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.

Quantitative analysis of protein–protein interactions (PPIs) using biolayer interferometry (BLI) requires effective suppression of nonspecific binding (NSB) between analytes and biosensors. In particular, the study of weak interactions (i.e., K D > 1 μM) requires high concentrations of analytes, which substantially increases NSB. However, there are only a few so-called NSB blockers compatible with biomolecules, which limits the use of BLI in the accurate analysis of weak interactions. The present study aims to identify a new NSB blocker for the quantitative analysis of weak PPIs using BLI. We find that saccharides, especially sucrose, are potent NSB blockers and demonstrate their compatibility with other blocking additives. We also demonstrate the effects of the new NSB blocker by characterizing the binding between nonstructural protein 1 of the influenza A virus and human phosphoinositide 3-kinase. We anticipate that the new NSB-blocking admixture will find broad applications in studying weak interactions using BLI.

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.

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