Crystal structures of human coronavirus NL63 main protease at different pH values

Four crystal structures of human coronavirus NL63 main protease (M^pro) in the apo form at different pH values are reported at resolutions of up to 1.78 Å. Comparison with M^pro from other human betacoronaviruses such as SARS-CoV-2 and SARS-CoV reveals common and distinct structural features in the different genera and extends knowledge of the diversity, function and evolution of coronaviruses.

Abstract

Human coronavirus NL63 (HCoV-NL63), which belongs to the genus Alphacoronavirus, mainly infects children and the immunocompromized and is responsible for a series of clinical manifestations, including cough, fever, rhinorrhoea, bronchiolitis and croup. HCoV-NL63, which was first isolated from a seven-month-old child in 2004, has led to infections worldwide and accounts for 10% of all respiratory illnesses caused by etiological agents. However, effective antivirals against HCoV-NL63 infection are currently unavailable. The HCoV-NL63 main protease (M^pro), also called 3C-like protease (3CL^pro), plays a vital role in mediating viral replication and transcription by catalyzing the cleavage of replicase polyproteins (pp1a and pp1ab) into functional subunits. Moreover, M^pro is highly conserved among all coronaviruses, thus making it a prominent drug target for antiviral therapy. Here, four crystal structures of HCoV-NL63 M^pro in the apo form at different pH values are reported at resolutions of up to 1.78 Å. Comparison with M^pro from other human betacoronaviruses such as SARS-CoV-2 and SARS-CoV reveals common and distinct structural features in different genera and extends knowledge of the diversity, function and evolution of coronaviruses.

1. Introduction  

Coronaviruses (CoVs) are a family of enveloped, single-stranded, plus-sense RNA viruses possessing a large genomic RNA of approximately 30 kb in size (Zumla et al., 2016 ▸; de Wilde et al., 2018 ▸; Paules et al., 2020 ▸; Hartenian et al., 2020 ▸; Masters, 2006 ▸). CoVs, which are classified into the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus (Paules et al., 2020 ▸; Hartenian et al., 2020 ▸; Corman et al., 2018 ▸; Chan et al., 2013 ▸), have been shown to cause acute and chronic respiratory, enteric and neurological diseases in both mammals and birds (Zumla et al., 2016 ▸; Masters, 2006 ▸; van der Hoek et al., 2006 ▸; Weiss & Leibowitz, 2011 ▸; Weiss & Navas-Martin, 2005 ▸). It is acknowledged that the genera Alphacoronavirus and Betacoronavirus can infect humans, while the genera Gammacoronavirus and Deltacoronavirus primarily infect birds (Corman et al., 2018 ▸; Chan et al., 2013 ▸). Along with SARS-CoV-2, which was newly identified in late December 2019, there are currently seven human coronaviruses, among which HCoV-NL63 and HCoV-229E belong to the genus Alphacoronavirus according to the gene sequence, while the other five CoVs (HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV and SARS-CoV-2) belong to the genus Betacoronavirus (Wang et al., 2016 ▸; Dai et al., 2020 ▸; Qiao et al., 2021 ▸). Unlike SARS-CoV, MERS-CoV and SARS-CoV-2, which can cause deadly viral infections in humans, the other four HCoVs (HCoV-NL63, HCoV-HKU1, HCoV-OC43 and HCoV-229E) cause relatively mild common colds (Corman et al., 2018 ▸; Annan et al., 2016 ▸; Owusu et al., 2014 ▸), accounting for 20–30% of adult upper respiratory tract infections (Paules et al., 2020 ▸; Wang et al., 2016 ▸).

