Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization

Abstract

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 Phe241 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.

Structure of the N-RNA/P interface reveals mode of L/P attachment to the nucleocapsid of human metapneumovirus

“…In one instance we observed the dissociation and rebinding of one of the simulated peptides. This is in line with the disordered character of P, but also with previous reports in RSV characterizing the interaction as giving rise to a fuzzy complex 37,42 .…”

“…P is a flexible protein with extensive intrinsically disordered regions, and the N-RNA/P interaction has been suggested to be transient, complicating structural characterization 31,32,37 . Using the deep-learning based Metapredict server 38 , we generated disorder-score plots for both HMPV N and P. In agreement with previous work, N disorder scores are globally low, while P contains significantly disordered regions (Fig.…”

4,4′-Dicyano- versus 4,4′-Difluoro-BODIPYs in Chemoselective Postfunctionalization Reactions: Synthetic Advantages and Applications

“… 12 Nevertheless, recent investigations showed that 4,4′-dicyano-BODIPYs are significantly more resistant to trifluoroacetic acid than the corresponding difluoro-BODIPYs. 13 This enhanced chemical stability toward acid on dicyano-BODIPYs 6b , 14 was attributed to a strengthening of the B–N bonds, owing to the higher aromaticity of the former. 12b…”