Evolutionary couplings detect side-chain interactions.

Patterns of amino acid covariation in large protein sequence alignments can inform the prediction of de novo protein structures, binding interfaces, and mutational effects. While algorithms that detect these so-called evolutionary couplings between residues have proven useful for practical applications, less is known about how and why these methods perform so well, and what insights into biological processes can be gained from their application. Evolutionary coupling algorithms are commonly benchmarked by comparison to true structural contacts derived from solved protein structures. However, the methods used to determine true structural contacts are not standardized and different definitions of structural contacts may have important consequences for interpreting the results from evolutionary coupling analyses and understanding their overall utility. Here, we show that evolutionary coupling analyses are significantly more likely to identify structural contacts between side-chain atoms than between backbone atoms. We use both simulations and empirical analyses to highlight that purely backbone-based definitions of true residue-residue contacts (i.e., based on the distance between Cα atoms) may underestimate the accuracy of evolutionary coupling algorithms by as much as 40% and that a commonly used reference point (Cβ atoms) underestimates the accuracy by 10-15%. These findings show that co-evolutionary outcomes differ according to which atoms participate in residue-residue interactions and suggest that accounting for different interaction types may lead to further improvements to contact-prediction methods.