Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity
| Autor: | Mary K. Lewinski, John C. Guatelli, Aaron L. Oom, Charlotte A. Stoneham, Daniela V. Castro, Haley Siddiqi, Ben A. Croker, Stanley J. Opella, Sang Ho Park, Alex E. Clark, Aaron F. Carlin, Anna A. De Angelis |
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| Přispěvatelé: | Lee, Benhur |
| Rok vydání: | 2021 |
| Předmět: |
RNA viruses
Coronaviruses Protein Conformation Cell Membranes Spectrum analysis techniques Biochemistry Ion Channels Amiloride Protein structure Chlorocebus aethiops Macromolecular Structure Analysis 2.2 Factors relating to the physical environment Biology (General) Aetiology Integral membrane protein Lung Pathology and laboratory medicine Drug discovery Chemistry Respiratory infection Medical microbiology Transmembrane domain Infectious Diseases Virion assembly 5.1 Pharmaceuticals Medical Microbiology Viruses SARS CoV 2 Pathogens Cellular Structures and Organelles Development of treatments and therapeutic interventions Infection Research Article Protein Binding Protein Structure SARS coronavirus QH301-705.5 Nuclear Magnetic Resonance 1.1 Normal biological development and functioning Protein domain Immunology Antiviral Agents Microbiology Chemical shift index Vaccine Related Coronavirus Envelope Proteins NMR spectroscopy Protein Domains Underpinning research Biodefense Virology Genetics Animals Humans Integral Membrane Proteins Binding site Nuclear Magnetic Resonance Biomolecular Molecular Biology Vero Cells Medicine and health sciences Binding Sites Biology and life sciences SARS-CoV-2 Virus Assembly Prevention Organisms Viral pathogens Proteins Membrane Proteins Polypeptides COVID-19 Cell Biology Pneumonia RC581-607 Microbial pathogens COVID-19 Drug Treatment Research and analysis methods Emerging Infectious Diseases Membrane protein Biophysics Parasitology Immunologic diseases. Allergy Peptides Biomolecular |
| Zdroj: | PLoS pathogens, vol 17, iss 5 PLoS Pathogens, Vol 17, Iss 5, p e1009519 (2021) PLoS Pathogens |
| Popis: | SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8–43) and a short cytoplasmic helix (residues 53–60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6–18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5’ position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein. Author summary The novel coronavirus SARS-CoV-2, the causative agent of the world-wide pandemic of COVID-19, has become one of the greatest threats to human health. While rapid progress has been made in the development of vaccines, drug discovery has lagged, partly due to the lack of atomic-resolution structures of the free and drug-bound forms of the viral proteins. The SARS-CoV-2 envelope (E) protein, with its multiple activities that contribute to viral replication, is widely regarded as a potential target for COVID-19 treatment. As structural information is essential for drug discovery, we established an efficient sample preparation system for biochemical and structural studies of intact full-length SARS-CoV-2 E protein and characterized its structure and dynamics. We also characterized the interactions of amilorides with specific E protein residues and correlated this with their antiviral activity during viral replication. The binding affinity of the amilorides to E protein correlated with their antiviral potency, suggesting that E protein is indeed the likely target of their antiviral activity. We found that residue asparagine15 plays an important role in maintaining the conformation of the amiloride binding site, providing molecular guidance for the design of inhibitors targeting E protein. |
| Databáze: | OpenAIRE |
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