key: cord-0795167-ww9fu8ey
authors: Gao, Xiaopan; Qin, Bo; Chen, Pu; Zhu, Kaixiang; Hou, Pengjiao; Wojdyla, Justyna Aleksandra; Wang, Meitian; Cui, Sheng
title: Crystal structure of SARS-CoV-2 papain-like protease
date: 2020-09-02
journal: Acta Pharm Sin B
DOI: 10.1016/j.apsb.2020.08.014
sha: 6a21cbe47bf30b889cf41ee990d01be2675c233e
doc_id: 795167
cord_uid: ww9fu8ey

The pandemic of coronavirus disease 2019 (COVID-19) is changing the world like never before. This crisis is unlikely contained in the absence of effective therapeutics or vaccine. The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays essential roles in virus replication and immune evasion, presenting a charming drug target. Given the PLpro proteases of SARS-CoV-2 and SARS-CoV share significant homology, inhibitor developed for SARS-CoV PLpro is a promising starting point of therapeutic development. In this study, we sought to provide structural frameworks for PLpro inhibitor design. We determined the unliganded structure of SARS-CoV-2 PLpro mutant C111S, which shares many structural features of SARS-CoV PLpro. This crystal form has unique packing, high solvent content and reasonable resolution 2.5 Å, hence provides a good possibility for fragment-based screening using crystallographic approach. We characterized the protease activity of PLpro in cleaving synthetic peptide harboring nsp2/nsp3 juncture. We demonstrate that a potent SARS-CoV PLpro inhibitor GRL0617 is highly effective in inhibiting protease activity of SARS-CoV-2 with the IC(50) of 2.2±0.3 μmol/L. We then determined the structure of SARS-CoV-2 PLpro complex by GRL0617 to 2.6 Å, showing the inhibitor accommodates the S3–S4 pockets of the substrate binding cleft. The binding of GRL0617 induces closure of the BL2 loop and narrows the substrate binding cleft, whereas the binding of a tetrapeptide substrate enlarges the cleft. Hence, our results suggest a mechanism of GRL0617 inhibition, that GRL0617 not only occupies the substrate pockets, but also seals the entrance to the substrate binding cleft hence prevents the binding of the LXGG motif of the substrate.

The 2019 novel coronavirus was officially named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (ICTV) 1 . This virus is the causative J o u r n a l P r e -p r o o f To purified recombinant PLpro and its variants, bacterial cells were pelleted by centrifugation and resuspended in lysis buffer (20 mmol/L Tris-HCl, pH 8.0, 300 mmol/L NaCl, 10 mmol/L imidazole, 10 mmol/L β-mercaptoethanol and 1 mmol/L PMSF). The cells were disrupted by ultrasonication. The cell debris was pelleted by centrifugation at 18,000×g for 50 min at 4 °C and the clarified supernatant was filtered through a 0.45 μm syringe filter and applied to Ni-NTA resin (QIAGEN, Shenzhen, China) . The resin was washed three times with 10 times column volume with the lysis buffer, and the target protein was then eluted with the elution buffer containing 300 mmol/L imidazole. The 6× His-SUMO tag was cleaved in the dialysis buffer (10 mmol/L HEPES pH=7.4, 100 mmol/L NaCl, and 10 mmol/L DTT) containing Ulp1 peptidase at 4 °C overnight. The resulting sample was loaded to Ni-NTA resin and the non-tagged PLpro was collected in flow through. The final purification was size-exclusion chromatography using the Superdex 200 10/300 column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) pre-equilibrated with buffer containing 10 mmol/L HEPES pH=7.4, 100 mmol/L NaCl, and 10 mmol/L DTT. The C-terminal His-tagged PLpro was purified with the same gel filtration column equilibrated with a buffer containing 20 mmol/L Tris-HCl pH=8.0, 100 mmol/L NaCl, and 2 mmol/L DTT.

