Biophysical and Dynamic Characterization of Fine-Tuned Binding of the Human Respiratory Syncytial Virus M2-1 Core Domain to Long RNAs

Abstract

The human Respiratory Syncytial Virus (hRSV) M2-1 protein functions as a processivity and antitermination factor of the viral polymerase complex. Here it is presented the first evidence that hRSV M2-1 core domain (cdM2-1) alone has an unfolding activity for long RNAs, as well as a biophysical and dynamic characterization of the cdM2-1/RNA complex. The main contact region of cdM2-1 with RNA was the α1–α2–α5–α6 helix bundle, which suffered local conformational changes and promoted the RNA unfolding activity. This activity may be triggered by base-pairing recognition. RNA molecules wrap around the whole cdM2-1, protruding their terminals over the domain. The α2–α3 and α3–α4 loops of cdM2-1 were marked by an increase in picosecond internal motions upon RNA binding even though they are not directly involved in the interaction. The results revealed that the cdM2-1/RNA complex originates from a fine-tuned binding, contributing to unraveling interaction aspects necessary to M2-1 activity. IMPORTANCE The main outcome is the molecular description of a fine-tuned binding of the cdM2-1/RNA complex and the evidence that the domain alone has an unfolding activity for long RNAs. This binding mode is essential in the understanding of the function in the full-length protein. Orthopneumovirus, as the human Respiratory Syncytial Virus (hRSV), stands out for the unique role of M2-1 as a transcriptional antitermination factor able to increase the RNA polymerase processivity.

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids

“…The effect of salt (NaCl) and inorganic phosphate (Pi) on the N-NTD:ssDNA complex formation was investigated at 25 °C. The binding isotherms were fitted using the following equation after data treatment [34] : where F is the measured fluorescence intensity in the presence of DNA, F 0 is the fluorescence in the absence of DNA, F S is the fluorescence emission of the protein saturated with DNA, K d is the dissociation constant, [ L T ] is the total ligand concentration (DNA), and [ P T ] is the total protein concentration (N-NTD or N-NTD-SR). The K d values were calculated from the fitting process by nonlinear least-squares optimization using Levenberg–Marquardt interactions in the software OriginPro 2021.…”

Structure insights, thermodynamic profiles, dsDNA melting activity, and liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid N-terminal domain binding to DNA

“…The effect of salt (NaCl) and inorganic phosphate (Pi) on the N-NTD:ssDNA complex formation was investigated at 25 °C. The binding isotherms were fitted using the following equation after data treatment (25): where F is the measured fluorescence intensity in the presence of DNA, F 0 is the fluorescence in the absence of DNA, F S is the fluorescence emission of the protein saturated with DNA, K d is the dissociation constant, [ L T ] is the total ligand concentration (DNA), and [ P T ] is the total protein concentration (N-NTD or N-NTD-SR). The K d values were calculated from the fitting process by nonlinear least-squares optimization using Levenberg–Marquardt interactions in the software OriginPro 2021.…”

“…The effect of salt (NaCl) and inorganic phosphate (Pi) on the N-NTD:ssDNA complex formation was investigated at 25 °C. The binding isotherms were fitted using the following equation after data treatment (25):…”