key: cord-1031951-0ztkz0id authors: Yang, Kai S.; Kuo, Syuan-Ting Alex; Blankenship, Lauren R.; Sheng, Yan J.; Sankaran, Banumathi; Li, Pingwei; Fierke, Carol A.; Russell, David H.; Yan, Xin; Xu, Shiqing; Liu, Wenshe Ray title: A Novel Y-Shaped, S-O-N-O-S-Bridged Crosslink between Three Residues C22, C44, and K61 Is a Redox Switch of the SARS-CoV-2 Main Protease date: 2022-04-29 journal: bioRxiv DOI: 10.1101/2022.04.29.490044 sha: 944849b94dc21b2b409b0a659251125eec0f3e92 doc_id: 1031951 cord_uid: 0ztkz0id As the COVID-19 pathogen, SARS-CoV-2 relies on its main protease (MPro) for pathogenesis and replication. During the crystallographic analyses of MPro crystals that were exposed to the air, a uniquely Y-shaped, S-O-N-O-S-bridged posttranslational crosslink that connects three residues C22, C44, and K61 at their side chains was frequently observed. As a novel posttranslational modification, this crosslink serves as a redox switch to regulate the catalytic activity of MPro, a demonstrated drug target of COVID-19. The formation of this linkage leads to a much more opened active site that can be potentially targeted for the development of novel SARS-CoV-2 antivirals. The inactivation of MPro by this crosslink indicates that small molecules that lock MPro in the crosslinked form can be potentially used with other active site-targeting molecules such as paxlovid for synergistic effects in inhibiting the SARS-CoV-2 viral replication. Therefore, this new finding reveals a unique aspect of the SARS-CoV-2 pathogenesis and is potentially paradigm-shifting in our current understanding of the function of MPro and the development of its inhibitors as COVID-19 antivirals. SARS-CoV-2 is the viral pathogen of COVID-19 that has ravaged the whole world for more than two years. Effective vaccines that target the membrane Spike protein of SARS-CoV-2 have been developed and widely adopted for human immunization. 1 However, the continuous emergence of new SARS-CoV-2 variants with mutations at Spike has led to viral evasion of vaccines and consequently infection surges. 2 The situation has called for the search for SARS-CoV-2 antivirals and led to the recent success of the development of Pfizer's paxlovid. 3, 4 SARS-CoV-2 is an RNA virus with a positive-sense RNA genome. It contains a big 20-kb open reading frame ORF1ab that is translated alternatively to form two large polypeptides pp1a and pp1ab in infected human cells. 5, 6 These two polypeptides need to be hydrolyzed to form 16 nonstructural proteins (nsps) that are key to viral biology including the formation of the RNA-dependent RNA polymerase complex for the replication of the viral genome and subgenomic RNAs. The maturation of pp1a and pp1ab is catalyzed by two internal nsp fragments. One of them is main protease (M Pro ) that processes 13 out of the total 16 nsps. 7 Paxlovid is a combination therapy with two chemical components. One component is nirmatrelvir that potently inhibits M Pro to prevent viral replication in infected human cells. 4 As an essential enzyme for SARS-CoV-2, M Pro is an established target for the development of SARS-CoV-2 antivirals. 8-12 Many academic research groups and a number of pharmaceutical companies have been working on the development of M Pro inhibitors. 13- 24 A large international COVID Moonshot project for the development of M Pro inhibitors has also been organized. 25 As a key tool for the structure-based drug discovery, X-ray protein crystallography has been frequently employed to determine structures of M Proinhibitor complexes to assist further inhibitor optimization. This is also the approach that we have adopted for the development of M Pro inhibitors. 26 To facilitate a quick determination of M Pro -inhibitor complexes, we have been crystalizing the apo form of M Pro and then soaking the crystals with different inhibitors for the ensuing X-ray protein crystallographic analysis. [26] [27] [28] Our obtained apo-M Pro crystals have a C121 or I121 space group that contains an M Pro dimer or monomer, respectively, in an asymmetric unit. In the I121 space group, two monomers from two asymmetric units form a tightly bound dimer. In both C121 and I121 space groups, a tightly bound dimer interacts with other M Pro dimers within the crystals at two regions, aa53-60 and aa216-222. The aa216-222 region is located in the noncatalytic C-terminal domain. Although the aa53-60 region is within the catalytic N-terminal domain, it is distant to the active site ( Figure 1A ). 