Crystallization trials of C-terminal His-tagged C111S PLpro mutant was performed in a hanging drop vapor diffusion system at 18 °C. C111S mutant was concentrated to 10 mg/mL before crystallization. The crystallization was conducted by mixing 1 μL sample and 1 μL reservoir buffer containing 3% dextran sulfate sodium salt, 0.1 mol/L Bicine pH=8.5, and 15% PEG 20,000. To crystalize SARS-CoV-2 PLpro GRL0617 (MedChemExpress, Monmouth Junction, NJ, USA) complex, wild-type PLpro was concentrated to 1 mg/mL before adding GRL0617. The molar ratio of PLpro:GRL0617 was 1:10. After incubating at 4 °C overnight, the mixture was concentrated to approximately 10 mg/mL. The crystallization was achieved by mixing 1 μL sample with 1 μL reservoir buffer containing 0.1 mol/L sodium citrate pH=5.6, 0.24 mol/L ammonium acetate and 24% PEG 4000. Subsequently, the crystals were soaked in reservoir buffer supplemented with 20% ethylene glycol (or 15% glycerol for GRL0617 complex crystals) and the crystals were flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K at the Shanghai synchrotron radiation facility (SSRF) Microplate reader (Molecular Devices, San Jose, CA, USA). Each reaction mixture (100 μL) contains 10 mmol/L HEPES pH=7.4, 100 mmol/L NaCl, 10 mmol/L DTT, 1 μmol/L SARS-CoV-2 PLpro and the indicated amount of peptide substrate. The reactions were performed in a 96-well plate at 30 °C. The fluorescent signals were recorded with the excitation at 340 nm and emission at 500 nm. The relationship between relative fluorescence unit (RFU) and peptide concentration was calibrated with Edans standard. The reactions were monitored every 30 s, and the initial hydrolysis rate was plotted as the function of substrate concentration. The plot was then fitted with the Michaelis-Menten equation using software GraphPad (La Jolla, CA, USA) to yield kinetic parameters.

Each reaction mixture contains 10 mmol/L HEPES pH=7.4, 100 mmol/L NaCl, 10 mmol/L DTT, 1 μmol/L SARS-CoV-2 PLpro and increasing amount of inhibitor. The reaction was initiated by adding 11 µmol/L fluorogenic peptide and the mixtures were incubated at 37 °C for 10 min. The initial hydrolysis rate was plotted as the function of inhibitor concentration and the plot was fitted by the equation Y=Bottom+(Top-Bottom)/(1+(X/IC 50 )) using software GraphPad.

To gain structural and biochemical insight into SARS-CoV-2 PLpro domain (nsp3 746-1063aa, Supporting Information Fig. S1 ), we expressed two PLpro variants. One contains a C-terminal His-tag, the other harbors an N-terminal His-SUMO tag and the tag was subsequently removed by Ulp1 proteinase (Fig. 1A and B) , thus yielded the non-tagged PLpro. The activity of both variants was evaluated using a fluorogenic peptide based in vitro protease assay as previously described 27 . The synthetic peptide substrate (13-mer) was derived from the proteolytic processing site between nsp2/nsp3 of pp1a polyprotein, which contains substrate recognition LKGG motif. Both variants exhibited proteolytic activity ( Fig. 1C and Supporting Information Fig. S2 ). The catalytic efficiency (k cat /K m ) of the C-terminal His-tagged and the non-tagged PLpro proteins were 1074.6±261.9 and 1339.1±362.3 L/mol·s, which is similar to that of SARS-CoV PLpro (Supporting Information Table S2 ). The catalytically null mutant C111S served as the negative control. The results demonstrate that the C-terminal His-tag has subtle effect on PLpro activity, therefore we used the non-tagged protein for further biochemical characterization and inhibitor evaluation in this study.