First isolated from a seven-month-old baby with bronchiolitis and conjunctivitis in the Netherlands (van der Hoek et al., 2004 ▸), HCoV-NL63 has been found to circulate globally (Owusu et al., 2014 ▸; van der Hoek et al., 2004 ▸; Zhou et al., 2013 ▸; Matoba et al., 2015 ▸). Young children, the elderly and the immunocompromized are susceptible to HCoV-NL63, and infection often manifests as cough, fever, rhinorrhoea, bronchiolitis and croup (Abdul-Rasool & Fielding, 2010 ▸; Lee & Storch, 2014 ▸). 10% of all respiratory illnesses can be attributed to HCoV-NL63 infection (Abdul-Rasool & Fielding, 2010 ▸). Unfortunately, there is no effective treatment for HCoV-NL63 infection at present. Similar to other corona­viruses, the HCoV-NL63 genome encompasses 27 553 nucleotides with a poly-A tail (van der Hoek et al., 2004 ▸; Abdul-Rasool & Fielding, 2010 ▸; Pyrc et al., 2007 ▸). The 3′-terminal third of the HCoV-NL63 genome encodes four main structural proteins (envelope protein, membrane protein, spike protein and nucleocapsid protein) and other accessory proteins. The 5′-terminal two-thirds of the HCoV-NL63 genome encodes two replicase polyproteins (pp1a and pp1ab), which are digested into 16 nonstructural proteins (nsp1–nsp16) that are essential for viral replication by papain-like proteases (PLPs) and chymotrypsin-like protease (3CL^pro or M^pro; van der Hoek et al., 2006 ▸; Dai et al., 2020 ▸; Jin et al., 2020 ▸). Its indispensable role in viral replication as well as the lack of closely related cellular homologues make M^pro a promising target for antiviral therapy (Yang et al., 2005 ▸; Pavlova et al., 2021 ▸; Zhang et al., 2020 ▸; Kneller et al., 2020 ▸).

To date, apo-state structures of M^pro from several human coronaviruses have been solved (Wang et al., 2016 ▸; Yang et al., 2003 ▸; Anand et al., 2003 ▸; Xue et al., 2008 ▸; Zhao et al., 2008 ▸, 2021 ▸), including that of HCoV-NL63 M^pro at pH 5.5 (PDB entry 3tlo; C. P. Chuck & K. B. Wong, unpublished work). Here, we report crystal structures of HCoV-NL63 M^pro at different pH values and compare them with the available structures.

2. Materials and methods  

2.1. Protein purification and expression  

The cDNA for HCoV-NL63 M^pro was codon-optimized, synthesized (Tsingke, People’s Republic of China) and subcloned into pET-28a in-frame with a 6×His tag at the N-terminus. The newly generated plasmid was then transformed into Escherichia coli strain BL21 (DE3) for protein expression. The bacteria were cultured in Luria Broth (LB) medium containing 50 µg ml⁻¹ kanamycin at 310 K. When the optical density at 600 nm reached 0.6, a final concentration of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added, with subsequent culture for 12 h at 291 K to induce the expression of M^pro. The bacteria were then harvested by centrifugation, resuspended in lysis buffer (20 mM Tris–HCl, 250 mM NaCl, 10 mM imidazole, 5% glycerol pH 7.5) and crushed using a Union-Biotech UH-06 homogenizer. The lysate of the bacteria was centrifuged at 12 000 rev min⁻¹ at 277 K for 35 min and the precipitate was discarded. The supernatant containing the target protein was incubated with Ni–NTA resin (GE Healthcare) and transferred into a disposable gravity column for purification. After the column had successively been washed with buffer A (20 mM Tris–HCl, 300 mM NaCl, 10 mM imidazole pH 7.5) and buffer B (20 mM Tris–HCl, 300 mM NaCl, 50 mM imidazole pH 7.5), the His-tagged protein was eluted using buffer C (20 mM Tris–HCl, 300 mM NaCl, 300 mM imidazole pH 7.5). The eluted protein was concentrated and further purified by subsequent size-exclusion chromatography (SEC) on a Superdex 200 Increase 10/300 GL column (GE Healthcare) with SEC buffer (20 mM Tris–HCl, 200 mM NaCl, 2 mM DTT pH 7.5). The purity of M^pro was evaluated by SDS–PAGE. High-purity M^pro fractions were pooled and concentrated to 10 mg ml⁻¹ using a membrane concentrator (Millipore). Finally, the desired protein was flash-frozen in liquid nitrogen and stored at 193 K for protein crystallization. Macromolecule-production information is given in Table 1 ▸.

