key: cord-0915421-35bu98qc
authors: Lin, Meng-Hsuan; Huang, Yi-Ping; Chang, Chi-Fon; Hsu, Chun-Hua
title: NMR assignments of the macro domain from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
date: 2021-01-24
journal: Biomol NMR Assign
DOI: 10.1007/s12104-020-09996-x
sha: b4a3080f3e9276a43d5831f5c5db51137b56c665
doc_id: 915421
cord_uid: 35bu98qc

SARS-CoV-2 is a novel pathogen causing pneumonia named COVID-19 and leading to a severe pandemic since the end of 2019. The genome of SARS-CoV-2 contains a macro domain that may play an important role in regulating ADP-ribosylation in host cells and initiating viral replication. Here, we report the (1)H, (13)C, and (15)N resonance assignments of the SARS-CoV-2 macro domain. This work provides the ground for further structural deciphering and biophysical investigation in protein function and antiviral agent design. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12104-020-09996-x) contains supplementary material, which is available to authorized users.

A novel virus, SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2, also called 2019-nCoV), was identified as the pathogen that caused the pandemic of emerged pneumonia-like disease, COVID-19, since the December of 2019. Based on the genome analysis of SARS-CoV-2, a macro domain was found in nonstructural protein 3 (NSP3). Viral macro domains had been reported possessing multifunction, for instance, interactions with ADP-ribose (ADPR) (Cho et al. 2016; Egloff et al. 2006; Makrynitsa et al. 2019) , poly-ADPR (Egloff et al. 2006) or adenine-rich RNAs (Tsika et al. 2019 ); ADPR-1″ phosphate dephosphorylation (Egloff et al. 2006; Saikatendu et al. 2005) ; and enzyme activity as an ADPR-protein hydrolase (Li et al. 2016) . Accumulated evidence about viral macro domains indicated a critical relevance to host cellular ADP-ribosylation, one of post-translational modification which correlated to DNA repair, transcription, and innate immune response (Alhammad and Fehr 2020; Fehr et al. 2020 ). Moreover, according to studies about viral macro domains from the mouse hepatitis virus (Eriksson et al. 2008) and Sindbis virus (Park and Griffin 2009) , viral replications would be depressed while ADPR binding abilities being disrupted by introducing mutations into these viral macro domains.

SARS-CoV-2 harbored a macro domain in its NSP3, so that, SARS-CoV-2 macro domain might play important roles in modulating host ADP-ribosylation and in viral replication. Indeed, there remained many mysteries about the function of the viral macro domain. However, this viral protein obviously is a possible target of antiviral agents. Here, we present the resonance assignment of the SARS-CoV-2 macro domain by a series of NMR experiments. This work would pave the way to the elucidation of the SARS-CoV-2 macro domain solution structure, which may be the base of the COVID-19 antiviral drug design targeting to SARS-CoV-2 macro domain.

The online version of this article (https ://doi.org/10.1007/s1210 4-020-09996 -x) contains supplementary material, which is available to authorized users. 

The protein production of the SARS-CoV-2 macro domain is similar to our previous work on MERS-CoV macro domain. Briefly speaking, the DNA fragment of the SARS-CoV-2 macro domain was synthesized and cloned into the pET-28a (+) vector (Novagen) between the NdeI and XhoI sites. This resulting plasmid was then transformed into E. coli BL21 (DE3), grown at 37 °C up to OD 600 0.6, using medium M9 with 1 g/l of NH 4 Cl and D-glucose. After inducing with 1 mM isopropyl-β-D-thiogalactoside (IPTG) and incubation overnight at 16 °C, E. coli cells would be gathered by centrifugation at 6000 rpm 10 min and resuspended by lysis buffer (25 mM phosphate buffer, pH 7.0, 100 mM NaCl) followed by 20 min sonication. The supernatant was then separated from the pellet by centrifugation at 13,000 rpm and 4 °C for 20 min. The recombinant SARS-CoV-2 macro domain with N-terminal His-tag was purified by Ni 2+ -NTA column with 300 mM imidazole elution. The purified protein was dialyzed against lysis buffer with 0.5 mM dithiothreitol (DTT). The N-terminal His-tag was removed by thrombin cleavage incubating at 10 °C overnight. The protein product with four additional residues (GSHM) at the N-terminus was further purified by gel filtration chromatography with column Superdex 75 increase 16/60 (GE healthcare).