26 The M Pro dimer in these crystals has an open active site whose structure rearrangements are not expected to be limited by the protein packaging in crystals (Supplementary Figure 1) . Therefore, it is optimal for a soaking-based structural determination of M Pro -inhibitor complexes. This relatively open form of M Pro is considered more desired than a closed form in representing M Pro in solutions as well since M Pro dimers in solutions are not expected to interact with each other. Therefore, we have been using these apo-M Pro crystals and soaking them with different inhibitors for the determination of crystal structures of more than 30 M Proinhibitor complexes. One unique observation that we have made for almost all of our determined M Pro -inhibitor structures was a poorly defined conformation for the aa46-50 region. This region is part of the M Pro active site, serving as a cap of the S2 site to bind the P2 residue in a peptide substrate ( Figure 1A ). 26, 29, 30 The electron density of this region was very weak in all determined M Pro -inhibitor structures indicating a highly flexible conformation ( Figure 1B and Supplementary Figure 2) . There have been a number of M Proinhibitor complexes whose crystal structures have been determined and deposited into the Residues that form the binding sites for P1 and P2 residues in a substrate are labeled. (B) The low electron density map around aa46-50 in a representative M Pro -inhibitor complex. The ligand MPI8 that is covalently bound to C145 is shown in a color code in which carbon atoms are shown in red. For the protein, carbon atoms are shown in light teal. The aa46-50 flexible region is indicated in a dashed square. The electron density was contoured at 1 . Protein Data Bank. [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] Many have a clearly defined conformation for aa46-50. However, crystals for most of these structures either had a closed form around the active site due to the protein packaging in crystals or were obtained by co-crystallization with ligands. We reason that in the closed form of the M Pro crystals the protein packaging limits the structure rearrangement of aa46-50 and prevents it from adopting a flexible conformation. However, in solutions this limitation does not apply. This structural rearrangement is apparently related to the soaking process since the apo-M Pro crystals that did not undergo soaking show a well-defined confirmation for aa46-50 and M Pro -inhibitor complexes in co-crystalized crystals did not show this rearrangement either. 8-10,26 M Pro -MPI8 was an early determined M Pro -inhibitor complex structure. 26 In this crystal structure, besides a structurally undefined aa46-50 region, we noticed the uniquely connected electron density at residues C22, C44 and K61. As shown in Figure 2A , 2Fo-Fc electron density contoured at 1 around the ends of side chains from all three residues clearly merged indicating that the three residues were covalently connected to form a Yshaped tri-residue crosslink. Directly connecting three residues at their terminal heteroatoms to form a Y-shaped S-N-S crosslink did not fit the calculated electron density. Although there was no literature report about a Y-shaped crosslink between one lysine and two cysteine residues in a protein, a recent Nature article by Wensien et al. Table S1. crosslink between K61 and two cysteine residues C22 and C44. We suspected that the inhibitor binding process might have triggered the formation of the crosslink, however, a careful search of our other determined structures revealed four apo-M Pro structures that contained this crosslink as well. As shown in Figures 1F-H , all four structures showed clearly connected electron density at the ends of three residues C22, C44 and K61. These four apo-M Pro structures were determined from apo-M Pro crystals that were soaked with chemical fragments but no bound ligands were observed. So far, all structures that showed this crosslink were determined from crystals that were exposed to the air due to the soaking process. We also determined X-ray crystal structures for several apo-M Pro crystals that were not exposed to the air. They all had a structurally defined aa46-50 region and exhibited no crosslink between C22, C44 and K61. One of these structures was previously deposited into the Protein Data Bank (PDB entry: 7JPY). 