Evolution of nonspectral rhodopsin function at high altitudes

Abstract

High-altitude environments present a range of biochemical and physiological challenges for organisms through decreases in oxygen, pressure, and temperature relative to lowland habitats. Proteinlevel adaptations to hypoxic high-altitude conditions have been identified in multiple terrestrial endotherms; however, comparable adaptations in aquatic ectotherms, such as fishes, have not been as extensively characterized. In enzyme proteins, cold adaptation is attained through functional trade-offs between stability and activity, often mediated by substitutions outside the active site. Little is known whether signaling proteins [e.g., G protein-coupled receptors (GPCRs)] exhibit natural variation in response to cold temperatures. Rhodopsin (RH1), the temperature-sensitive visual pigment mediating dim-light vision, offers an opportunity to enhance our understanding of thermal adaptation in a model GPCR. Here, we investigate the evolution of rhodopsin function in an Andean mountain catfish system spanning a range of elevations. Using molecular evolutionary analyses and site-directed mutagenesis experiments, we provide evidence for cold adaptation in RH1. We find that unique amino acid substitutions occur at sites under positive selection in high-altitude catfishes, located at opposite ends of the RH1 intramolecular hydrogen-bonding network. Natural high-altitude variants introduced into these sites via mutagenesis have limited effects on spectral tuning, yet decrease the stability of dark-state and light-activated rhodopsin, accelerating the decay of ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates-properties of rhodopsin that mediate rod sensitivity and visual performance. Our results support a role for natural variation in enhancing the performance of GPCRs in response to cold temperatures.H igh-altitude environments impose a suite of biochemical and physiological constraints on organisms, such as hypoxia, low atmospheric pressure, and decreasing temperatures (1-3). At the biochemical level, proteins adapted to such conditions are of particular interest to evolutionary biologists (1, 4). Studies of high-altitude-adapted organisms, especially endotherms, have focused primarily on adaptation to hypoxia, including modification of proteins involved in oxygen metabolism (2, 5), and hemoglobin structure and function (6). In contrast, our understanding of biochemical adaptations for high altitude in ectothermic organisms, for which cold temperatures impose unique constraints (7-9), remains limited. Studies in prokaryotes have highlighted how cold adaptation in enzymes is attained by functional trade-offs between protein stability and activity (10), a trade-off also characterized in enzymes from ectothermic vertebrates, such as teleost fishes, where single mutations altering intramolecular hydrogen bonding networks (HBNs) decrease protein stability and ligand binding affinity to optimize kinetic rates for cold environments (11). It is currently...

Convergent selection pressures drive the evolution of rhodopsin kinetics at high altitudes via nonparallel mechanisms

“…Rh1 sequences originating from Andean catfishes (Siluriformes: Astroblepus and Ancistrus ) were the same as those collected and processed in our previous study (Castiglione et al. ) and are publicly available (see Table S4). Rh1 alignments were generated using PRANK codon alignment (Löytynoja and Goldman ) followed by manual verification.…”

“…; Castiglione et al. ; Hauser et al. ), suggesting that similar ecological shifts may produce similar changes in site‐specific d N / d S .…”

“…), there are other mutations affecting stability that produce little to no effect on λ MAX (Castiglione et al. ; Dungan and Chang , Hauser et al. ), suggesting that in some environments it is protein stability, rather than spectral absorbance, that is specifically targeted by selection.…”

“…), was under positive selection relative to lowland catfishes at sites near key structural motifs known to control kinetic rates (Castiglione et al. ). Rare natural variants substituted into these sites accelerated both thermal‐ and light‐activated decay rates of rhodopsin in vitro, likely by disrupting two distinct hubs of the intramolecular hydrogen‐bond network that stabilizes dark‐state and light‐activated rhodopsin (Castiglione et al.…”

“…Rare natural variants substituted into these sites accelerated both thermal‐ and light‐activated decay rates of rhodopsin in vitro, likely by disrupting two distinct hubs of the intramolecular hydrogen‐bond network that stabilizes dark‐state and light‐activated rhodopsin (Castiglione et al. ). This effect was consistent with cold adaptation in enzyme proteins that display adaptive mutations decreasing stability to improve activity relative to thermostable homologs at low temperatures (Siddiqui and Cavicchioli ; Fields et al.…”