Crystallographic structure of xanthorhodopsin, the light-driven proton pump with a dual chromophore

Abstract

Homologous to bacteriorhodopsin and even more to proteorhodopsin, xanthorhodopsin is a light-driven proton pump that, in addition to retinal, contains a noncovalently bound carotenoid with a function of a light-harvesting antenna. We determined the structure of this eubacterial membrane protein-carotenoid complex by X-ray diffraction, to 1.9-A resolution. Although it contains 7 transmembrane helices like bacteriorhodopsin and archaerhodopsin, the structure of xanthorhodopsin is considerably different from the 2 archaeal proteins. The crystallographic model for this rhodopsin introduces structural motifs for proton transfer during the reaction cycle, particularly for proton release, that are dramatically different from those in other retinal-based transmembrane pumps. Further, it contains a histidine-aspartate complex for regulating the pK(a) of the primary proton acceptor not present in archaeal pumps but apparently conserved in eubacterial pumps. In addition to aiding elucidation of a more general proton transfer mechanism for light-driven energy transducers, the structure defines also the geometry of the carotenoid and the retinal. The close approach of the 2 polyenes at their ring ends explains why the efficiency of the excited-state energy transfer is as high as approximately 45%, and the 46 degrees angle between them suggests that the chromophore location is a compromise between optimal capture of light of all polarization angles and excited-state energy transfer.

Fig. 1.

Sequence alignment of green light-absorbing proteorhodopsin (PR), xanthorhodopsin (XR), and bacteriorhodopsin (BR), reevaluated from the one shown in ref. by using information gained from the diffraction structure. Red, conserved residues in all three; purple, conserved residues in xanthorhodopsin and bacteriorhodopsin; yellow, conserved residues in xanthorhodopsin and proteorhodopsins; blue, residues involved with carotenoid binding. Top row of numbers refer to the xanthorhodopsin sequence; bottom row to the bacteriorhodopsin sequence. Underlining indicates residues in transmembrane helices. Proteorhodopsin sequence refers to a species from Monterey Bay, MBP1 (protein accession No. AAG10475).

Fig. 2.

Location of salinixanthin (orange) and retinal (magenta) in xanthorhodopsin. (A) The extended carotenoid is tightly bound on the transmembrane surface of xanthorhodopsin, traversing nearly the entire bilayer, with an inclination of 54° to the membrane normal. Its keto-ring binds in a pocket between helices E and F, very near the β-ionone ring of the retinal. The angle between the chromophore axes is 46°. The angle between the planes of their π-systems is 68°. Horizontal lines indicate the approximate boundaries of the lipid bilayer. Helices E and F are marked. (B) The binding pocket of the keto ring is formed by Leu-148, Gly-156, Phe-157, Thr-160, Met-208, and Met-211, as well as the retinal β-ionone ring. (C) The keto-ring of the carotene is rotated 82° out of plane of the salinixanthin-conjugated system and is in van der Waals distance of the retinal β-ionone and the phenolic side chain of Tyr-207.

Fig. 3.

Displacements of the B–C and F–G interhelical segments expose a deep cavity that extends from the extracellular side halfway toward Schiff base. (A) Comparison of xanthorhodopsin and bacteriorhodopsin. The antiparallel β-sheet of the B–C segment of xanthorhodopsin (gray) packs against the N terminus (Bottom Right), whereas in bacteriorhodopsin (magenta) this segment packs against the F–G loop (Bottom Left). Helices A–E are viewed from the front; F and G are in the back, as marked. (B) The resulting hydrophilic cavity in xanthorhodopsin extends from the extracellular surface to Arg-93 and other buried functional groups. In bacteriorhodopsin, this region is occupied by the protein and includes the proton release group composed of Glu-194, Glu-204, and 3 ordered water molecules, absent in xanthorhodopsin. Buried residues with functional roles in transport are shown to illustrate their proximity to the aqueous interface.

Fig. 4.

Structure of the retinal, the Schiff base counterion, and the extracellular region. The counterion to the Schiff base is an aspartate–histidine complex. The network of water molecules that leads to the extracellular surface in bacteriorhodopsin is missing, and Arg-93 interacts primarily with protein side chains.

Fig. 5.

Protonation states of the aspartate–histidine counterion complex. The pH of the crystallization (5.6), is below the observed (8) spectral transition between the protonated and deprotonated forms of the Schiff base counterion (presumably neutral/zwitterionic and anionic). Thus, it seems likely that the crystallographic structure contains the neutral/zwitterionic counterion.

Fig. 6.

Cytoplasmic region, with the proton donor Glu-107 and its link via Wat-502 to the retinal region. As in the other microbial rhodopsins, Wat-501 is hydrogen-bonded tightly between the tryptophan just above the retinal (Trp-200) and the main-chain carbonyl of the residue in the π-bulge of helix G (Ala-239). Helices B, C, F, and G are marked.