Table 1. Macromolecule-production information.

Source organism	Human coronavirus NL63
DNA source	Gene synthesis
Cloning sites	BamHI–XhoI
Cloning vector	pET-28a
Expression vector	pET-28a
Expression host	E. coli strain BL21 (DE3)
Complete amino-acid sequence of the construct produced	SGLKKMAQPSGCVERCVVRVCYGSTVLNGVWLGDTVTCPRHVIAPSTTVLIDYDHAYSTMRLHNFSVSHNGVFLGVVGVTMHGSVLRIKVSQSNVHTPKHVFKTLKPGDSFNILACYEGIASGVFGVNLRTNFTIKGSFINGACGSPGYNVRNDGTVEFCYLHQIELGSGAHVGSDFTGSVYGNFDDQPSLQVESANLMLSDNVVAFLYAALLNGCRWWLCSTRVNVDGFNEWAMANGYTSVSSVECYSILAAKTGVSVEQLLASIQHLHEGFGGKNILGYSSLCDEFTLAEVVKQMYGVNLQ

2.2. Crystallization  

The concentration of HCoV-NL63 M^pro with a 6×His tag at the N-terminus used for crystallization was 10 mg ml⁻¹. We used the sitting-drop method at 293 K to preliminarily screen the crystals using commercial kits (Hampton Research). After two days, small lump-like crystals were obtained under two conditions: (i) 0.1 M sodium citrate tribasic dihydrate supplemented with 2%(v/v) Tacsimate and 16%(w/v) polyethylene glycol 3350 (pH 5.6) and (ii) 0.2 M potassion formate supplemented with 20%(w/v) polyethylene glycol 3350 (pH 7.5). We then further optimized the crystallization conditions and obtained another two crystals at pH 5.0 and pH 5.2. The optimized crystals were larger than the previous crystals. All crystals obtained under these four conditions were used for diffraction. The crystals were cryoprotected in a mixture of well solution and glycerol in a 1:1 volume ratio and were then subjected to ultrafast cooling in liquid nitrogen. Detailed crystallization information is shown in Table 2 ▸.

Table 2. Crystallization.

	pH 5.0	pH 5.2	pH 5.6	pH 7.5
Method	Sitting-drop vapor diffusion	Sitting-drop vapor diffusion	Sitting-drop vapor diffusion	Sitting-drop vapor diffusion
Plate type	48-well plates	48-well plates	48-well plates	48-well plates
Temperature (K)	293	293	293	293
Protein concentration (mg ml−1)	10	10	10	10
Buffer composition of protein solution	20 mM Tris–HCl pH 7.5, 200 mM NaCl, 2 mM DTT	20 mM Tris–HCl pH 7.5, 200 mM NaCl, 2 mM DTT	20 mM Tris–HCl pH 7.5, 200 mM NaCl, 2 mM DTT	20 mM Tris–HCl pH 7.5, 200 mM NaCl, 2 mM DTT
Composition of reservoir solution	0.1 M sodium citrate tribasic dihydrate, 16%(w/v) polyethylene glycol 3350, pH 5.0	0.1 M sodium citrate tribasic dihydrate, 16%(w/v) polyethylene glycol 3350, pH 5.2	0.1 M sodium citrate tribasic dihydrate, 2%(v/v) Tacsimate, 16%(w/v) polyethylene glycol 3350, pH 5.6	0.2 M potassion formate, 20%(w/v) polyethylene glycol 3350, pH 7.5
Volume and ratio of drop	3 µl, 2:1	3 µl, 2:1	3 µl, 2:1	3 µl, 2:1
Volume of reservoir (µl)	80	80	80	80

2.3. Data collection and processing  

All diffraction data were collected on macromolecular crystallography beamline 17U1 (BL17U1) at Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, People’s Republic of China at 100 K with a wavelength of 0.97918 Å. The collected data were processed by the HKL-2000 software package. Data-collection and processing statistics are shown in Table 3 ▸.

