Vertebrate ultraviolet visual pigments: protonation of the retinylidene Schiff base and a counterion switch during photoactivation

Abstract

For visual pigments, a covalent bond between the ligand (11-cis-retinal) and receptor (opsin) is crucial to spectral tuning and photoactivation. All photoreceptors have retinal bound via a Schiff base (SB) linkage, but only UV-sensitive cone pigments have this moiety unprotonated in the dark. We investigated the dynamics of mouse UV (MUV) photoactivation, focusing on SB protonation and the functional role of a highly conserved acidic residue (E108) in the third transmembrane helix. On illumination, wild-type MUV undergoes a series of conformational changes, batho --> lumi --> meta I, finally forming the active intermediate meta II. During the dark reactions, the SB becomes protonated transiently. In contrast, the MUV-E108Q mutant formed significantly less batho that did not decay through a protonated lumi. Rather, a transition to meta I occurred above approximately 240 K, with a remarkable red shift (lambda(max) approximately 520 nm) accompanying SB protonation. The MUV-E108Q meta I --> meta II transition appeared normal but the MUV-E108Q meta II decay to opsin and free retinal was dramatically delayed, resulting in increased transducin activation. These results suggest that there are two proton donors during the activation of UV pigments, the primary counterion E108 necessary for protonation of the SB during lumi formation and a second one necessary for protonation of meta I. Inactivation of meta II in SWS1 cone pigments is regulated by the primary counterion. Computational studies suggest that UV pigments adopt a switch to a more distant counterion, E176, during the lumi to meta I transition. The findings with MUV are in close analogy to rhodopsin and provides further support for the importance of the counterion switch in the photoactivation of both rod and cone visual pigments.

Fig. 1.

The primary sequence of MUV (A, cytoplasmic side up) and a similarity-based model of the MUV binding site, with selected atoms of the retinal chromophore numbered (B, extracellular side up). The secondary structure elements (e.g., transmembrane segments) shown are based on the crystal structure of bovine rhodopsin. The five amino acids shown in larger yellow circles correspond to S85 in TM2; E108 in TM3, at the same position as the rhodopsin counterion (E113); E176 and S181 in β-strands 3 and 4 (EII, extracellular loop 2), respectively; and K291 in TM7, the retinal binding site (K296 in rhodopsin).

Fig. 2.

(A) Formation of the primary photostationary state of E108Q. The difference spectra between the photostationary state (PSS305) obtained after 15, 30, 45, and 60 min of illumination at 75 K minus the dark state spectrum are shown and labeled 15 m, 30 m, 45 m, and 60 m, respectively. (B) Thermal decay of the primary photostationary state of E108Q. The difference spectra calculated by subtracting the PSS305 formed at 75 K from the spectra equilibrated at 100, 160, 180, and 200 K are labeled accordingly. (C) Formation of the later intermediates of E108Q. Light minus dark difference spectra obtained after illumination at 243 K. The curve labeled PSS305 was the photostationary state obtained after illumination of the pigment with 305 nm light for 135 min. The remaining spectra were successive illumination of the PSS305 with 330 nm light for 15, 30, 60, 90, and 120 min (PSS330). (D) Thermal decay of the PSS330 generated at 243 K. Difference spectra were calculated by subtracting the PSS330 equilibrated at 253, 258, and 263 K from that equilibrated at 248 K plotted.

Fig. 3.

The pure electronic spectra of the MUV-E108Q intermediates obtained via the decomposition of the temperature-trapped photostationary state spectra are shown in the corresponding panels labeled with the intermediate and temperature at which it is stabilized and the absorption maximum. The spectra and absorption maxima of the wild-type MUV intermediates are shown in gray. Note that the E108Q mutant does not produce any lumi intermediate, whereas MUV does. The absorption maxima are indicated in nanometers and are accurate to ±2 nm.

Fig. 4.

(A) Transducin activation by MUV and its counterion mutant, E108Q. The reaction was illuminated for 1 min, and then aliquots were removed and assayed for GTPγS bound to transducin. The solid lines represent the regression fit to the initial rate for each curve. (B) The decay of the active state of MUV and E108Q after illumination. The pigments were exposed to 1 min of illumination in the absence of transducin. Aliquots were added to transducin with a delay of 1–30 min and incubated for an additional 10 min in dark before assaying; a delay of 0 min was achieved by illuminating in the presence of transducin.

Fig. 6.

Schematic diagram of the key molecular changes in the mouse UV binding site in the dark state, batho, lumi, and meta I states. The arrows in A and C indicate the key conformational or chemical changes associated with transformation of the dark state to batho and lumi to meta I. The chromophore geometry in the batho state is all-trans, but the conformational details regarding the single bonds remains unknown. The relative location of the residues is approximate and does not reflect possible movement during the thermal relaxations (see Discussion). Molecular orbital (mozyme) calculations indicate that E176, when protonated (neutral), is freely rotating, and we use * to indicate that there are two tyrosine residues, Y187 and Y263, that alternate in hydrogen bonding to E176, depending on rotational state. Only in the meta I state do Y187 and Y263 both strongly hydrogen bond to E176 (D).

Fig. 5.

Molecular models of the lumi (A) and meta I (B) intermediates of mouse UV based on the assumption that a counterion switch occurs during the lumi (E108 counterion) to meta I (E176 counterion) transformation. The chromophore, binding site residues, and water were allowed to seek minimum energy, but the protein backbone and outer residues were fixed. The electronic spectra of the two intermediates are simulated by using MNDO-PSDCI molecular orbital theory in the C and D, and the calculated absorption band maxima are indicated in nanometers. The dipole moments of the binding site residues, μ_bs, are given in the upper right of A and B and the dipole moment vector is shown by using red-to-blue arrows. The MNDO-PSDCI calculations included all 36 single and 666 double excitations within the π electron manifold of the chromophore. The heights of the vertical bars in C and D are proportional to the oscillator strengths of the π* ← π transitions, where solid blue bars indicate B_u⁺-like states, dashed blue bars indicate B_u^–-like states, solid red bars indicate A_g⁺-like states, and dashed red bars indicate A_g^–-like states. The simulated chromophore spectra were generated assuming Gaussian profiles with full-widths at half-maxima of 4,000 cm^–1.