An empirical test of convergent evolution in rhodopsins

Abstract

Rhodopsins are photochemically reactive membrane proteins that covalently bind retinal chromophores. Type I rhodopsins are found in both prokaryotes and eukaryotic microbes, whereas type II rhodopsins function as photoactivated G-protein coupled receptors (GPCRs) in animal vision. Both rhodopsin families share the seven transmembrane α-helix GPCR fold and a Schiff base linkage from a conserved lysine to retinal in helix G. Nevertheless, rhodopsins are widely cited as a striking example of evolutionary convergence, largely because the two families lack detectable sequence similarity and differ in many structural and mechanistic details. Convergence entails that the shared rhodopsin fold is so especially suited to photosensitive function that proteins from separate origins were selected for this architecture twice. Here we show, however, that the rhodopsin fold is not required for photosensitive activity. We engineered functional bacteriorhodopsin variants with novel folds, including radical noncircular permutations of the α-helices, circular permutations of an eight-helix construct, and retinal linkages relocated to other helices. These results contradict a key prediction of convergence and thereby provide an experimental attack on one of the most intractable problems in molecular evolution: how to establish structural homology for proteins devoid of discernible sequence similarity.

Keywords: bacteriorhodopsin; convergence; fold; homology; lysine; permutation.

Fig. 1.

Type I and type II rhodopsin fold architecture. The protein chains are colored blue to red proceeding from the N-terminus to the C-terminus. The seven transmembrane helices are labeled alphabetically from A to G. The covalently bound retinal chromophore is depicted as white sticks in the center of each protein. (A) Halobacterium salinarum bacteriorhodopsin (PDBID: 1UAZ). (B) Bovine rhodopsin (PDBID: 3C9L). See also supplementary table S1, Supplementary Material online.

Fig. 2.

Bacteriorhodopsin transmembrane helix permutation constructs. For each permutation construct, a secondary structure schematic is shown above emphasizing differing connectivities. Below is the primary sequence structure, with transmembrane helices colored as in figure 1. Panels B–E represent the noncircular permutations. Panels F and G represent the circular permutations with the eighth additional “WALP21” helix shown in gray. (A) Wild-type bR, (B) GBCDEFA, (C) CDEFGBA, (D) GFABCDE, (E) CDEFABG, (F) FGWABCDE, (G) DEFGWABC, (H) BCDEFGWA. See also supplementary table S2 and figure S1, Supplementary Material online.

Fig. 3.

Activity and λ_max of permutation constructs. Rates are given relative to WT Ht bR (0.076 H⁺ per second per bR molecule). Reported rates are the averages of 3–5 replicates, with relative standard deviation of approximately 50%. See also supplementary figure S2, Supplementary Material online.

Fig. 4.

Lysine swap mutations. The bR protein is shown as viewed from the cytoplasmic side of the membrane, looking down the helices. Cytoplasmic inter-helical loops have been omitted for clarity. Helices are colored as in figure 1. Pink spheres indicate the β-carbons of key residues involved in swapping the lysine–Schiff base position. The C15 of the retinal chromophore is shown as a white sphere. The “counterion” D85 is shown as sticks for reference.

Fig. 5.

Proposed evolutionary relationships of GPCRs and rhodopsins. The relationships shown for GPCR are based on previous work by others (Feuda et al. 2012; Krishnan et al. 2012). Node a represents the divergence between glutamate and cAMP GPCRs, node b represents the divergence of class A from cAMP GPCRs, and node c represents our proposed position for the common ancestor of rhodopsins. The placement of node c allows for the most parsimonious acquisition of photosensitive function and retinal binding, avoiding convergent loss or gain of these features.