Table 3. Data collection and processing.

Values in parentheses are for the highest resolution shell.

	pH 5.0	pH 5.2	pH 5.6	pH 7.5
PDB code	7e6l	7e6n	7e6r	7e6m
Diffraction source	BL17U1, SSRF	BL17U1, SSRF	BL17U1, SSRF	BL17U1, SSRF
Wavelength (Å)	0.97918	0.97918	0.97918	0.97918
Temperature (K)	100	100	100	100
Space group	P1211	P1211	P1211	P1211
a, b, c (Å)	63.07, 82.40, 63.92	63.37, 83.21, 64.00	63.15, 82.91, 63.84	62.58, 80.70, 63.31
α, β, γ (°)	90, 108.47, 90	90, 108.64, 90	90, 108.70, 90	90, 108.26, 90
Total reflections	373806	312372	285265	315996
Unique reflections	59245	54115	49375	52288
Resolution (Å)	1.78 (1.83–1.78)	1.84 (1.89–1.84)	1.90 (1.94–1.90)	1.83 (1.88–1.83)
Rmerge (%)	4.6 (17.3)	4.6 (16.2)	7.5 (39.8)	6.0 (28.5)
〈I/σ(I)〉	19.9 (2.1)	20.2 (2.0)	16.5 (2.2)	18.5 (2.4)
Completeness (%)	99.7 (98.6)	99.2 (93.8)	99.6 (97.8)	99.9 (99.9)
Multiplicity	6.3 (6.2)	5.8 (4.4)	5.8 (4.3)	6.0 (6.7)

2.4. Structure refinement  

The structures of HCoV-NL63 M^pro were determined by molecular replacement using the HCoV-NL63 M^pro structure with PDB code 3tlo as the search model. The original model was reconstructed using Coot (Emsley et al., 2010 ▸), and Phenix (Liebschner et al., 2019 ▸) was employed to refine the model. Structure-refinement statistics are summarized in Table 4 ▸. All coordinates have been deposited in the PDB with accession codes 7e6l, 7e6m, 7e6n and 7e6r. PyMOL (Schrödinger) was applied to generate diagrams and perform structural analysis.

Table 4. Structure refinement.

	pH 5.0	pH 5.2	pH 5.6	pH 7.5
PDB code	7e6l	7e6n	7e6r	7e6m
Resolution range (Å)	59.82–1.78	60.69–1.84	60.52–1.90	60.11–1.83
Rwork/Rfree (%)	18.10/20.16	19.96/23.00	18.70/22.24	19.13/22.53
No. of atoms	4897	4851	4870	4685
Mean B factor (Å2)	28.8	32.0	30.9	34.9
R.m.s.d., bond lengths (Å)	0.006	0.009	0.007	0.006
R.m.s.d., bond angles (°)	0.83	0.960	0.88	0.855
Ramachandran favored (%)	97.98	97.62	98.15	98.32
Ramachandran allowed (%)	2.02	2.38	1.85	1.68
Ramachandran outliers (%)	0.00	0.00	0.00	0.00