Comparing to the activity of SARS-CoV-2 PLpro in processing ISGylated or ubiquitinated substrates (k cat /K m for deISGylating 30,210 L/mol·s) 10 , the activity in cleaving short peptide is much less efficient. This substrate specificity is in line with SARS-CoV PLpro; deubiquitination activity of SARS-CoV PLpro is ~220 folds more efficient than cleaving small peptide substrate 14 . Recent structural investigations revealed the J o u r n a l P r e -p r o o f distinct binding sites for ISG15 outside the substrate binding cleft (S1-S4 pockets) 10 , which offered the structural basis for PLpro preference towards the ISGylated substrate.

Next, we characterized SARS-CoV-2 PLpro using crystallographic methods. In the absence of ligand, only the C-terminal His-tagged PLpro C111S mutant yielded measurable crystals. The crystals belonged to the space group of C2 and diffracted the X ray to 2.5 Å. Comparing to several SARS-CoV PLpro structures with C2 space group (PDB ID: 2FE8, 3MJ5 and 4OW0), the current crystal form of SARS-CoV-2 PLpro has larger cell dimensions (Table S1 ), higher solvent content (56%) and unique crystal packing. Instead of containing a crystallographic trimer in the asymmetry unit (ASU) in apo SARS-CoV PLpro structure, our structure has two SARS-CoV-2 PLpro dimers in the ASU (Supporting Information Fig. S3A-S3C) . Intriguingly, the dimer interface involves an intermolecular disulfate-bond bridging two C270 residues of adjacent PLpro copies (Fig.   S3A ). The unique packing interaction, large solvent space and reasonable resolution (2.5 Å) of the current crystal form offer several advantages for fragment-based screening using crystallographic approach: (1) Although the active site cysteine mutation C111S of this crystal form prevents the screening of cysteine reactive inhibitor/fragment, it is still useful for identifying non-covalent inhibitor targeting substrate binding pockets S1-S4 and allosteric sites. For example, several crystal structures of SARS-CoV-2 PLpro C111S mutant complexed by various non-covalent inhibitors have been recently deposited in Protein Data Bank (PDB ID: 7JIT, 7JIR and 7JIV), which support our assertion. (2) PLpro is challenging to crystalize in the absence of ligand/inhibitor. The frequent observed crystal forms containing crystallographic trimer (2FE8 for SARS-CoV PLpro and 6W9C for SARS-CoV-2 PLpro) are difficult for compound soaking, probably due to the tight packing between PLpro monomers. Our crystal form has relatively higher solvent content and looser packing ( Fig. S3B and S3C ), thus may facilitate rapid diffusion of compound into crystals.

Given that SARS-CoV-2 and SARS-CoV PLpro proteases share ~82% amino acid sequence identity, most structural features of the orthologs are conserved (Supporting Information Fig. S1 ). We searched the unliganded SARS-CoV-2 PLpro structure against all entries in the Protein Data Bank using DALI server. The top hit was a structure of SARS-CoV PLpro inhibitor complex (PDB: 3E9S, Z-score=41.2, RMSD=1.0 Å). To facilitate structural comparison, we labeled the secondary structural elements of SARS-CoV-2 PLpro following the same scheme for SARS-CoV PLpro (Supporting Information Fig. S1 ). SARS-CoV-2 PLpro is divided into four sub-domains, the N-terminal ubiquitin-like domain (Ubl, β1-3), the α-helical Thumb domain (α2-7), the β-stranded Finger domain (β4-7) and the Palm domain (β8-13) ( Fig. 2A) . In the Finger sub-domain, four conserved cysteine (C189 and C192 on the loop between β4-5, C224 and C226 on the loop between β6-7) form a zinc finger belonging to the "zinc ribbon" fold group 28 .

