key: cord-0962621-6ibc7avw authors: Agback, Tatiana; Dominguez, Francisco; Frolov, Ilya; Frolova, Elena I.; Agback, Peter title: 1H, 13C and 15N resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution date: 2021-05-05 journal: bioRxiv DOI: 10.1101/2021.05.05.442725 sha: 69ceb9cfd74f0cdff0171d6dce3bd92ba3120ae9 doc_id: 962621 cord_uid: 6ibc7avw Structural characterization of the SARS-CoV-2 full length nsp1 protein will be an essential tool for developing new target-directed antiviral drugs against SARS-CoV-2 and for further understanding of intra- and intermolecular interactions of this protein. As a first step in the NMR studies of the protein, we report the 1H, 13C and 15N resonance backbone assignment as well as the Cβ of the apo form of the full-lengthSARS-CoV-2 nsp1 including folded domain together with the flaking N- and C-terminal intrinsically disordered fragments. The 19.8 kD protein was characterized by high-resolution NMR. Validation of assignment have been done by using two different mutants, H81P and K129E/D48E as well as by amino acid specific experiments. According to the obtained assignment, the secondary structure of the folded domain in solution was almost identical to its previously published X-ray structure, but some discrepancies have been detected. In the solution SARS-CoV-2 nsp1 exhibited disordered, flexible N- and C-termini with different dynamic characteristics. The short peptide in the beginning of the disordered C-terminal domain adopted two different conformations distinguishable on the NMR time scale. We propose that the disordered and folded nsp1 domains are not fully independent units but are rather involved in intramolecular interactions. Studies of the structure and dynamics of the SARS-CoV-2 mutant in solution are on-going and will provide important insights into the molecular mechanisms underlying these interactions. Within the recent 1.5 years, the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-24 CoV-2) has spread all over the world and devastated the economies of essentially all countries (1, 2). To 25 date, more than one hundred million people have contracted the disease that led more than 3 M deaths 26 (https://www.worldometers.info/coronavirus). Despite the enormous public health threat of this and 27 previous CoV infections, no efficient therapeutic means have been developed against coronaviruses 28 (CoV) before the COVID-19 pandemics. One of the major reasons for this was a lack of detailed 29 knowledge of the mechanism of CoV replication and interaction with host cells. downregulation of cellular translation and is a major -CoV-specific virulence factor (6, 9, 10). It 43 interacts with the 40S ribosomal subunit, blocks the RNA channel and inhibits initiation of translation 44 of cellular, but not viral, RNA templates (6, 8, 11-17). SARS-CoV-1 and MERS nsp1 proteins are also 45 indirectly involved in endonuclease degradation of cellular mRNAs; however, the mechanism of this 46 function remains to be determined (18, 19) . It is still unknown whether SARS-CoV-2 nsp1 can mediate degradation of cellular RNAs. Nsp1 of both SARS-CoV-1 and SARS-CoV-2 were also implicated in 48 inhibition of nuclear-cytoplasmic traffic, albeit by different mechanisms (20, 21). The above activities 49 appear to play critical roles in the downregulation of the innate immune response that can mount during 50 SARS-CoV-2 infection, and thus, control the infection spread. Nsp1 proteins of -CoVs also facilitate 51 cell cycle arrest, which is clearly detectable during viral infection and nsp1 expression (9, 22). Importantly, the previous studies demonstrated that the deletion of nsp1 gene in the genome of other - CoVs makes them nonviable (23). This strongly indicated the direct involvement of the latter protein in (~aa 10 to 124 in SARS-CoV2 nsp1), which was proposed to be critical for degradation of cellular 65 mRNAs. The first 10 aa and the C-terminal fragment (aa125-180) in SARS-Cov-2 nsp1 are predicted to 66 be intrinsically disordered. However, the last 26-aa-long peptide in this C-terminal fragment has been 67 shown to fold into two short -helixes upon binding to 40S ribosome subunit (7, 8, 17). The structure of 68 folded N-terminal domain of SARS-CoV nsp1 has been determined by NMR (PDB:2HSX), and two X- a near-complete backbone resonance assignment of the SARS-CoV-2 nsp1 was reported (31). The latter 84 protein was analysed in an acidic buffer, pH 6.5. As a first step towards characterizing the structure and dynamics of the full-length SARS-CoV-86 2 nsp1 in neutral buffer by NMR spectroscopy, we herein report the almost complete 1 H, 13 C and 15 N 87 backbone and 13 C side chain assignment of the wild type protein and two of its mutants: a single mutant Table 1 . Table 1 . To verify assignment of the aa located in the disordered fragment of SARS-CoV-2 nsp1, 13 C 146 observed CON experiment with IPAP scheme for virtual decoupling (37, 38) The chemical shifts of the full-length SARS-CoV-2 nsp1 were analyzed with TALOS+ 159 software (41). As input for TALOS+ analysis, the experimentally derived chemical shifts of 1 HN, 15 N, 160 13 Cα, 13 Cβ, 13 C´ and 1 H  nuclei for every aa were used. In case of absence of the chemical shifts, 161 TALOS+ uses a database of sequences to predict the secondary structure (41). The secondary X-ray based structures were extracted with the UCSF Chimera program(42) 163 using the PDB entry: 7K7P (30). In the text and figures, the standard nomenclature for amino acids of the carbon atoms was used, where 165 13 Cα is the carbon next to the carbonyl group 13 C´ and 3 Cβ is the carbon next to 13 Cα (43). which is shown in Fig.2A . Indeed, spectra of the TROSY-MUSIC presented