2.5. Enzyme-activity assay  

The effects of pH on enzyme activity were measured by a fluorescence resonance energy transfer (FRET) protease assay. The fluorogenic substrate DABCYL-KTSAVLQSGF­RKM-Glu-EDANS was purchased from GL Biochem Ltd. The assay was performed in a total volume of 20 µl with an HCoV-NL63 M^pro concentration of 36 µg ml⁻¹. The pH of the Tris buffer was first set to pH 5.0, pH 5.2, pH 5.6, pH 7.5 or pH 8.0. 1 µl recombinant HCoV-NL63 M^pro was then mixed with 17 µl assay buffer (50 mM Tris, 1 mM EDTA) and incubated at room temperature (RT) for 30 min. 18 µl assay buffer (50 mM Tris, 1 mM EDTA) without HCoV-NL63 M^pro was used as a blank control. Subsequently, 2 µl of a fluorogenic substrate at a concentration of 20 µM was added to the above mixtures in a 384-well plate and the reaction solution was incubated at RT for 20 min. Afterwards, the fluorescence signal with excitation at 360 nm and emission at 490 nm was recorded seven times using a Paradigm microplate reader at intervals of 10 min. For the HCoV-NL63 M^pro activity assay, three replicates were measured for each group to determine the fluorescence values. The luminescence data were analyzed using GraphPad Prism 7.0.

3. Results and discussion  

3.1. Purification and crystallization of HCoV-NL63 M^pro  

As shown in Fig. 1 ▸(a), the elution volume of HCoV-NL63 M^pro from size-exclusion chromatography (SEC) is approximately 14 ml, and the molecular weight of the target protein is estimated as 74 kDa according to the standard curve of protein peak volumes (Fig. 1 ▸ c). Given that the molecular weight of monomeric HCoV-NL63 M^pro with a 6×His tag at the N-terminus is about 37 kDa, the recombinant M^pro is a dimer. SDS–PAGE analysis of HCoV-NL63 M^pro after SEC further showed that the purity of recombinant M^pro was greater than 95% (Fig. 1 ▸ b), which meets the requirements for crystallization. High-purity M^pro fractions were pooled and concentrated to 10 mg ml⁻¹ for protein crystallization. As shown in Figs. 2 ▸(a) and 2 ▸(b), two crystals with few differences in morphology were obtained after preliminary screening under two conditions: (i) 0.1 M sodium citrate tribasic dihydrate, 2%(v/v) Tacsimate, 16%(w/v) polyethylene glycol 3350 (pH 5.6) and (ii) 0.2 M potassion formate, 20%(w/v) polyethylene glycol 3350 (pH 7.5). By optimization, we obtained two further crystals at pH 5.0 and pH 5.2, as shown in Figs. 2 ▸(c) and 2 ▸(d). The resolutions of these four crystals obtained at four different pH values range from 1.78 to 1.90 Å, as shown in Table 3 ▸.

Figure 1.

Purification of recombinant HCoV-NL63 M^pro. (a) Size-exclusion chromatography of HCoV-NL63 M^pro. HCov-NL63 M^pro eluted as a single peak from a Superdex 200 Increase 10/300 GL column. The peak volume is about 14 ml. The calculated molecular weight is approximately 74 kDa using the standard curve in (c), suggesting a dimeric form. (b) SDS–PAGE analysis of purified HCoV-NL63 M^pro. The monomer runs as a single band and the purity is greater than 95%. According to the protein standard markers, the molecular mass is close to 37 kDa. (c) A standard curve for SDS–PAGE (a) was generated using known protein standards. MW represents the molecular weight of the corresponding protein. x represents the peak volume. MW (kDa) = 10^{(−0.2177x + 5.0182)}.

Figure 2.

Crystals of HCoV-NL63 M^pro at four different pH values. (a) 0.1 M sodium citrate tribasic dihydrate, 2%(v/v) Tacsimate, 16%(w/v) polyethylene glycol 3350, pH 5.6. (b) 0.2 M potassion formate, 20%(w/v) polyethylene glycol 3350, pH 7.5. (c) 0.1 M sodium citrate tribasic dihydrate, 16%(w/v) polyethylene glycol 3350, pH 5.0. (d) 0.1 M sodium citrate tribasic dihydrate, 16%(w/v) polyethylene glycol 3350, pH 5.2.