26 As shown in Figure 3A , the superposition of 7JPY over an apo-M Pro structure with the Y-shaped crosslink reveals a large structural rearrangement at the region aa43-52 between two structures. In 7JPY, the side chain of C44 points toward the active site. The small aa46-51 helix tucks C44 toward the active site and makes it a key component to form the S2 binding pocket for a substrate P2 residue. The thiol group of C44 is 9.1Å away from the K61 side chain amine ( Figure 3A in lemon). This distance is too far to form any possible direct interaction between the two residues. In order for C44 to physically meet K61 for a covalent interaction, the C44 backbone αcarbon moved 4.1Å closer to K61 and rotated its side chain almost 180˚ to adopt a conformation to form a covalent adduct with the K61 side chain amine as shown in the at aa43-52. In 7JPY, C22 and K61 are in a close distance to each other. This close distance will likely make them form an N-O-S-bridged crosslink first and then a structurally flipped C44 will be engaged to generate the second N-O-S bridge. Since all our M Pro structures that contained the Y-shaped posttranslational crosslink were determined from crystals that were exposed to the air, the molecular oxygen was most likely the reagent that generated the Y-shaped S-O-N-O-S bridge. Although how exactly the molecular oxygen oxidizes the three residues to form the crosslink needs to be further explored and confirmed, we propose a likely mechanism as shown in Figure 3B . In this mechanism, the C22 thiolate reacts with oxygen to form a peroxysulfane that then reacts with lysine to generate the first S-O-N crosslink. After C44 flips toward K61, its thiolate can react with oxygen to form the second peroxysulfane that then reacts with the first S-O-N crosslink to form the Y-shaped, S-O-N-O-S-bridged tri-residue crosslink. Since the crosslink formation generates a much more opened active site in M Pro , it presumably weakens the binding of a peptide substrate to M Pro and consequently leads to a less active enzyme. To test this prospect, we freshly expressed M Pro and purified it without adding a reducing reagent in lysis and purification buffers. 1 mM DTT was previously used to maintain N. gonorrhoeae transaldolase in its reduced state presumably due to its reduction of the N-O-S bond. 31 We added 1 mM DTT to the freshly purified M Pro as well for 30 min to reduce its potentially generated Y-shaped, S-O-N-O-S-bridged trisubstrate crosslink. We then tested the activities of the enzyme in two conditions, one with the addition of 1 mM DTT and one without. The results, as shown in Figure 4A have experienced damage to their lungs that induces hypoxia. 32 A hypoxic condition that provides a reducing environment will favor more active M Pro that drives the SARS-CoV-2 reproduction in infected human lungs. Our discovery may partially explain the severe lung damage in COVID-19 patients since the more active M Pro enhances SARS-CoV-2 reproduction and therefore aggravates the symptom. SARS-CoV-2 patients will typically develop inflammation and redox imbalance with altered levels of metabolites and antioxidants. 33 There have been many proposals to use redox-based therapeutics to potentially treat COVID-19 patients. One typical suggestion is to take antioxidants. However, the intake of antioxidants could further activate M Pro . 34 The authors declare no competing financial interests. The pET28a-His-SUMO-M Pro expression and purification are according to our previous report. 35 The µL of each substrate solution, final concentration from 4 µM to 500 µM with 5% DMSO, was added to each well to initiate the assay. The first 0-200 seconds were analyzed by linear regression for initial slope analyses with GraphPad Prism 8.0. The production of crystals of apo M Pro and M Pro -inhibitor complexes was following the previous protocols with the crystal growing condition of 0.1 M Bis-tris, pH6.5, 16% w/v PEG10k. 35 The data of M Pro with MPI12 was collected on a Rigaku R-AXIS IV++ image plate detector, the data of M Pro with MPI19 was collected at the Advanced Light Source Sketcher from the CCP4 suite were employed for the generation of PDB and geometric restraints for the inhibitors. The inhibitors were built into the Fo-Fc density by using Coot 38 . Refinement of all the structures was performed with Real-space Refinement in Phenix 37 . Details of data quality and structure refinement are summarized in Table S1 . All structural figures were generated with PyMOL (https://www.pymol.org). Native mass spectrometry (nMS) analysis was performed on a Q Exactive UHMR Hybrid Quadruple-Orbitrap Mass Spectrometer (ThermoFisher) with m/z range was set from 1,000 to 10,000 and with resolution set to 12,500 (at m/z 400). 10 µL of the sample was loaded to a borosilicate glass capillary tip (Sutter, CA) with 1100 to 1500 V spray voltage supplied by an inserted platinum wire. Important parameters to reduce non-specific adducts include: capillary temperature 100oC, in-source trapping and activation -10 V, ion transfer high m/z, collision-induced dissociation (CID) 10 eV, and higher energy dissociation (HCD) 30 V. In variable-temperature electrospray ionization (vT-ESI) experiment, the temperature of solution was controlled as previously described. 39 The temperature was varied from 25oC to 40oC with 5oC increments and the equilibrium time at each temperature was 5 minutes. 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