Delicate structural coordination of the Severe Acute Respiratory Syndrome coronavirus Nsp13 upon ATP hydrolysis

Abstract

To date, an effective therapeutic treatment that confers strong attenuation toward coronaviruses (CoVs) remains elusive. Of all the potential drug targets, the helicase of CoVs is considered to be one of the most important. Here, we first present the structure of the full-length Nsp13 helicase of SARS-CoV (SARS-Nsp13) and investigate the structural coordination of its five domains and how these contribute to its translocation and unwinding activity. A translocation model is proposed for the Upf1like helicase members according to three different structural conditions in solution characterized through H/D exchange assay, including substrate state (SARS-Nsp13-dsDNA bound with AMPPNP), transition state (bound with ADP-AlF4−) and product state (bound with ADP). We observed that the 19– 20 loop on the 1A domain is involved in unwinding process directly. Furthermore, we have shown that the RNA dependent RNA polymerase (RdRp), SARSNsp12, can enhance the helicase activity of SARSNsp13 through interacting with it directly. The interacting regions were identified and can be considered common across CoVs, which provides new insights into the Replication and Transcription Complex (RTC) of CoVs. INTRODUCTION The emergence of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2003 was the first opportunity to allow investigation of a coronavirus (CoV) that was a severe human pathogen. A decade later, a similar coronavirus termed Middle East Respiratory Syndrome Coronavirus (MERS-CoV) emerged, but alarmingly this virus has higher case-fatality rates than SARS-CoV. Thus, there is a refocussing of the world’s attention onto CoVs. The fact that no therapeutic treatments are available for CoVs is a serious concern (1,2). It is therefore necessary to study the life cycle of CoVs to develop new ideas for effective vaccines or drugs. SARS-CoV belonging to the genus Betacoronavirus in the family Coronaviridae has one of the largest known RNA genomes (∼29.7 kb) among RNA viruses. Two large polyproteins pp1a and pp1ab are encoded by this genome. After being proteolytically processed, 16 non-structural proteins (Nsps) are produced including primase (Nsp8), RNA-dependent RNA polymerase (Nsp12) and helicase (Nsp13). These three enzymes and other Nsps are components of a replication and transcription complex (RTC) which is essential for the life cycle of SARS-CoV (3,4). Helicase SARS-CoV Nsp13 (SARS-Nsp13) plays a vital role in catalyzing the unwinding of duplex oligonucleotides into single strands in an NTP-dependent manner. Importantly, SARS-Nsp13 has been identified as an ideal target for the development of anti-viral drugs due to its sequence conservation and indispensability across all CoV species (5– 7). SARS-Nsp13 has been characterized as belonging to superfamily 1 (SF1) of the six helicase superfamilies which are *To whom correspondence should be addressed. Tel: +86 10 62771493; Fax: +86 10 62773145; Email: [email protected] †The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. C © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] D ow naded rom http/academ ic.p.com /nar/advance-articleoi/10.1093/nar/gkz409/5498756 by gest on 29 M ay 2019 2 Nucleic Acids Research, 2019 classified on the basis of several conserved motifs and can unwind both RNA and DNA duplexes in the 5′ to 3′ direction (8). The associated NTPase activity can target all natural nucleotides and deoxynucleotides as substrates (9,10). Moreover, it has been shown that SARS-Nsp12 can enhance the helicase activity of SARS-Nsp13 by increasing the step size of nucleic acid (dsRNA or dsDNA) unwinding by 2-fold (11). However, how the SARS-Nsp12 increase its helicase activity and if the NTPase activity is also influenced remains unclear. Structures of helicases from SF1 are available, amongst which the Upf1, eukaryotic RNA helicase essential for nonsense-mediated mRNA decay (NMD) signal pathway and Nsp10, the helicase of equine arteritis virus (EAV) share many structural features (12,13). SARS-CoV Nsp13 is also a Upf1-like helicase. However, until recently when the MERS-CoV Nsp13 was solved, no structural information for this coronavirus helicase was available despite biochemical characterization and the determination of kinetic parameters associated with its helicase or NTPase activity (14). The structure of MERS-CoV helicase in the absence of nucleotide and substrate was reported to have four domains, an N-terminal