutron-scattering length of +6.671 fm and, thus, is observed as peaks in nuclear density maps. Because deuterium atoms scatter neutrons just as well as other protein atoms, they can be directly detected in neutron structures at moderate resolutions as low as 2.5–2.6 Å (38Gerlits O. Weiss K.L. Blakeley M.P. Veglia G. Taylor S.S. Kovalevsky A. Zooming in on protons: neutron structure of protein kinase A trapped in a product complex.Science Adv. 2019; 5 (30906862)eaav048210.1126/sciadv.aav0482Crossref PubMed Scopus (14) Google Scholar, 39Banco M.T. Mishra V. Ostermann A. Schrader T.E. Evans G.B. Kovalevsky A.Y. Ronning D.R. Neutron structures of the Helicobacter pylori 5´-methylthioadenosine nucleosidase highlight proton sharing and protonation states.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27856757): 13756-1376110.1073/pnas.1609718113Crossref PubMed Scopus (22) Google Scholar). Notably, sulfur has a coherent neutron-scattering length of +2.847 fm, less than half the magnitude of that for carbon, oxygen, nitrogen, and deuterium. Consequently, deprotonated thiol groups (S–) in Cys and side-chain sulfur atoms in Met residues are often not easily visible in nuclear density maps. We grew neutron-quality crystals of the ligand-free SARS–CoV-2 3CL Mpro at pH 6.6, allowing us to obtain a room-temperature neutron structure of the enzyme refined jointly with a room-temperature X-ray data set collected from the same crystal (Fig. 1) (40Adams P.D. Mustyakimov M. Afonine P.V. Langan P. Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules.Acta Crystallogr. D. 2009; 65 (19465771): 567-57310.1107/S0907444909011548Crossref PubMed Scopus (130) Google Scholar). We accurately determined the locations of exchangeable hydrogen atoms that were observed as deuterium attached to electronegative atoms such as oxygen, nitrogen, or sulfur atoms. We discovered that the catalytic dyad comprising residues Cys145 and His41 is in the reactive zwitterionic state having deprotonated negatively charged Cys145 and doubly protonated positively charged His41. Our experimental observations identified the protonation states of all other ionizable amino acid residues, allowing us to accurately map the electric charges and hydrogen-bonding networks in the SARS–CoV-2 3CL Mpro active-site cavity and throughout the enzyme structure. Neutron diffraction data were collected from a hydrogen/deuterium–exchanged SARS–CoV-2 3CL Mpro crystal at pD 7.0 (pD = pH + 0.4) to 2.5 Å resolution (Fig. 3). The electron density for the catalytic site and the nearby residues of SARS–CoV-2 3CL Mpro is shown in Fig. 3A Although hydrogen-bonding interactions can be inferred from the distances between the heavy atoms, the locations of hydrogen atoms and the protonation states of the amino acid residues can only be assumed. Instead, the nuclear density map shown in Fig. 3B displays the actual positions of exchanged deuterium atoms, accurately visualizing hydrogen-bond donors and acceptors. In the neutron structure, we observed that the catalytic Cys145 thiol is in the deprotonated, negatively charged thiolate state. In contrast, located 3.9 Å away from Cys145, the catalytic residue His41 is protonated on both Nδ1 and Nε2 nitrogen atoms of the imidazole side chain and is therefore positively charged (Fig. S1). As a result, the catalytic site natively adopts the zwitterionic reactive state required for catalysis (19Huang C. Wei P. Fan K. Liu Y. Lai L. 3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism.Biochemistry. 2004; 43 (15078103): 4568-457410.1021/bi036022qCrossref PubMed Scopus (163) Google Scholar, 20Solowiej J. Thomson J.A. Ryan K. Luo C. He M. Lou J. Murray B.W. Steady-state and pre-steady-state kinetic evaluation of severe acute respiratory syndrome coronavirus (SARS–CoV) 3CLpro cysteine protease: development of an ion-pair model for catalysis.Biochemistry. 2008; 47 (18237196): 2617-263010.1021/bi702107vCrossref PubMed Scopus (30) Google Scholar, 41Kneller D.W. Phillips G. O'Neill H.M. Tan K. Joachimiak A. Coates L. Kovalevsky A. Room-temperature X-ray crystallography reveals the oxidation and reactivity of cysteine residues in SARS–CoV-2 3CL Mpro: insights into enzyme mechanism and drug design.IUCrJ. 2020; 7 (33063790): 1028-103510.1107/S2052252520012634Crossref Scopus (26) Google Scholar). His41 is strongly hydrogen-bonded to a water molecule (D2Ocat) that presumably plays the role of the third catalytic residue (18Anand K. Ziebuhr J. Wadhwani P. Mesters J.R. Hilgenfeld R. Coronavirus main protease (3CLpro) structure: basis for design of anti-SARS drugs.Science. 2003; 300 (12746549): 1763-176710.1126/science.1085658Crossref PubMed Scopus (1275) Google Scholar, 25Kneller D.W. Phillips G. O'Neill H.M. Jedrzejczak R. Stols L. Langan P. Joachimiak A. Coates L. Kovalevsky A. Structural plasticity of SARS–CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography.Nat. Commun. 2020; 11 (32581217)320210.1038/s41467-020-16954-7Crossref PubMed Scopus (222) Google Scholar) from a canonical catalytic triad, stabilizing the charge and position of the His41 imidazolium ring. The Nδ1-D…OD2O distance is 1.7 Å. The position of the D2Ocat molecule is stabilized by several more, but possibly weaker, hydrogen bonds with His41 main chain, His164, and Asp187. His164 is doubly protonated and positively charged; it donates a deuterium in a hydrogen bond with the Thr175 hydroxyl within the interior of the protein. As expected, Asp187 is not protonated, is negatively charged, and participates in a strong salt bridge with Arg40. His163 positioned near the catalytic Cys145 is singly protonated and uncharged, making a hydrogen bond with the protonated phenolic side chain of Tyr161, whose OD group is rotated away from the His163 imidazole (Fig. S1). The main-chain deuterium atoms of Gly143, Ser144, and Cys145 comprising the oxyanion hole are also readily visible in the nuclear density (Fig. 3B). The P1 group of a substrate, usually Gln, binds in a rather peculiar substrate-binding subsite S1 (Fig. 4A). From one side, it is flanked by residues 140–144, making a turn that creates the oxyanion hole and, on the opposite side, by Met165, Glu166, and His172. The back wall
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