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

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.

Keywords: apo structure; coronaviruses; crystallization; human coronavirus NL63; main protease.

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.

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.

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.

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).