Delicate structural coordination of the Severe Acute Respiratory Syndrome coronavirus Nsp13 upon ATP hydrolysis - PubMed (original) (raw)

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

Zhihui Jia et al. Nucleic Acids Res. 2019.

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 Upf1-like 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 β19-β20 loop on the 1A domain is involved in unwinding process directly. Furthermore, we have shown that the RNA dependent RNA polymerase (RdRp), SARS-Nsp12, can enhance the helicase activity of SARS-Nsp13 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.

© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.

Figure 1.

Overall structure of SARS-Nsp13. (A) Ribbon structure of SARS-Nsp13 is composed of ZBD (lime), stalk (yelloworange), 1B (salmon), 1A (aquamarine) and 2A (palecyan) domains. Three zinc atoms are shown as dark red spheres and schematic diagram of the domain organization of SARS-Nsp13. (B) Up, the crystal packing arrangement of two SARS-Nsp13 molecules. Down, the crystal packing arrangement of two MERS-Nsp13 molecules (5WWP). (C) The ATPase activity of SARS-Nsp13. The final concentration of SARS-Nsp13 was 25 nM. The gradient concentration of substrate ATP were 0.08, 0.1, 0.25, 0.5, 1.0, 1.25, 2.0 mM. The calculated _V_0 corresponding to each ATP concentration was plotted against the ATP concentration fitting the Michaelis-Menten function. The final _V_max is 0.4845 ± 0.01311 μM/min. _K_m = 0.1552 ± 0.01693 mM. (D) The unwinding activity of SARS-Nsp13. Up, 20 nM SARS-Nsp13 was incubated with dsDNA for 1, 5 and 10 min. Down, SARS-Nsp13 of different concentrations were incubated with dsDNA for 1 min. The dsDNA (5′-CAGACATTTTAGAGG-3′-CY3, 5′-AATGTCTGACGTAAAGCCTCTAAAATGTCT-3′) used in the assay is labelled with CY3.

Figure 2.

Figure 2.

The active pocket composed of ATPase related residues. (A) Left, superposition between Yeast-Upf1-ADP-AlF4− (2XZL) (24) in grey and SARS-Nsp13 in green. Right, The stick model of all the ATPase related residues. The ADP-AlF4− is from the Upf1-ADP-AlF4− complex structure not the SARS-Nsp13 structure. AlF4− is presented in cyan while the ADP molecule in salmon. All the residues are presented in color by element with S atoms in orange, O atoms in red, N atoms in blue and H atoms in tints. (B) ATPase activity of all the ATP hydrolysis related mutants. The initial ATP concentration is 150 μM and the protein concentration is 25 nM. The changes of percentage of hydrolyzed ATP over time is demonstrated. The fitting function is one-phase association in the GraphPad Prism program. (C) Initial ATP hydrolysis velocities of WT-Nsp13 and six mutants under 150 μM ATP concentration are 0.3952 ± 0.05841 μM/s (WT-Nsp13), 0.1926 ± 0.01509 μM/s (K288A), 0.1884±0.01409 μM/s (S289A), 0.1725 ± 0.00748 μM/s (D374A), 0.1753 ± 0.0072 μM/s (E375A), 0.1716 ± 0.00947 μM/s (Q404A) and 0.1661 ± 0.005 μM/s (R567A) respectively. (D) The time-course changing of dsDNA unwound fraction for the WT-Nsp13. The initial dsDNA concentration is 250 nM and the protein concentration is 20 nM. The fitting function is one-phase association in the GraphPad Prism program. (E) The time-course changing of dsDNA unwound fraction for the three mutants including K288A, S289A, D374A. The initial dsDNA concentration is 250 nM and the protein concentration is 20 nM. The fitting function is one-phase association in the GraphPad Prism program. (F) The time-course changing of dsDNA unwound fraction for the three mutants including E375A, Q404A, R567A. The initial dsDNA concentration is 250 nM and the protein concentration is 20 nM. The fitting function is one-phase association in the Graphpad Prism program. (G) Initial unwinding velocities of WT-Nsp13 and six mutants under the dsDNA substrate concentration of 250 nM are 1.801 ± 0.2308 nM/s (WT-Nsp13), 0.037 ± 0.001212 nM/s (K288A), 0.04637 ± 0.008041 nM/s (S289A), 0.03097 ± 0.0049 nM/s (D374A), 0.02903 ± 0.007 nM/s (E375A), 0.04497 ± 0.00208 nM/s (Q404A) and 0.0407 ± 0.006129 nM/s (R567A) respectively.

