Biophysical characterization of the interaction between M2-1 protein of hRSV and quercetin

Abstract

hRSV is the major causative agent of acute respiratory infections. Among its eleven proteins, M2-1 is a transcription antiterminator, making it an interesting target for antivirals. Quercetin is a flavonol which inhibits some virus infectivity and replication. In the present work, the M2-1 gene was cloned, expressed and the protein was purified. Thermal stability and secondary structure were analyzed by circular dichroism and the interaction with Quercetin was evaluated by fluorescence spectroscopy. Molecular docking experiments were performed to understand this mechanism of interaction. The purified protein is mainly composed of α-helix, with a melting temperature of 328.6K (≈55°C). M2-1 titration with Quercetin showed it interacts with two sites, one with a strong constant association K1 (site 1≈1.5×10M) by electrostatic interactions, and another with a weak constant association K2 (site 2≈1.1×10M) by a hydrophobic interaction. Ligand's docking shows it interacts with the N-terminus face in a more polar pocket and, between the domains of oligomerization and RNA and P protein interaction, in a more hydrophobic pocket, as predicted by experimental data. Therefore, we postulated this ligand could be interacting with important domains of the protein, avoiding viral replication and budding.

Biophysical and dynamic characterization of a fine-tuned binding of the human Respiratory Syncytial Virus M2-1 core domain to long RNAs

“…DSC data showed that the melting temperature of the hRSV cdM2-1 (68.7 °C) is at least 13 °C higher than those reported for the full-length M2-1 (~55 °C) (15). Similar results were reported for other proteins in the literature, such as the C-terminal domain (CH2) of the human utrophin calponin-homology domain (24) and the N-terminal domain (C2A) of the cytosolic C2 domains in tandem of synaptotagmin I (25).…”

“…The structural models of the cdM2-1/RNA complex were generated using the 3dRPC webserver (20). The structural restrains of the complex were defined from the chemical shift perturbations due to the NMR titrations of [U- 15 The fluorescence quenching experiments showed that the binding of the hRSV cdM2-1 to RNA has nano to micromolar dissociation constants (Table 1) nucleic acids with a length between 10 and 20 nucleotides with sequence specificity, being that the first group determined values in 10-100 micromolar range using CSP by NMR spectroscopy and the second, in the hundred-nanomolar range using EMSA (Electrophoretic Mobility Shift Assay) and fluorescence anisotropy (7,10). Studies reported that the interaction of the full-length M2-1 with nucleic acids present dissociation constants of tenths of nanomolar that are at least 10fold lower than those recorded for the cdM2-1: 30 nM for long RNA without sequence specificity (22), ~19 nM for poly-A 13mer RNA (8), and ~15 nM for 20mer RNA with sequence specificity (10).…”

“…The core domain (residues 58-177) of hRSV M2-1 (cdM2-1) was expressed in E. coli BL21 (DE3) RIL cells with a pD441-NHT vector (ATUM, USA) in M9 minimal medium containing 15 NH 4 Cl as the sole nitrogen source for production of isotopically labeled protein, as described previously (32). The fluorescence quenching data of the cdM2-1/RNA interaction were analyzed using the following equation 34:…”

“…The 15 N hetNOE values were calculated from resonance intensity ratios obtained from the NOE and control spectra, with uncertainties estimated from the background noise of the spectra.…”

Insights into Interactions of Flavanones with Target Human Respiratory Syncytial Virus M2-1 Protein from STD-NMR, Fluorescence Spectroscopy, and Computational Simulations

“…M 2-1 can be considered as the target protein to the potential antiviral activity of small molecules, such as flavonoids. Previous studies showed for example that the quercetin [30] and with its acetylated derivatives [31] can bind to the antitermination factor M 2-1 . Therefore, the present work aimed to investigate the binding interaction of HST and HSD with the hRSV M 2-1 protein by using experimental techniques of STD-NMR and fluorescence spectroscopy in association with computational tools of molecular docking and molecular dynamics.…”