Abstract
Trade-offs between protein stability and activity can restrict access to evolutionary trajectories, but widespread epistasis may facilitate indirect routes to adaptation. This may be enhanced by natural environmental variation, but in multicellular organisms this process is poorly understood. We investigated a paradoxical trajectory taken during the evolution of tetrapod dim-light vision, where in the rod visual pigment rhodopsin, E122 was fixed 350 million years ago, a residue associated with increased active-state (MII) stability but greatly diminished rod photosensitivity. Here, we demonstrate that high MII stability could have likely evolved without E122, but instead, selection appears to have entrenched E122 in tetrapods via epistatic interactions with nearby coevolving sites. In fishes by contrast, selection may have exploited these epistatic effects to explore alternative trajectories, but via indirect routes with low MII stability. Our results suggest that within tetrapods, E122 and high MII stability cannot be sacrificed-not even for improvements to rod photosensitivity.
Highlights
Nature-inspired strategies are increasingly recruited toward engineering objectives in protein design (Khersonsky and Fleishman, 2016; Jacobs et al, 2016; Goldenzweig and Fleishman, 2018, a central challenge of which is to successfully manipulate backbone structure to modulate stability without introducing undesirable pleiotropic effects on protein activity (Khersonsky and Fleishman, 2016; Goldenzweig and Fleishman, 2018; Starr and Thornton, 2017; Tokuriki and Tawfik, 2009)
We found that E122 has been fixed in tetrapod RH1 since the most recent common ancestor ~350 million years ago (MYA) (Hedges et al, 2015), where it appears along the ancestral branch leading to tetrapods (Figure 2A; Table 3) following the diversification from lungfishes (I122, codon ATA, Figure 2A; Supplementary file 1) and the coelacanth (Q122, codon CAA, Figure 2A; Supplementary file 1)
Our experiments demonstrate that N123, which is not, to our knowledge, observed within any vertebrate rhodopsin other than the Characiphysi (Tables 12, Supplementary files 1–3) is required for a complete rescue of MII stability within the long-wave sensitive opsins (LWS) cone opsin amino acid variant (COV) I122 background, where it has opposite functional effects depending on E vs. I122 backgrounds (Figure 4D–E; Figure 5)
Summary
Nature-inspired strategies are increasingly recruited toward engineering objectives in protein design (Khersonsky and Fleishman, 2016; Jacobs et al, 2016; Goldenzweig and Fleishman, 2018, a central challenge of which is to successfully manipulate backbone structure to modulate stability without introducing undesirable pleiotropic effects on protein activity (Khersonsky and Fleishman, 2016; Goldenzweig and Fleishman, 2018; Starr and Thornton, 2017; Tokuriki and Tawfik, 2009). Engineering protein stability and activity requires an understanding of a protein’s sequence-function relationship, or landscape (Pal and Papp, 2017; Wu et al, 2016; Starr et al, 2017), where billions of possible pair-wise and third-order interactions can exist between amino acids (Starr and Thornton, 2017; Storz, 2016), and only a limited number of amino acid combinations will confer the function of interest (Wu et al, 2016; Starr et al, 2017; McMurrough et al, 2014; Mateu and Fersht, 1999; Tarvin et al, 2017).
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