Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with viral genome replication. In this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can initially bind in a comparatively stabilized state to the viral RdRp active site, as it primarily forms base stacking with the template uracil nucleotide (nt +1), which under freely fluctuations supports a low free energy barrier of the RDV-TP insertion (∼1.5 kcal mol −1 ). In comparison, the corresponding natural substrate ATP binds initially to the RdRp active site in Watson–Crick base pairing with the template nt, and inserts into the active site with a medium low free energy barrier (∼2.6 kcal mol −1 ), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be specifically stabilized by motif C-S759, S682 (near motif B) with the base, and motif G-K500 with the template backbone. Although the RDV-TP insertion can be hindered by motif F-R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif A-D623, respectively.
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Enhanced TROSY Effect in [2‐ 19 F, 2‐ 13 C] Adenosine and ATP Analogs Facilitates NMR Spectroscopy of Very Large Biological RNAs in Solution
Large RNAs are central to cellular functions, but characterizing such RNAs remains challenging by solution NMR. We present two labeling technologies based on [2‐19F, 2‐13C]‐adenosine, which allow the incorporation of aromatic19F‐13C spin pairs. The labels when coupled with the transverse relaxation optimized spectroscopy (TROSY) enable us to probe RNAs comprising up to 124 nucleotides. With our new [2‐19F, 2‐13C]‐adenosine‐phosphoramidite, all resonances of the human hepatitis B virus epsilon RNA could be readily assigned. With [2‐19F, 2‐13C]‐adenosine triphosphate, the 124 nt pre‐miR‐17‐NPSL1‐RNA was produced via in vitro transcription and the TROSY spectrum of this 40 kDa [2‐19F, 2‐13C]‐A‐labeled RNA featured sharper resonances than the [2‐1H, 2‐13C]‐A sample. The mutual cancelation of the chemical‐shift‐anisotropy and the dipole‐dipole‐components of TROSY‐resonances leads to narrow linewidths over a wide range of molecular weights. With the synthesis of a non‐hydrolysable [2‐19F, 2‐13C]‐adenosine‐triphosphate, we facilitate the probing of co‐factor binding in kinase complexes and NMR‐based inhibitor binding studies in such systems. Our labels allow a straightforward assignment for larger RNAs via a divide‐and‐conquer/mutational approach. The new [2‐19F, 2‐13C]‐adenosine precursors are a valuable addition to the RNA NMR toolbox and will allow the study of large RNAs/RNA protein complexes in vitro and in cells.
more » « less- NSF-PAR ID:
- 10491886
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- Volume:
- 63
- Issue:
- 9
- ISSN:
- 1433-7851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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