NMR experiments were collected on Bruker Avance 600 and 800 MHz spectrometers at 310K with 5 mm triple resonance cryoprobe and Z-gradient. The collected data were acquired and processed using the software Top-spin2.1 (Bruker, Germany) and further analyzed using SPARKY (Lee et al. 2015) . 1 H chemical shifts were externally referenced to 0 ppm using standard chemical 2,2-dimethyl-2-silapentane-5-sulfonate. 15 N and 13 C chemical shifts were indirectly referenced to IUPAC recommendations (Markley et al. 1998) . Protein backbone assignments were based on triple resonance experiments: HNCACB, CBCA(CO)NH, HNCA, HNCO, and HN(CA) CO. Side-chain assignments were based on 13C-HCCH-TOCSY and 13C-(H)CCH-TOCSY.

The recombinant macro domain of SARS-CoV-2 with a molecular weight of 18.8 kDa contains 171 amino acids and 4 additional N-terminal residues (to which the number − 3, − 2, − 1, 0 are assigned). The backbone assignments of the SARS-CoV-2 macro domain were almost completed under the experimental conditions (pH 6.0 at 298K). Completeness of the backbone and side-chain resonances assignments, estimated by CYANA3.98 (Guntert 2004) , is 91.6%. Except for five prolines (P30, P72, P96, P123, P134), 98.8% of backbone amides (168/170) were assigned while the unassigned residues were G-3and S-2. The 2D 1 H-15 N HSQC spectrum and amide resonance assignments are shown in Fig. 1 . The side-chain assignments were also completed. 98.5% of 1 Hβ, 100% of 13 Cβ, 83.3% of 1 Hγ, and 66.6% of 13 Cγ were assigned. The methyl region of the 2D 1 H-13 C HSQC spectrum with the side-chains assignments of residues are shown in Fig. 2 .

The secondary structural population of SARS-CoV-2 macro domain was predicted by deviations between Cα and Cβ chemical shift (Δδ 13 Cα-Δδ 13 Cβ), and TALOS+ (Shen et al. 2009 ). The results indicated that the SARS-CoV-2 macro domain consisted of seven β-strands and six α-helices (Fig. 3) . The chemical shift assignments Fig. 1 1 H-15 N HSQC spectrum of SARS-CoV-2 macro domain recorded at 600 MHz with a cryogenic-probe with phosphate buffer pH 6.0 at 298K. Backbone amide 1 H and 15 N cross-peaks are presented. The horizontal lines connect pairs of the side-chain protons from amino acids Asn and Gln ◂ of the SARS-CoV-2 macro domain at 298K and pH 6.0 were deposited in the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu) under accession number 50422. Fig. 2 1 H-13 C HSQC methyl correlation spectrum of SARS-CoV-2 macro domain recorded at 600 MHz with a cryogenic-probe with phosphate buffer pH 6.0 at 298K. The assigned methyl cross peaks are labeled ◂ Fig. 3 The secondary structure of SARS-CoV-2 macro domain is predicted by CαCβ chemical shift difference, and TALOS+. Upper panel is the parameter ∆δCα − ∆δCβ shows the deviation of Cα and Cβ experimental values from the corresponding random coil values. Positive and negative values suggest α-helix and β-strand structure, respectively. Lower panel is TALOS + index showing the prediction of secondary structure distribution based on backbone N, H, Cα, Hα, C, and side-chain Cβ chemical shift values. Negative and positive values suggest α-helix (in pink) and β-strand (in green) structure, respectively. Chemical shift analysis resulting in secondary structure elements of the macro domain is represented

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The NMR spectra were obtained at the High-field NMR Center, Academia Sinica (AS-CFII-108-112). This work is supported by National Taiwan University (NTU CC-109L893501 and NTU 109L7734) and the Ministry of Science and Technology, Taiwan  (MOST 108-