Functional trade-offs and environmental variation shaped ancient trajectories in the evolution of dim-light vision

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.

Keywords: biochemistry; chemical biology; evolutionary biology; intramolecular epistasis; none; protein evolution; rhodopsin.

Figure 1.. Natural variation at site 122 determines rhodopsin function and stability.

(A) Amino acid consensus residues at site 122 across vertebrate rod opsins (rhodopsin; RH1) and the cone opsins (long-wave (LWS), short-wave (SWS1 and SWS2) and middle-wave (RH2) sensitive). Modified from (Lamb et al., 2007). (B) Relative stability of the rod and cone opsin active-conformation (MII) in different vertebrates (Imai et al., 2005). (C) Schematic representation of naturally occurring cone opsin variants (COVs) and other amino acids across vertebrate RH1 (see Figure 1—figure supplements 1–2; Tables 1–2, Supplementary files 1–2). E122 is invariant in all Tetrapod RH1 genes sequenced to date. Natural deep-sea amino acid variants (Hunt et al., 2001; Yokoyama et al., 1999) are identified with an asterisk (*; Table 2). (D) Introduction of the ancestral cone opsin (LWS) variant I122 blue shifts tetrapod RH1 spectral absorbance and accelerates decay of the MII light-activated conformation.

Figure 1—figure supplement 1.. Schematic of RH1 site 122 variation across the vertebrate phylogeny.

All known tetrapod RH1 is conserved as E122. Asterisks indicate the presence of deep-dwelling species within a clade (Hunt et al., 2001; Yokoyama et al., 1999) (Table 2).

Figure 1—figure supplement 2.. Vertebrate phylogeny used in computational analyses.

Tetrapods (green) and Teleost fishes (blue) along with outgroups (black) relationships were constructed according to species relationships (see Materials and methods).

Figure 2.. Local coevolutionary forces govern the evolution of site 122 differentially between tetrapods and fish (teleost) RH1.

(A) Extant and reconstructed codon variation at site 122 (Materials and methods). Despite a variety of residues at site 122 across the Coelacanth (Q122), Lungfish (I122; Ceratodontiformes), and Tetrapods (E122), GAA codons encoding for E122 are nevertheless predicted as the ancestral state with high posterior probabilities (shown in parentheses). E122 (GAA/GAG) is also likely to have been present in the last common ancestor of Cypriniformes and the Characiphysi, although with low posterior probabilities and therefore high uncertainty. I122 codon ATC is fixed in all Characiphysi rhodopsin to our knowledge (Supplementary file 3). Approximate divergence times are from (Hedges et al., 2015). (B) Mutual information (MI) analyses (MISTIC [Simonetti et al., 2013]) reveal all sites coevolving with site 122 are within 6 Å. Significance thresholds were determined by reference to the highest MI z-score from all sites across analyses of randomized datasets (n = 150; z-score cut-off = 21.6), as previously described (Ashenberg and Laub, 2013). (C) Sites within this radius displayed decreased amino acid variation in tetrapod and characiphysi RH1, where E122 and I122 are fixed, respectively (asterisks). (D) In tetrapods and characiphysi RH1, reduction in amino acid variation (relative to teleosts) at positions within the 6 Å radius were driven by increases in purifying selection on non-synonymous codons. Statistically significant gene-wide increases in purifying selection (*) between lineages were detected by likelihood ratio tests of alternative (Clade model C [Bielawski and Yang, 2004]) and null (M2a_REL [Weadick and Chang, 2012]) model analyses of codon substitution rates (d_N/d_S) ((p<0.001); Tables 3–5). Sites estimated to be under this increase in purifying selection (*) were those identified in the divergent site class of the CmC model analyses through a Bayes empirical Bayes analysis as previously described (Castiglione et al., 2017. Site-specific d_N/d_S estimates are from M8 analyses on phylogenetically pruned datasets (Tables 8–10; Figure 1—figure supplement 2; Figure 2—figure supplement 1).