The geometry of SARS-CoV-2 PLpro active site resembles that of SARS-CoV PLpro. All catalytically important residues are invariant ( Fig. 2B and Supporting Information Fig. S1 ). The catalytic triads include residues C111, H272 and D286. Residue C111 (mutated to serine in apo structure) is located 3.6 Å away from the catalytic histidine H272; the corresponding distance SARS-CoV PLpro is 3.7 Å. Residue H272 donates a hydrogen bond to D286 with the length of 3.0 Å; the corresponding distance in SARS-CoV PLpro is 2.7 Å.

The hydrogen bond between D108 and W93 (2.8 Å) strengthens the conformation of oxygen anion hole; the corresponding hydrogen bond in SARS-CoV PLpro is 3.0 Å in length.

To gain insight of substrate binding mechanism, we superimposed the unliganded SARS-CoV-2 PLpro structure to an inhibitor bound SARS-CoV-2 PLpro structure (PDB ID: 6WX4). The conformational change of the BL2 loop is remarkable. The BL2 loop is located between β11-12 strands, spanning residues 267-271. This is a flexible loop that recognizes P2-P4 of the LXGG motif of substrate. In the unliganded PLpro (Fig.   2C ), the BL2 loop adopts a relatively close confirmation. Whereas in the presence of the tetrapeptide inhibitor VIR251, the loop moves outward by ~3.2 Å to provide enough room for the tetrapeptide. Similar conformational changes were observed for SARS-CoV PLpro 20 , suggesting that the substrate recognition mechanism is well preserved.

To expedite the development of COVID-19 therapeutics, drug repurposing is a welcomed strategy. We inhibits the activity of SARS-CoV-2 PLpro 17 . In revising our manuscript, two independent group also reported the crystal structure of SARS-CoV-2 PLpro in complex with GRL0617 in preprints 48, 49 . We first confirmed that GRL0617 inhibits the activity of SARS-CoV-2 PLpro with an IC 50 of 2.2±0.3 μmol/L (Fig. 3A) , which is similar to its efficacy against SARS-CoV PLpro (IC 50 = 0.6±0.1 μmol/L, Supporting Information Table S3 ).

To reveal the structural basis for GRL0617 inhibition, we next determined the crystal structure of SARS-CoV-2 PLpro complexed by GRL0617. The non-tagged wild-type protein was used in co-crystallization. The crystals of the GRL0617 complex diffracted the X ray to 2.6 Å. It belonged to the space group of P2 1 and contained 4 PLpro copies in ASU. GRL0617 binds to all PLpro copies in ASU and the inhibitor is associated with well-defined electron density (Fig. 3B) . Using software phenix.polder, we calculated a polder map with GRL0617 itself omitted. Positive electron density clearly delineates the shape of the inhibitor, which confirmed the presence of GRL0617 in crystals (Supporting Information Fig. S5 ). The binding mode of GRL0617 with SARS-CoV-2 PLpro is nearly identical to that of SARS-CoV PLpro (Supporting Information Fig. S6A ). Briefly, GRL0617 accommodates in the substrate cleft formed between the BL2 loop and the loop connecting α3 and α4 (α3-to-α4 loop), where it occupies the S3 and S4 pockets.

Two highly specific hydrogen bonds are critical to PLpro-GRL0617 interaction (Fig. 3C) . The carboxylate side chain of residue D164 accepts one hydrogen bond (angle=145°, length=2.8 Å) from N2 nitrogen of GRL0617, and the O7 oxygen of the inhibitor accepts another hydrogen bond (angle=158°, length=2.4 Å) from the NH group of Q269. It is worth noting that comparing to apo structure, the peptide backbone of Q269 is flipped out by ~180° (Supporting Information Fig. S6B ) in GRL0617-PLpro complex, so that the NH group of Q269 is oriented in an optimal conformation hydrogen bonding with the inhibitor. Importantly, these hydrogen bonds essentially anchor the flexible BL2 loop to the body of PLpro and fix the loop in a closed conformation.

