期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 24, 期 1, 页码 -出版社
MDPI
DOI: 10.3390/ijms24010569
关键词
respiratory syncytial virus; phosphoprotein; nucleoprotein; replication complex; fuzzy complex; protein-protein interaction; PPI inhibition; nuclear magnetic resonance; fluorescence anisotropy; B-cyano BODIPYs
The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is crucial for the formation of the RSV polymerase. We investigated the binding affinities and dynamics of this interaction using nuclear magnetic resonance and fluorescence polarization. We optimized parameters for a robust RSV N-P inhibition assay and validated it with the M76 molecule for further chemical library screening.
The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe(241) residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries.
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