Figure 2—figure supplement 1.. Phylogeny used in PAML computational analyses of Characiphysi rhodopsin coding-sequences, along with outgroups (Table 10; Supplementary file 3).

Relationships were constructed according to species relationships (see Materials and methods).

Figure 3.. Coevolving sites form the LxxEIA and FxxINS motifs.

(A) Overview of tetrapod RH1 MII rhodopsin crystal structure (Choe et al., 2011 with coevolving sites. The green highlight and dashed line indicate the stabilizing hydrogen bond between E122-H211. (B) Reconstruction of residues at site 122 (Figure 2—figure supplement 1) and coevolving positions for ancestral characiphysi, tetrapod and outgroup rhodopsins indicates the entrenchment of two structural motifs centering around site 122 (Materials and methods). The LxxEIA (or LEIA) motif was also predicted as present within the ancestral Osteichthyes. Approximate divergence times are from (Hedges et al., 2015.

Figure 4.. Coevolving sites modulate the pleiotropic functional effects of site 122.

The LEIA and FINS motifs are convergent solutions for high tetrapod RH1 active state (MII) stability but with different spectral absorbances. (A) The introduction of the ancestral cone opsin variant into tetrapod RH1 (E122I) blue-shifts rhodopsin absorbance λ_MAX and dramatically destabilizes the MII active-conformation (Figure 4—figure supplement 1; Table 6). Bar graphs show retinal release half-life values. (B) Substituting FINS motif residues into coevolving sites have varied effects on rhodopsin spectral tuning and the stability of the active-conformation. (C) Within the E122I background, FINS motif substitutions at coevolving sites have marked effects on spectral tuning, but no rescue effect on MII active-conformation stability. (D) Partial incorporation of the FINS motif within tetrapod rhodopsin produces further blue-shifting effects and has a significant but small stabilizing effect within the E122I background. (E) Full incorporation of the FINS motif into tetrapod RH1 maintains the absorbance blue-shift while fully rescuing the destabilizing effects of E122I on tetrapod RH1. Statistically significant differences in MII stability were calculated using two-tailed t-tests with unequal variance, with standard error reported in bar graphs (*p<0.05; **p<0.01; ***p<0.001). The number of biological replicates (i.e. separate elutions and/or purifications of rhodopsin) are described in Table 6.

Figure 4—figure supplement 1.. Absorbance spectra of dark-state WT and mutant rhodopsins with wavelength of maximum absorbance (λ_MAX) shown.

Figure 5.. LEIA and FINS motifs are alternative solutions for high tetrapod RH1 MII stability within a limited sequence-function landscape.

Spectral absorbance (λ_MAX) and stability of the active-conformation (MII) of wild type and mutant tetrapod RH1 with E122 (green) and I122 (blue), respectively. The only natural intermediate between the wild-type tetrapod consensus motif (LEIA) and the wild-type Characiphysi motif (FINS) is ‘FIIA’ from Lungfish RH1. The mutation I123N has opposite effects on MII stability depending on background sequence (sign-epistasis), which may have closed the LEIA to FINS motif evolutionary trajectory (dashed line) for tetrapod RH1.Although reflecting a limited experimental dataset, these epistatic effects may have created indirect routes to the high MII stability of the FINS motif via intermediates with low MII stability.

Figure 5—figure supplement 1.. Compensatory effects at coevolving sites are mediated by a diversity of possible structural mechanisms.

(A) Overview of a homology modeled mutant rhodopsin MII structure containing the FINS motif (Materials and methods) (Choe et al., 2011. F119 is membrane-facing (blue). (B) Zoom-in of F119 and buried-site N123, which may form novel hydrogen bonds with nearby conserved residues N78 and T160. These interactions may alter the conformation of residues near S124, as shown in (C) where D83, S298 and N302 participate in a hydrogen bond network stabilizing the active-conformation (Choe et al., 2011). Cascading structural alterations from F119, to N123 to S124 (green) may therefore increase active-conformation stability by integrating into existing stabilizing motifs, therefore compensating for the loss of the E122-H211 hydrogen bond (red).