Amino acid covariation, where the identities of amino acids at different sequence positions are correlated, is a hallmark of naturally occurring proteins. This covariation can arise from multiple factors, including selective pressures for maintaining protein structure, requirements imposed by a specific function, or from phylogenetic sampling bias. Here we employed flexible backbone computational protein design to quantify the extent to which protein structure has constrained amino acid covariation for 40 diverse protein domains. We find significant similarities between the amino acid covariation in alignments of natural protein sequences and sequences optimized for their structures by computational protein design methods. These results indicate that the structural constraints imposed by protein architecture play a dominant role in shaping amino acid covariation and that computational protein design methods can capture these effects. We also find that the similarity between natural and designed covariation is sensitive to the magnitude and mechanism of backbone flexibility used in computational protein design. Our results thus highlight the necessity of including backbone flexibility to correctly model precise details of correlated amino acid changes and give insights into the pressures underlying these correlations.
Proteins generally fold into specific three-dimensional structures to perform their cellular functions, and the presence of misfolded proteins is often deleterious for cellular and organismal fitness. For these reasons, maintenance of protein structure is thought to be one of the major fitness pressures acting on proteins. Consequently, the sequences of today's naturally occurring proteins contain signatures reflecting the constraints imposed by protein structure. Here we test the ability of computational protein design methods to recapitulate and explain these signatures. We focus on the physical basis of evolutionary pressures that act on interactions between amino acids in folded proteins, which are critical in determining protein structure and function. Such pressures can be observed from the appearance of amino acid covariation, where the amino acids at certain positions in protein sequences are correlated with each other. We find similar patterns of amino acid covariation in natural sequences and sequences optimized for their structures using computational protein design, demonstrating the importance of structural constraints in protein molecular evolution and providing insights into the structural mechanisms leading to covariation. In addition, these results characterize the ability of computational methods to model the precise details of correlated amino acid changes, which is critical for engineering new proteins with useful functions beyond those seen in nature.