Epistasis among clustered lineage-specific amino acid substitutions in the Drosophila Trio protein

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Abstract Intramolecular epistasis is increasingly recognized as a key factor shaping patterns of evolutionary rate variation among protein sites and constraining adaptive evolution. While genome-wide analyses have revealed that intramolecular epistatic interactions can drive the spatial clustering of amino acid substitutions, direct empirical evidence for such interactions and their evolutionary consequences remains limited. Using a population genetic screen for spatially-clustered and lineage-specific adaptive amino acid substitutions in Drosophila proteins, we systematically identify experimentally tractable candidates for functional analysis. As proof of concept, we focus on the Trio protein, a Rho guanine nucleotide exchange factor that exhibits three spatially-clustered putatively adaptive amino acid substitutions in the D. melanogaster lineage. By systematically reconstructing evolutionary intermediates in vivo using genome editing, we find that all possible intermediate states exhibit reduced viability and/or locomotor defects, providing strong evidence for epistatic constraints on evolutionary trajectories. Notably, these deleterious effects are recessive, suggesting that intermediate combinations of epistatically interacting amino acid substitutions can accumulate in heterozygotes prior to fixation, thereby circumventing apparent constraints imposed by maladaptive intermediate states. Together, these findings provide a rare empirical view of the fitness landscape shaped by intramolecular epistasis and establish a framework for investigating the constraints on adaptive protein evolution in diploid multicellular organisms. Author Summary Proteins fold into three-dimensional structures that are essential for their function. Because these structures depend on interactions among amino acids, the fitness effect of a mutation at one site can depend on the amino acid states at other sites. Such dependencies constrain the paths that protein evolution can take, whether evolution proceeds neutrally or adaptively. Although intramolecular epistasis has been demonstrated in microbial systems and in vitro , direct experimental evidence for such constraints in diploid multicellular organisms in vivo is rare. Here, we identify Drosophila proteins that exhibit clusters of closely-spaced putatively adaptive amino acid substitutions and focus experimental analyses on one example, Trio, a Rho guanine nucleotide exchange factor. Using genome editing to reconstruct evolutionary intermediates in the D. melanogaster lineage, we find that all intermediate versions of Trio reduce viability or impair locomotor function. Importantly, these harmful effects are recessive, suggesting that they could be masked when paired with an ancestral version of the protein. This implies that individual amino acid changes may persist in heterozygotes within populations and later combine to contribute to adaptation. If the recessivity of deleterious intermediates along adaptive evolutionary paths proves to be widespread, it could have important implications for our mechanistic understanding of adaptive protein evolution.

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