Figure 3.

Figure 3.

Nucleic acids binding regions. Images B, C, D and E represent results of the H/D exchange experiments recognizing the nucleic acids binding regions. There are four shift patterns for each peptide in different samples where the first and second row represents the shift patterns of SARS-Nsp13 incubated with 7-fold molar and 3-fold molar excess of dsDNA respectively, the third row represents the shift pattern of SARS-Nsp13 and the last row represents the unexchanged pattern of SARS-Nsp13. The x-axes displays the mass-to-charge ratio of each peptide. The dashed vertical lines indicate the mass-to-charge ratio for each peptide in different samples. When the mass-to-charge ratio of SARS-Nsp13 incubated with nucleic acids shifts to right compared to that of SARS-Nsp13, more H/D exchanges in the peptides happen and vice versa. (A) Regions in red are the predicted nucleic acids binding related peptides based on the complex model, where the dsDNA is highlighted in blue. (B) Shift patterns for peptides 153–179. (C) Shift patterns for peptides 209–224. (D) Shift patterns for peptides 331–357. (E) Shift patterns for peptides 523–542.

Figure 4.

Figure 4.

Unwinding activity of double mutants relevant for nucleic acids binding. (A) The rectangle indicates the location of amino acid residues involved in nucleic acids binding and the black arrow points to the β19–β20 loop. Residues in salmon belong to the 1B domain. Residues in cyan belong to the 1A domain. Residues in palecyan belong to the 2A domain. All residues are presented in color by elements with S atoms in orange, O atoms in red, N atoms in blue and H atoms in tints. (B) The binding affinity to dsDNA of SARS-Nsp13 and six double mutants are demonstrated through EMSA. The protein concentration is 5 μM and the dsDNA concentration is 50 nM. Protein was incubated with dsDNA for 30 min at room temperature. (C) The time-course changing of dsDNA unwound fraction for WT-Nsp13 and six double mutants. The initial dsDNA concentration is 400 nM and the protein concentration is 20 nM. The fitting function is one-phase association in the Graphpad Prism program. (D) Initial unwinding velocities of WT-Nsp13 and six double mutants under the dsDNA concentration of 400 nM are 1.657 ± 0.6578 nM/s (WT-Nsp13), 0.9663 ± 0.3265 nM/s (N179A/R212A), 0.3105 ± 0.1036 nM/s (R337A/R339A), 0.1184 ± 0.04126 nM/s (K345A/K347A), 0.3797 ± 0.06969 nM/s (R507A/K508A), 0.04992 ± 0.2609 nM/s (K524A/Q531A) and 0.7764 ± 0.4421 nM/s (S539A/Y541A) respectively.

Figure 5.

Figure 5.

Different states of Nsp13 with different small molecules as indicated by H/D exchange experiment results (Supplementary Table S1). A, B, C and D represent four different conformation states of SARS-Nsp13 with or without small molecules and they form a cycle in which ATP is hydrolysed by step. Regions in red suggest it experienced less H/D exchanges while regions in blue suggest it experienced more compared to the previous state in the cycle. (A) The initial state where Nsp13 is bound with dsDNA. (B) The substrate state where Nsp13-dsDNA is bound with ATP analog (AMPPNP). (C) The transition state where Nsp13-dsDNA is bound with ADP-AlF4−. (D) The product state where Nsp13-dsDNA is bound with ADP.

Figure 6.