CH domain, two helicase core domains RecA1 and RecA2 and an inserted domain 1B. In addition, there is a ‘stalk’ region which connects the CH domain and 1B domain. However, how the five domains cooperate to contribute to the helicase function remains undefined. Here, we first present the structure of the full-length SARS-CoV Nsp13 (SARS-Nsp13). The five domains including zinc-binding domain, stalk domain, 1B domain, 1A domain and 2A domain are shown to coordinate with each other to complete the final unwinding process. Helicases have been characterized as translocases as the unwinding activity can be the result of it translocating on singlestranded oligonucleotides (15). We demonstrate how the 1A and 2A domains coordinate with each other when SARSNsp13 translocates on ssDNA through observing the H/D exchanges conditions of three states of SARS-Nsp13 with different ligands bound including ATP analog (AMPPNP), ADP-AlF4− and ADP. Moreover, we show that SARSNsp13 can interact with SARS-Nsp12 with high affinity and identified the key interaction domain on SARS-Nsp13, which provides us with insight into the RTC of SARS-CoV. MATERIALS AND METHODS Protein expression and purification The full-length helicase SARS-Nsp13 (1–601aa) was encoded by nucleotides (GenBank accession no. AY291315) of the SARS-CoV genome from strain Frankfurt 1 and was inserted into the modified pET-28a vector at NcoI/XhoI restriction sites with a hexa-histidine tag attached at its Nterminal end. BL21(DE3) cells were then transformed by introduction of this plasmid. After enlarging the reproduction volume of competent cells, the target gene was overexpressed. Cells were grown at 37◦C and induced with 200 M IPTG when the OD value reached ∼0.8. Thereafter, the induced cells were transferred to 18◦C to grow for 12–16 h. Cells were harvested at 4500 rpm by centrifugation at 4◦C. After ultrasonification and centrifugation at 14 000 rpm, the supernatant was run through a Ni-affinity column and the protein eluted with 200 mM imidazole. The eluate was then further purified by ion exchange column Hitrap S and sizeexclusion chromatography (Superdex 200, GE Healthcare). Crystallization, data collection and structure determination The protein solution was collected and concentrated to 6.7 mg/ml and then incubated with 25 thymine single-stranded DNA(dT25) at a molar ratio of 1:1.2 and then incubated with 2 mM AMPPNP at 4◦C for 3 h. The hanging-drop vapor-diffusion method was used to grow the Nsp13 crystals. The conditions for optimal crystal growth were 12% (w/v) polyethylene glycol 20 000, 2 M ammonium sulfate and 0.1 M MES monohydrate pH 6.5 at 16◦C. The protein and this crystallization buffer were mixed in equal volumes. All diffraction data sets were collected on beamline BL19U at Shanghai Synchrotron Radiation Facility (SSRF). Data was indexed, integrated and scaled with XDS (16). Single-wavelength anomalous data were collected at the zinc absorption edge and SHELXD was used to locate the six zinc atoms (17). The density map was improved with solvent flattening module of PHENIX program (18). The initial model was manually built in COOT (19) and further refined in PHENIX. The final 153 residues (443–596) were fitted using molecular replacement module in PHENIX with the equivalent residues of MERS-Nsp13. The structure was refined to 2.8 Å resolution. Data collection and processing statistics are summarized in Table 1. Surface plasmon resonance (SPR) assay 100 l of 20 g/ml SARS-Nsp12 in sodium acetate buffer pH 4.5 was prepared to be amino coupled onto channel 2 of a CM5 chip and fixed through addition of 100 l ETA in water. A gradient of SARS-Nsp13 was set up from 0.39 M to 3.12 M for four cycles of binding data measurements. 5 mM NaOH buffer was used for regeneration of the chip. The contact time and dissociation time were each set to 60 s. The experimental data and fitting data were processed with GraphPad Prism. Nucleic acid unwinding assay As for the helicase activity, dsDNA (5′-AATGTCTGAC GTAAAGCCTCTAAAATGTCTG-3′-BHQ, CY3-5′-CA GACATTTTAGAGG-3′) was used where the excitation wavelength was set to 547 nm and emission wavelength was set to 562 nm to detect fluorescence of CY3. 200 nM Nsp13 (final concentration) was added to the reaction buffer (50 mM HEPES 7.5, 20 mM NaCl, 4 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA) to incubate with dsDNA and 20 M trap ssDNA for 5 min. Then 2 mM ATP (final concentration) was added to initiate the helicase activity and the fluorescence value was recorded by Perkin-Elmer Envision.