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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High-throughput single-molecule quantification of individual base stacking energies in nucleic acids
Base stacking interactions between adjacent bases in DNA and RNA are important for many biological processes and in biotechnology applications. Previous work has estimated stacking energies between pairs of bases, but contributions of individual bases has remained unknown. Here, we use a Centrifuge Force Microscope for high-throughput single molecule experiments to measure stacking energies between adjacent bases. We found stacking energies strongest between purines (G|A at −2.3 ± 0.2 kcal/mol) and weakest between pyrimidines (C|T at −0.5 ± 0.1 kcal/mol). Hybrid stacking with phosphorylated, methylated, and RNA nucleotides had no measurable effect, but a fluorophore modification reduced stacking energy. We experimentally show that base stacking can influence stability of a DNA nanostructure, modulate kinetics of enzymatic ligation, and assess accuracy of force fields in molecular dynamics simulations. Our results provide insights into fundamental DNA interactions that are critical in biology and can inform design in biotechnology applications.
more » « less- Award ID(s):
- 1651877
- NSF-PAR ID:
- 10395665
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract The structures of zinc carbene ZnCH2and zinc carbyne HZnCH, and the conversion transition states between them are optimized at B3LYP/aug‐cc‐pVTZ, MP2/aug‐cc‐pVTZ, and CCSD/aug‐cc‐pVTZ levels of theory. The thermodynamic energies with CCSD(T) method are further extrapolated to basis set limit through a series of basis sets of aug‐cc‐pVXZ (X=D, T, Q, 5). The Zn−C bonding characteristics are interpreted by molecular plots, Laplacian of density plots, the integrated delocalization indices, net atomic charges, and derived atomic hardness. On the one hand, the studies demonstrated the efficiency of DFT method in structure optimizations and the accuracy of CBS method in obtaining thermodynamic energies; On the other hand, the density analysis of CCSD/aug‐cc‐pVDZ density demonstrates that both the sharing interaction and ionic interaction are important in ZnCH2ad HZnCH. The3B1state of ZnCH2is the global minimum and formed in visible light, but its small bond dissociation energy (47.0 kcal/mol) cannot keep the complex intact under UV light (79.4–102.1 kcal/mol). However, the3Σ−state of HZnCH can survive the UV light due to the greater Zn−C dissociation energy (100.7 kcal/mol). The delocalization indices of Zn…C in both3B1of ZnCH2(0.777) and the3Σ−state of HZnCH (0.785) are close to the delocalization index of the single C−C bond of ethane (0.841), i. e. the nomenclature of Zinc carbene and Zinc carbyne is incorrect. The stronger Zn−C bond in the3Σ−state of HZnCH than in the3B1state of ZnCH2can be attributed to the larger charge separation in the former. It was found that the Zn−C bonds in related Zinc organic compounds were also single bonds no matter whether the organic groups are CR, CR2, or CR3. The ionic interactions were discussed in terms of the atomic hardness that were in turn related to ionization energy and electron affinity. The unique combination of covalent and ionic characteristics in the Zn−C bonds of organic Zinc compounds could be the origin of many interesting applications of organic Zinc reagents.
