An experimental assay of the interactions of amino acids from orthologous sequences shaping a complex fitness landscape

Characterizing the fitness landscape, a representation of fitness for a large set of genotypes, is key to understanding how genetic information is interpreted to create functional organisms. Here we determined the evolutionarily-relevant segment of the fitness landscape of His3, a gene coding for an enzyme in the histidine synthesis pathway, focusing on combinations of amino acid states found at orthologous sites of extant species. Just 15% of amino acids found in yeast His3 orthologues were always neutral while the impact on fitness of the remaining 85% depended on the genetic background. Furthermore, at 67% of sites, amino acid replacements were under sign epistasis, having both strongly positive and negative effect in different genetic backgrounds. 46% of sites were under reciprocal sign epistasis. The fitness impact of amino acid replacements was influenced by only a few genetic backgrounds but involved interaction of multiple sites, shaping a rugged fitness landscape in which many of the shortest paths between highly fit genotypes are inaccessible. Author summary An intuitive understanding of protein evolution dictates that, with the exception of adaptive substitutions, amino acid states should be freely exchangeable between the same gene from different species. However, the extent to which this assertion holds true has not been tested in a controlled experiment. Here, we show that whether an amino acid state can be exchanged between orthologues depends on other amino acid states in the same protein. Furthermore, we show that the mode of interaction of amino acid states is multidimensional. Assuming that amino acid replacements influence the protein in several independent ways substantially improves our ability to predict the effect of an amino acid state in a protein sequence that has not been observed in nature.