3.2. Overall structure of HCoV-NL63 M^pro  

HCoV-NL63 M^pro forms a dimer consisting of two protomers (A and B) in the crystal, as expected, and protomers A and B are oriented at right angles with respect to each other, similar to the crystal structures of the main protease from other coronaviruses (Yang et al., 2003 ▸; Anand et al., 2003 ▸; Xue et al., 2008 ▸; Zhao et al., 2008 ▸, 2021 ▸). Each of the two protomers consists of three domains that are common to CoV M^pros (Fig. 3 ▸ a). Domain I (residues 8–100) and domain II (residues 101–183) of HCoV-NL63 M^pro together compose a chymotrypsin-like fold, while domain III (residues 200–303) includes a globular antiparallel α-helical cluster which links to domain II via a long loop region of 16 residues. The α-helical domain III is unique to CoV M^pro and is responsible for dimerization. The substrate-binding site is located in a cleft between domains I and II, and Cys144 and His41 in the central position of the substrate-binding site make up the catalytic dyad (Fig. 3 ▸ b), as has been observed in other CoV M^pro structures.

Figure 3.

Structure of HCoV-NL63 M^pro. (a) Overview of homodimers shown as cartoons. The coloring is as follows: pH 5.0, orange; pH 7.5, cyan; pH 5.2, magenta; pH 5.6, yellow. (b) Structural alignment of protomer A in M^pro (pH 5.0, cyan; pH 7.5, magenta; pH 5.2, yellow; pH 5.6, orange) with that in the previous reported apo enzyme (gray; PDB entry 3tlo). The backbone is presented in cartoon representation. (c) Comparison of protomer A in structures of apo-form HCoV-NL63 M^pro (orange, PDB entry 7e6r) and HCoV-NL63 M^pro bound to the inhibitor N3 (gray; PDB entry 5gwy). N3 is shown as green sticks.

3.3. Comparison of HCoV-NL63 M^pro with previously reported structures  

Superimpositions of the M^pro structure at pH 5.5 (PDB entry 3tlo) with the M^pro structures at pH 5.0 (PDB entry 7e6l), pH 7.5 (PDB entry 7e6m), pH 5.2 (PDB entry 7e6n) and pH 5.6 (PDB entry 7e6r) show root-mean-square deviation (r.m.s.d.) values of 0.471, 0.392, 0.521 and 0.503 Å over the 511, 509, 518 and 511 best-aligned C^α atoms, respectively (Fig. 3 ▸ b). Moreover, the catalytic dyad and substrate-binding pocket are identical. A structural comparison of protomer A in the structure of apo-form HCoV-NL63 M^pro and that with the inhibitor N3 reveals some structural differences (Fig. 3 ▸ c). On the binding of N3, the binding pocket of HCoV-NL63 M^pro undergoes a huge conformational change to better accommodate the N3 molecule. N3, a synthetic peptido­mimetic compound with a Michael acceptor, and Cys144 of the catalytic dyad form an irreversible covalent bond to achieve mechanism-based enzyme inactivation. The differences in protein structure between apo M^pro and the M^pro–N3 complex have been discussed in detail (Wang et al., 2016 ▸).

