Biomolecular recognition often involves conformational changes as a prerequisite for binding (i.e., conformational selection) or concurrently with binding (i.e., induced-fit). Recent advances in structural and kinetic approaches have enabled the detailed characterization of protein motions at atomic resolution. However, to fully understand the role of the conformational dynamics in molecular recognition, studies on the binding transition state are needed. Here, we investigate the binding transition state between nonstructural protein 1 (NS1) of the pandemic 1918 influenza A virus and the human p85β subunit of PI3K. 1918 NS1 binds to p85β via conformational selection. We present the free-energy mapping of the transition and bound states of the 1918 NS1:p85β interaction using linear free energy relationship and ϕ-value analyses. We find that the binding transition state of 1918 NS1 and p85β is structurally similar to the bound state with well-defined binding orientation and hydrophobic interactions. Our finding provides a detailed view of how protein motion contributes to the development of intermolecular interactions along the binding reaction coordinate.

Elucidating how individual mutations affect the protein energy landscape is crucial for understanding how proteins evolve. However, predicting mutational effects remains challenging because of epistasis—the nonadditive interactions between mutations. Here, we investigate the biophysical mechanism of strain-specific epistasis in the nonstructural protein 1 (NS1) of influenza A viruses (IAVs). We integrate structural, kinetic, thermodynamic, and conformational dynamics analyses of four NS1s of influenza strains that emerged between 1918 and 2004. Although functionally near-neutral, strain-specific NS1 mutations exhibit long-range epistatic interactions with residues at the p85β-binding interface. We reveal that strain-specific mutations reshaped the NS1 energy landscape during evolution. Using NMR spin dynamics, we find that the strain-specific mutations altered the conformational dynamics of the hidden network of tightly packed residues, underlying the evolution of long-range epistasis. This work shows how near-neutral mutations silently alter the biophysical energy landscapes, resulting in diverse background effects during molecular evolution.

The mechanisms underlying how individual mutations affect the protein energy landscape are crucial for understanding how proteins evolve. However, predicting mutational effects remains challenging because of epistasis—the nonadditive interactions between mutations. Here, we investigate the biophysical mechanism of strain-specific epistasis in the nonstructural protein 1 (NS1) of the influenza A virus (IAV). To understand the molecular basis of epistasis, we conducted comprehensive analyses of four NS1s of IAV strains that emerged between 1918 and 2004. We find that strain-specific mutations of NS1s are near-neutral with respect to the association with the p85β subunit of PI3K. However, the individual residues on the p85β-binding interface show long-range epistatic interactions with strain-specific mutations. We reveal that strain-specific mutations reshaped the energy landscape of NS1, leading to long-range epistasis. Our findings offer a high-resolution mechanism of how near-neutral mutations silently alter the biophysical energy landscapes, resulting in diverse background effects during molecular evolution.