The closure of the BL2 loop induced by GRL0617 is remarkable. Superimposing apo structure to the tetrapeptide and GRL0617 bound structures clearly show that while the binding of GRL0617 narrows the substrate cleft between the BL2 loop and the α3-to-α4 loop, the binding of the tetrapeptide VIR251 widen the cleft (Fig. 3D) . The BL2 loop in the closed conformation would clash with the tetrapeptide. Multiple conformations of the BL2 loop were observed in the unliganded and inhibitor-bound SARS-CoV PLpro structures 7,19,50,51 . The intrinsic plasticity of the BL2 loop suggests an induced-fit mechanism adopted by PLpro in substrate recognition as well as its ability to accommodate structurally different substrates.

Depending on the size of inhibitor, the BL2 loop can adopt either more close or more open conformations. The mechanism of the larger inhibitor might be binding competition for substrate pockets, hence inhibits the protease activity. Taken together, our results suggest a possible mechanism for PLpro inhibition: GRL0617 J o u r n a l P r e -p r o o f not only occupies S3-S4 pockets, but also seals the substrate binding cleft in the narrowest conformation hence prevents the entering of the LXGG motif of the substrate.

More than 15-year CoV PLpro inhibitor development, mainly targeting SARS-CoV PLpro, has identified numerous compounds. Given high similarity SARS-CoV-2 PLpro shares with the PLpro of SARS and other CoVs, prior knowledge is certainly useful to COVID-19 drug development. Known PLpro inhibitors include not only the catalytic C111 reactive compounds but also a large collection of non-covalent compounds. Those range from the inhibitors discovered from the yeast-based screening, thiopurine compounds, natural compounds to the naphthalene-based compounds. Of those, the naphthalene-based compounds are of particular interests for its potent efficacy in proteinase inhibition and low molecular mass. GRL0617 and GRL0667 are two of the most potent naphthalene-based inhibitors despite they occupy the S4/S3 pockets that is ~8 Å away from the catalytic triad. As demonstrated by our structural characterizations, while the naphthalene portion of GRL0617 accommodates the S4 pocket of SARS-CoV-2 PLpro, the benzene ring fits the S3 pocket. Because the S4 pocket determines the specificity of the LXGG motif via recognizing the P4 leucine side chain, the naphthalene occupying the S4 pocket is good candidate for inhibitor design. By contrast, the S3 pocket recognizes the backbone of the P3, thus accepts any residues; therefore, the 1-naphthyl substitution and position of the hydrogen donating nitrogen should be fixed and the S3 is variable in future drug design and modification. Very recently, Osipiuk and colleagues 48 synthesized six novel compounds, of which five are benzamine functionalized derivatives of GRL0617 and one is a GRL0617 variant lacking the chirality center, and all six retained the naphthalene portion. Using crystallographic approaches, they revealed that compounds 1-3 bind to the same site as GRL0617. However, the inhibitory effects of these compounds on the proteinase activity in vitro are not connected to their efficacy in suppressing SARS-CoV-2 replication in cells. The weakest proteinase inhibitor compound 5 performed unexpectedly well in SARS-CoV-2 replication assays, whereas the potent proteinase inhibitors compounds 2 and 3 failed to show any antiviral activity. Their results suggest that improving cell permeability, solubility as well as minor modification of the compounds are important for the next-stage optimization.

Two important structures of SARS-CoV-2 PLpro protease were determined. The unliganded structure has novel crystal packing, high solvent content and reasonable resolution; thus, it offers a good foundation for fragment-based screening targeting the enzyme. The GRL0617 bound structure provides valuable insight into the inhibition mechanism at atomic level. Given that GRL0617 is one of the most promising inhibitors of CoV PLpro, our findings will aid further optimization of the inhibitor, which may contribute to speed up therapeutic development of COVID-19.

The atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession codes:7CJD and 7CMD. 

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The authors thank the staff of PX III beamline at the Swiss Light Source, Paul Scherrer Institute (Villigen, Switzerland) for assistance in data collection. 

The authors declare no conflicts of interest.