Structural determinants underlying photoprotection in the photoactive orange carotenoid protein of cyanobacteria

Abstract

The photoprotective processes of photosynthetic organisms involve the dissipation of excess absorbed light energy as heat. Photoprotection in cyanobacteria is mechanistically distinct from that in plants; it involves the orange carotenoid protein (OCP), a water-soluble protein containing a single carotenoid. The OCP is a new member of the family of blue light-photoactive proteins; blue-green light triggers the OCP-mediated photoprotective response. Here we report structural and functional characterization of the wild type and two mutant forms of the OCP, from the model organism Synechocystis PCC6803. The structural analysis provides high resolution detail of the carotenoid-protein interactions that underlie the optical properties of the OCP, unique among carotenoid-proteins in binding a single pigment per polypeptide chain. Collectively, these data implicate several key amino acids in the function of the OCP and reveal that the photoconversion and photoprotective responses of the OCP to blue-green light can be decoupled.

FIGURE 1.

Photoactivity of the isolated OCP preparations. Shown are absorbance spectra of the orange (gray line) and red (heavy black line) forms of OCP isolated from the overexpressing His-tagged WT OCP strain (overWT), from the overexpressing His-tagged W110S OCP strain (overW110S), from the overexpressing His-tagged W110F OCP strain (overW11F), from the overexpressing His-tagged Y44S OCP strain (overY44S), from the overexpressing His-tagged R155L OCP strain (overR155L), and from the His-tagged WT strain (WT). To obtain the spectrum of the red, activated form, the isolated OCP was illuminated with 1200 μmol photons m⁻² s⁻¹ of blue light at 12 °C for 5 min.

FIGURE 2.

Structure of the Synechocystis OCP. A, stereo representation of the overall structure of the Synechocystis OCP. The N-terminal domain is colored blue; the C-terminal domain is shown in red, and the carotenoid is represented by sticks (orange). The linker region between the two domains is shown in gray. The side chains of conserved residues among the OCP orthologs are shown as gray sticks with polar atoms colored (oxygen in red; nitrogen in blue; sulfur in green). Structurally conserved water molecules are shown as yellow spheres. Water molecules discussed throughout the text are numbered. B, the N-terminal and central interfaces between the N- and C-terminal domains of the OCP, colored as in A. C, structure of the central interface in the R155L OCP mutant. The glycerol molecule is shown in yellow sticks with oxygen atoms colored red.

FIGURE 3.

Primary and secondary structure of the OCP. HMM sequence logo derived from the primary structure of OCP orthologs (reciprocal best BLAST hits to the sequence of Synechocystis OCP) with secondary structure elements based on the Synechocystis wild type structure. The linker region is composed of residues 166–192. αJ is found in the A. maxima OCP structure, but because of disorder in this region in the Synechocystis OCP electron density, it was not modeled.

FIGURE 4.

Carotenoid-protein interactions in the OCP and comparison of carotenoid conformations between A. maxima and Synechocystis OCP. A, the two molecules of Synechocystis wild type OCP in the crystallographic asymmetric unit are shown. The two domains are colored as in Fig. 2, and the molecule on the right contains the structurally conserved water molecules. Residues within 3.9 Å of the carotenoid are shown as gray sticks with polar atoms colored as in Fig. 2. Glycerol molecules are shown as yellow sticks. B, superposition of the ECN (orange) and 3′-hydroxyechinenone (blue) from the Synechocystis and A. maxima OCP structures. C, close-up of the carotenoid-protein interactions in the N-terminal domain; side chains and water molecules discussed throughout the text are labeled.

FIGURE 5.

Gel filtration of Synechocystis OCP. Shown are size exclusion chromatograms of OCP and standards (shifted up) labeled 1 for thyroglobulin (670 kDa), 2 for γ-globulin (158 kDa), 3 for ovalbumin (43 kDa), 4 for myoglobin (17.6 kDa), and 5 for bovine serum albumin (66 kDa; additional standard). OCP was eluted at 11.2 ml after ovalbumin (elution at 10.8 ml), which has a molecular mass of 43 kDa. Experiments were performed with and without OCP mixed with standards.

FIGURE 6.

Blue-green light-induced fluorescence quenching in single OCP mutants. Low light-adapted cells (80 μmol photons m⁻² s⁻¹) (at 3 μg/ml chlorophyll) of WT (closed circles) and of simple mutants Y44S (open squares), Y44F (closed squares), W110S (open triangles), W110F (closed triangles), and R155L (open circles) (A) and overexpressing WT OCP (closed circles), W110S OCP (open triangles), W110F OCP (closed triangles), Y44S OCP (open squares), and R155L OCP (open circles) (B) were illuminated with high intensities (740 μmol photons m⁻² s⁻¹) of blue-green light (400–550 nm). Fluorescence yield changes were detected with a PAM fluorometer, and saturating pulses were applied to measure maximal fluorescence levels (F_m′).