Figure 6.

The ZBD domain plays a critical role during the SARS-Nsp13 helicase activity cycle. (A) Key residues participating in zinc finger formation. ZF3 motif is highlighted in red. Three zinc atoms are presented in sphere in red. (B) How the CH domain of Upf1 is rotated away through interacting with Upf2 (18). The CH domain is highlighted in green. The Upf2 is highlighted in salmon. The left structure represents the Upf1 only while the right structure represents the Upf1–Upf2 complex. (C) Hydrophobic residues involved in stalk region packing against ZBD and 1A domains respectively. Residues in green belong to the ZBD domain. Residues in yelloworange belong to the stalk domain. Residues in salmon belong to the 1B domain. Residues in cyan belong to the 1A domain. All residues are presented in color by elements with S atoms in orange, O atoms in red, N atoms in blue and H atoms in tints. (D) The two residues N102 and K131 involved in hydrophilic interaction in the stalk domain. Residues in green belong to the ZBD domain. Residues in cyan belong to the 1A domain. The stalk domain is presented in yelloworange. All residues are presented in color by elements with S atoms in orange, O atoms in red, N atoms in blue and H atoms in tints. (E) The time-course changing of dsDNA unwound fraction for mutants N102A and K131A. The initial dsDNA concentration is 400 nM and the protein concentration is 20 nM. The fitting function is one-phase association in the Graphpad Prism program. (F) Initial unwinding velocities of WT-Nsp13 and two single mutants under the dsDNA concentration of 400 nM are 1.643 ± 0.1667 nM/s (WT-Nsp13), 0.5119 ± 0.06516 nM/s (N102A) and 0.3164 ± 0.05154 nM/s (K131A) respectively.

Figure 7.

Figure 7.

The interaction between SARS-Nsp13 and SARS-Nsp12. (A) The unwinding activity of SARS-Nsp13 incubated with SARS-Nsp12 and MERS-Nsp12 is presented as the time-course changing of the dsDNA unwound fraction. The initial dsDNA concentration is 250 nM and the protein concentration is 20 nM. The fitting function is one-phase association in the Graphpad Prism program. (B) Initial unwinding velocities of SARS-Nsp13, SARS-Nsp13 with SARS-Nsp12 and SARS-Nsp13 with MERS-Nsp13 under the dsDNA concentration of 400 nM are 2.078 ± 0.4675 nM/s (SARS-Nsp13), 3.199 ± 0.4153 nM/s (SARS-Nsp13 incubated with SARS-Nsp12), 2.905 ± 0.3589 nM/s (SARS-Nsp13 incubated with MERS-Nsp12) respectively. (C) ATPase activity of SARS-Nsp13, SARS-Nsp13 with SARS-Nsp12 and SARS-Nsp13 with MERS-Nsp13. The initial ATP concentration is 150 μM and the protein concentration is 25 nM. The changes of percentage of hydrolyzed ATP over time is demonstrated. The fitting function is one-phase association in the GraphPad Prism program. (D) ATP hydrolysis velocities of SARS-Nsp13 and SARS-Nsp13 incubated with SARS-Nsp12 or MERS-Nsp12 under 150 μM ATP concentration are 0.2401±0.01062 μM/s (SARS-Nsp13), 0.3572 ± 0.0329 μM/s (SARS-Nsp13 incubated with SARS-Nsp12) and 0.3373 ± 0.0314 μM/s (SARS-Nsp13 incubated with MERS-Nsp12) respectively. (E) Representative SPR sensorgrams for SARS-Nsp12 with 3.12 μM SARS-Nsp13 (blue), 1.56 μM SARS-Nsp13 (orange), 0.78 μM SARS-Nsp13 (pink) and 0.39 μM SARS-Nsp13 (green). Association time was 60 s and dissociation time was 60 s. The binding affinity is _K_D = 236 nM. (F) SARS-Nsp12 binding regions on ZF3 motif of ZBD and 1A domains are highlighted in red.

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