in Fig. 1 (a) These data were used to assign the resonances at 308 K. The 1 H-15 N-HSQC spectrum at 219 308K shows well-dispersed and narrow-line widths of the amide signals (Fig. 2 B, C) . At this 220 temperature, we have observed and assigned 158 aa, including prolines. Importantly, even at this 221 higher temperature (308K), amino acids 125K, 124R, 123L and 122L shows two sets of amides 1 HN -222 15 N cross peaks, which allowed us to conclude that the aa between the folded domain and the C- nuclei located in its proximity due to its unique structure and possibly enhance the stability of the loop. As it is shown in Fig. 3d , the CSP observed in 1 H-15 N HSQC spectra of wt nsp1 vs the H81P mutant 248 (red bars) are evident. As expected, the most significant CSPs were observed between aa 75 and 85. Noteworthy, the aa of the N-termini (10-17) and at the beginning of the C-terminal disordered 250 fragment (120-127aa) are affected as well. This finding led us to the conclusion that aa corresponding 251 to those three regions are in close proximity. CoV-2 nsp1 mutants will be published elsewhere. The resulting assignment of the full length of SARS-CoV-2 nsp1 was as following. domain of the SARS-CoV-2 nsp1, which was derived from the NMR data, and the previously 288 determined crystal structure (7K7P) shows that they are almost identical, and this has validated our 289 resonance assignment ( Fig.3b and a) . Nevertheless, a few important inconsistencies were identified. (Fig. 3C) ], the segment between aa 92-103 is dynamic. This prediction is 295 in agreement with our finding that amide protons between residues I95 and G98 were not observed at 296 308K and 298K, suggesting their involvement in multiple conformational exchange and exposure to 297 the solvent. We additionally performed an analysis of the 3D 1 H-15 N NOESY spectrum to determine 298 dipole-dipole contacts of NH-NH and NH-H  protons, which allows to detect hydrogen bonds between 299 two -strands (44). The β-sheet formed by strands 4 and 3, according to the X-ray structure, was 300 confirmed by observing NH-NH and NH-H NOEs between those strands, but not between the 4 and 301 5 strands. These data contradict the X-ray results, which suggest low mobility of the 5 strand due to 302 the additional hydrogen bonds between the 4 and 5 strands. Subtle differences between the X-ray and NMR secondary structures were also noticed for the 305 strands 1, 2, 6 and 7. According to our NMR data, in solution, these strands are extended by one 306 or two aa at their C-termini. Furthermore, the -helix 2 in solution is one aa shorter than in the X-ray 307 structure. Our data also predicted that the -helix has a break at aa H45. Another difference is that the 308 previously identified short -helix 3 is not predicted by the NMR data. Instead, it suggests the presence 309 of a long, disordered loop between aa 55 and 67, which, according to TALOS+ prediction, has 310 restricted mobility (Fig.3b, c) . Importantly, this region of SARS-CoV-2 nsp1 sequence is well 311 characterized by NMR through CS as well as NOE of NH-NH, NH -NH-H  protons contacts. This led 312 us to conclusion that these discrepancies between NMR and X-ray secondary structure predictions are 313 likely result from the crystallisation conditions. Two loop regions in the folded domain of SARS-CoV-2, (aa Q27-S34 and G112-A117) are 315 in agreement with the X-ray structure and have restricted mobility according to the higher order S 2 316 parameter predicted by TALOS+ (Fig 3c) . were identified based on the S2 order parameter predicted by TALOS+: S74-H83 and L92-E102 (Fig 319 3 b, c) . This prediction is in line with the lack of peaks or broadened 15 N/ 1 HN cross peaks, even at 320 T298K in the 1 H-15 N NMR spectra. We did not observe resonances for H81 and G82 in the first region 321 and for S100 and G101 in the second one. It can be explained either by broadening of the cross peaks 322 below detection limit or, which is more likely, by the involvement of these regions in slow 323 conformational exchange. The N-and C-termini, comprising aa M1-N9 and N124-G180, respectively, were identified 325 by CSI as fully unstructured, but having different predicted order parameters (S 2 ) through the 326 sequences. Dynamic regions with an order parameter S 2 below 0.6 were predicted for M1-F8, S135-327 Q158 and H165-G180. The increase in dynamic behaviour of those aa correlated with the changes in 328 the intensities of the 15 N/ 1 HN backbone cross peaks in the 1 H-15 N NMR spectra of nsp1. These cross 329 peaks have higher intensity compared to cross peaks belonging to the folded less dynamic aa. Based 330 on these data, we propose that in the full-length SARS-CoV-2 nsp1, the folded and disordered parts of 331 the protein behave not as fully independent units but are rather involved in intramolecular interactions. In conclusion, the near complete 15 N/ 13 C/ 1 H backbone resonance and part of side chain 334 assignment of the full-length SARS-CoV-2 nsp1 at pH 7.5 and physiological salt concentration has 335 been performed. Validation of assignment have been done by using two different nsp1 mutants as well 336 as MUSIC type experiments. Assignment revealed that the secondary structure of the rigid folded 337 domain is almost identical to that determined by X-ray. However, the existence of the short -strand 338 (aa 95 to 97), which is considered to be the significant structural difference between SARS-CoV-1 and 339 SARS-CoV-2 nsp1 proteins, was not confirmed. In solution, SARS-CoV-2 nsp1exhibits disordered, Acceleration Funds to E.F. and I.F. We thank Nikita Shiliaev for technical assistance. 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