Relocating the active-site lysine in rhodopsin and implications for evolution of retinylidene proteins

Abstract

Type I and type II rhodopsins share several structural features including a G protein-coupled receptor fold and a highly conserved active-site Lys residue in the seventh transmembrane segment of the protein. However, the two families lack significant sequence similarity that would indicate common ancestry. Consequently, the rhodopsin fold and conserved Lys are widely thought to have arisen from functional constraints during convergent evolution. To test for the existence of such a constraint, we asked whether it were possible to relocate the highly conserved Lys296 in the visual pigment bovine rhodopsin. We show here that the Lys can be moved to three other locations in the protein while maintaining the ability to form a pigment with 11-cis-retinal and activate the G protein transducin in a light-dependent manner. These results contradict the convergent hypothesis and support the homology of type I and type II rhodopsins by divergent evolution from a common ancestral protein.

Fig. 1.

The retinal-binding pocket of bovine rhodopsin (PDB ID code 1U19). 11-cis-retinal, orange; Glu113 counterion, red; Lys296, blue; positions at which a Lys was introduced (Gly90, Thr94, Ala117, Ser186, and Phe293), green.

Fig. 2.

Ability of rhodopsin mutants to form pigments with 11-cis-retinal. Normalized UV-visible absorption spectra for dark-adapted pigments in 0.02% (wt/vol) DDM at pH 7.5 at room temperature. Insets are an expanded view of the long-wavelength λ_max peak. Scale bars in upper left corner of each panel (upper right of Insets) represent 0.03 absolute absorbance.

Fig. 3.

[³⁵S]-GTPγS binding to transducin following light activation of select mutant pigments. Each reaction contained 5 nM rhodopsin, 1 μM transducin, 3 μM GTPγS in 10 mM Tris buffer at pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.1 mM EDTA, and 0.01% (wt/vol) DDM. Blue circles, WT; green squares, K296G/G90K; orange triangles, K296A/S186K; maroon diamonds, K296G/F293K; black inverted triangles, K296A/T94K; shading, reactions run in the dark. Error bars represent the SD (n = 4).

Fig. 4.

Constitutive activation of transducin by select mutant opsins, monitored by [³⁵S]-GTPγS binding to activated transducin. Each reaction contained 5 nM opsin, 1 μM transducin, 3 μM GTPγS in 10 mM Tris buffer at pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.1 mM EDTA, and 0.01% (wt/vol) DDM. Purple open triangles, K296A; light blue open squares, K296G; orange triangles, K296A/S186K; light blue +, K296A/A117K; green squares, K296G/G90K; black inverted triangles, K296A/T94K; maroon diamonds, K296G/F293K; light purple X, K296G/A117K; blue circles, WT. Error bars represent the SD (n = 4).