As mentioned above, we did not observe significant differences in the apo state of HCoV-NL63 M^pro at different pH values. However, it has been reported that changes in the pH greatly impacted the conformation of SARS-CoV M^pro, and as a result SARS-CoV M^pro has a varying activity at different pH values (Yang et al., 2003 ▸). In an enzyme-activity test experiment, SARS-CoV M^pro showed the highest proteolytic activity at pH 7.3–8.5, but showed only 50% activity at pH 6.0. Therefore, SARS-CoV M^pro has been proven to have a pH-triggered activation switch. However, we tested the activity of HCoV-NL63 M^pro at different pH values and did not obtain similar results to SARS-CoV M^pro. The activity of HCoV-NL63 M^pro at different pH values determined by FRET showed little difference (see Supplementary Fig. S1). Simultaneously, the structures of HCoV-NL63 M^pro at different pH values are almost identical. Consequently, the structure and activity of HCoV-NL63 M^pro are consistent. This also indicates that the structure of HCoV-NL63 M^pro is relatively stable over a comparatively wide pH range. The reason for the difference in the structure and activity of these two proteases at different pH values is currently unknown. It is worth continuing to explore the structural and functional changes of HCoV-NL63 M^pro at further pH values. Another point worth highlighting is that HCoV-NL63 and SARS-CoV belong to the genera Alphacoronavirus and Betacoronavirus, respectively, which may make a difference.

Superimposition of HCoV-NL63 M^pro at pH 5.0 (PDB entry 7e6l) onto SARS-CoV M^pro (PDB entry 1uj1; Yang et al., 2003 ▸) and SARS-CoV-2 M^pro (PDB entry 7c2q; Zhou et al., 2021 ▸) revealed larger structural deviations (Fig. 4 ▸). The r.m.s.d.s were 1.432 and 1.182 Å over 534 and 492 best-aligned C^α atoms, respectively. Interestingly, residues 45–51 of M^pro form a tight loop to maintain the S2 pocket in HCoV-NL63, while the same region in SARS-CoV-2 adopts an α-helical secondary structure similar to that in SARS-CoV (Wang et al., 2016 ▸). The loops (formed by residues 186–191) that link domain II and domain III in SARS-CoV and SARS-CoV-2 M^pro also differ from that in HCoV-NL63 M^pro by extending out from the binding pocket. Additionally, the imidazole ring of His41 flips over and moves towards the hydrophobic core.

Figure 4.

Superimposition of HCoV-NL63 M^pro with M^pro from SARS-CoV and SARS-CoV-2. The main chains of the three M^pro structures (HCoV-NL63, cyan, PDB entry 7e6l; SARS-CoV, green, PDB entry 1uj1; SARS-CoV-2, blue, PDB entry 7c2q) are superimposed and the backbones are represented as cartoons. Residues 45–51 and residues 186–191 are marked with a dashed yellow oval and a red oval, respectively.

According to the sequence alignment (Fig. 5 ▸), the amino-acid sequences of the seven CoV M^pros have significant homology, with a consensus of more than 70%. In addition, the conserved substrate-binding pockets of CoV M^pros, together with the vital role of M^pro in viral replication, contribute to our in-depth research on the coronavirus main protease.

Figure 5.

Alignment of the M^pro amino-acid sequences from seven CoVs. The sequence and secondary structure of HCoV-NL63 M^pro (PDB entry 7e6m) are used as a reference for the alignment. ClustalW was used for sequence alignment and ESPript 3.0 was used to generate the graphical representation. Secondary-structural elements shown include α-helices, β-strands and β-turns (TT).

In conclusion, we have resolved crystal structures of HCoV-NL63 M^pro at different pH values, which will improve our understanding of CoV M^pro to some extent. Simultaneously, further analysis of the structure of HCoV-NL63 M^pro and the comparison of these four apo-form structures with previously reported M^pro structures from human coronaviruses may contribute to the better design and optimization of antiviral drugs in the long run.

Supplementary Material

PDB reference: human coronavirus NL63 3C-like protease, pH 5.0, 7e6l

PDB reference: pH 5.2, 7e6n

PDB reference: pH 5.6, 7e6r

PDB reference: pH 7.5, 7e6m

Funding Statement

This work was funded by National Natural Science Foundation of China grant 81760408; National Key Research and Development Projects grant 2020YFC2005800; Natural Science Foundation of Jiangxi Province grant 20113BCB22005; Jiangxi Provincial Department of Science and Technology grant 20181BCG42001.