key: cord-0944208-qgklt8wa
authors: Huang, Yi-Ping; Cho, Chao-Cheng; Chang, Chi-Fon; Hsu, Chun-Hua
title: NMR assignments of the macro domain from Middle East respiratory syndrome coronavirus (MERS-CoV)
date: 2016-03-18
journal: Biomol NMR Assign
DOI: 10.1007/s12104-016-9676-9
sha: 61bf159b95a8827b5e21db833d2fc1974d91425a
doc_id: 944208
cord_uid: qgklt8wa

The newly emerging human pathogen, Middle East respiratory syndrome coronavirus (MERS-CoV), contains a macro domain in the highly conserved N-terminal region of non-structural protein 3. Intense research has shown that macro domains bind ADP-ribose and other derivatives, but it still remains intangible about their exact function. In this study we report the preliminary structural analysis through solution NMR spectroscopy of the MERS-CoV macro domain. The near complete NMR assignments of MERS-CoV macro domain provide the basis for subsequent structural and biochemical investigation in the context of protein function.

dispensable for coronavirus replication (Putic et al. 2005) , however it seems to play a role in the pathogenesis of mouse hepatitis virus infection (Eriksson et al. 2008 ). In addition, viral macro proteins may act via ADP-ribose binding to influence the cellular macro domains-regulated pathways either to promote virus replication or to inhibit host responses directed against the virus (Neuvonen and Ahola 2009 ). Since the biochemical, enzymatic and structural analysis of MERS-CoV macro domain is not available, here we present the expression, purification, and chemical shift assignments of MERS-CoV macro domain. The NMR chemical shift assignments serve as the basis for further structural characterization and ligand screening, which provides insight into the conformational properties of this domain in solution and contributes to understanding its function.

The DNA sequence containing MERS-CoV macro domain (aa. 1110-1274) was chemical synthesized and cloned into NdeI/XhoI site of pET-28a(?) vector system (Novagen). Bacteria of the E. coli strain BL21(DE3) transformed with the pET-28a (?)-macro domain plasmid were grown in 4 liters of LB medium at 37°C until the absorbance at 600 nm reach 1.0. Cells were then harvested by centrifugation and re-suspended in one liter of M9 minimal medium supplemented with 1 g/L of 15 NH 4 Cl and 2 g/L of 13 C 6 -glucose (Cambridge Isotope Laboratories). Isopropylb-D-thiogalactoside (IPTG, 1 mM) was added to induce His-tagged protein for 20 h at 16°C. The cell culture was harvested by centrifugation at 6000 rpm.

For purification, cell pellets were re-suspended in 50 mL buffer containing 25 mM sodium phosphate and 100 mM NaCl at pH 7.0 and then disrupted by sonication for 20 min. The cell extract was clarified by centrifugation at 12,500 rpm for 30 min at 4°C to remove debris. The supernatant was then applied to Ni-NTA column (GE, Healthcare) equilibrated with the same re-suspension buffer, and His-tagged protein was eluted with 200 mM imidazole. The purified His-tagged macro domain was then digested with thrombin for 6 h at 16°C to remove the His-tag. Finally, macro domain protein (20 kDa, with extra Gly, Ser, His and Met at N-terminus) was purified using size-exclusion Superdex75 XK 16/60 column (GE, Healthcare). The purified protein was concentrated to 0.1-0.5 mM in 20 mM sodium phosphate (pH 6.5) and 100 mM or 150 mM NaCl for NMR structural studies.

All NMR experiments were carried out at 293 K on Bruker Avance 600 MHz NMR or 800 MHz spectrometers equipped with 5 mm triple resonance cryoprobe and Z-gradient. The data was acquired and processed using the software Topspin2.1 (Bruker, Germany) and further analyzed using Sparky, version 3.114 (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco), following the procedures as described previously (Yang et al. 2010; Chen et al. 2014) . 1 H chemical shifts were externally referenced to 0 ppm of 2,2-dimethyl-2-silapentane-5-sulfonate, whereas 13 C and 15 N chemical shifts were indirectly referenced according to IUPAC recommendations (Markley et al. 1998) . Protein backbone resonance assignments were based on standard triple resonance experiments (Sattler et al. 1999 ): HNCACB, CBCA(CO)NH, HNCO and HN(CA)CO. Aliphatic side-chain assignments were primarily done by HCCH-TOCSY and HCCH-COSY with the help of HCC(CO)NH and HBHA(CO)NH experiments.

The backbone resonance assignments were nearly complete. Figure 1 illustrates the 2D ( 1 H-15 N) HSQC spectrum and assignments of the amide resonances. Except for the first four amino acids on N-terminal (Gly -2 , Ser -1 , His 0 and Met 1 ) and six Proline residues (Pro 1 , Pro 72 , Pro 96 , Pro 118 , Pro 123 , and Pro 134 ), amides of all other residues (158 out of 168) have been assigned under the experimental conditions (pH 6.5 at 293 K). Among these 158 residues, all other backbone resonances ( 1 Ha, 13 Ca, 13 Cb and 13 C) are 100 % completed. Completeness of 1 H resonances assignment, including side-chain, calculated by CYANA3.9 (Güntert 2004 ) is 86.7 %. Secondary structure elements of MERS-CoV macro domain were identified by calculating the chemical shift deviations of the Ca(DdCa) and Cb(DdCb) from the random coil values and was corroborated by analysis of the chemical shift data using the program TALOS? (Shen et al. 2009 ). Positive and negative values of the difference between DdCa and DdCb correspond to a-helix and b-sheet secondary structure, respectively and correlated well with TALOS? index (Fig. 2) . Six helices and seven b-strands could be deduced for MERS-CoV moacro domain protein based on the secondary chemical shift analysis, which results in residues 21-27, 47-54. 58-69, 104-115, 134-144 and 156-162 in a-helices and residues 5-10, 14-19, 32-25, 78-81, 89-94, 119-124 and 148-153 forming b-sheets. The resonance assignments have been deposited to the BioMagResBank (http://www.bmrb.wisc.edu/) under the accession number 26657. 

Backbone resonance assignments of the a sub-domain of Brevibacillus thermoruber Lon protease

Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the coronavirus study group

Mouse hepatitis virus liver pathology is dependent on ADPribose-1 00 -phosphatase, a viral function conserved in the alphalike supergroup

Coronaviruses: an overview of their replication and pathogenesis

Automated NMR protein structure calculation with CYANA

The macro domain protein family: structure, functions, and their potential therapeutic implications

Bats as reservoirs of severe emerging infectious diseases

Dissection of aminoterminal functional domains of murine coronavirus nonstructural protein 3

The macro domain is an ADPribose binding module

Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy

Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites

The hepatitis E virus ORF1 'X-domain' residues from a putative macrodomain protein/Appr-1 00 -pase catalyticsite, critical for viral RNA replication

ADP-ribose-1 00 -monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture

Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients

TALOS?: a hybrid method for predicting protein backbone torsion angle from NMR chemical shifts

Resonance assignments of human C35 (C17orf37) protein, a novel tumor biomarker

Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia

Acknowledgments The NMR spectra were obtained at High-Field Nuclear Magnetic Resonance Center (HF-NMRC) and GRC NMR Core Facility in Academia Sinica, Taiwan. This work was supported by the Ministry of Science and Technology, Taiwan (103-2113-M-002-009-MY2), and National Taiwan University (NTU-ERP-104R8600 and NTU-ICRP-104R7560-5) to C.-H. Hsu.