Local and global structural drivers for the photoactivation of the orange carotenoid protein

Photoprotective mechanisms are of fundamental importance for the survival of photosynthetic organisms. In cyanobacteria, the orange carotenoid protein (OCP), when activated by intense blue light, binds to the light-harvesting antenna and triggers the dissipation of excess captured light energy. Using a combination of small angle X-ray scattering (SAXS), X-ray hydroxyl radical footprinting, circular dichroism, and H/D exchange mass spectrometry, we identified both the local and global structural changes in the OCP upon photoactivation. SAXS and H/D exchange data showed that global tertiary structural changes, including complete domain dissociation, occur upon photoactivation, but with alteration of secondary structure confined to only the N terminus of the OCP. Microsecond radiolytic labeling identified rearrangement of the H-bonding network associated with conserved residues and structural water molecules. Collectively, these data provide experimental evidence for an ensemble of local and global structural changes, upon activation of the OCP, that are essential for photoprotection.Photosynthetic organisms have evolved a protective mechanism known as nonphotochemical quenching (NPQ) to dissipate excess energy, thereby preventing oxidative damage under high light conditions (1). In plants and algae, NPQ involves pH-induced conformation changes in membrane-embedded protein complexes and enzymatic interconversion of carotenoids (2, 3). Cyanobacteria, in contrast, use a relatively simple NPQ mechanism governed by the water soluble orange carotenoid protein (OCP). The OCP is composed of an all α-helical N-terminal domain (NTD) consisting of two discontinuous four-helix bundles and a mixed α/β C-terminal domain (CTD), which is a member of the widely distributed nuclear transport factor 2-like superfamily (Fig. S1A) (4, 5). There are two regions of interaction between the NTD and CTD (4, 5): the major interface, which buries 1,722 Å of surface area, and the interaction between the N-terminal alpha-helix (αA) and the CTD (minor interface) (Fig. S1A). A single noncovalently bound keto-carotenoid [e.g., echinenone (ECN)] spans both domains in the structure of the resting (inactive) form of the protein (OCP^O).Open in a separate windowFig. S1.Structure of the OCP. (A) Crystal structure of Synechocystis OCP (PDB ID code 3MG1) consisting of two domains, NTD and CTD as described in the main text introduction, which form major and minor interfaces. (B) Amino acid residues within 3.9 Å of the carotenoid are shown by sticks. (C) Surface-bound water molecules at the major interface are shown in slate-colored spheres in Synechocystis OCP (PDB ID code 3MG1). This layer of water molecules fully or partially eclipses other water molecules, which are either conserved or found to be at the same location (within 0.5 Å) in the crystal structures of A. maxima and Synechocystis OCP (Fig. 4 A and B. Removal of the slate-colored spheres, exposing partially buried water, is shown in orange in D. The fully buried waters (red spheres) are invisible in the surface diagram of OCP. (E) Cross-sectional view to show the position of fully and partially buried structural waters in OCP^O. (F) Details of water–protein H-bonding network in water cluster 1 at the major interface. The absolutely conserved R155 is closely surrounded (<3.2 Å, capable of forming H-bond) by a number of buried (HOH1151,1200, and 1671) water molecules, which are involved in dense residue-water interactions as discussed in the main text. Similar H-bonding networks are also observed in the water clusters 2 and 3 (6). Exposure to blue light converts OCP^O to the active (red) form, OCP^R (7). OCP^R is involved in protein–protein interactions with the phycobilisome (PB) (5) and the fluorescence recovery protein (FRP), which converts OCP^R back to OCP^O (8). The OCP^R form is therefore central to the photoprotective mechanism, and determining the exact structural changes that accompany its formation are critical for a complete mechanistic understanding of the reversible quenching process in cyanobacteria. Although crystal structures exist of both the (inactive) OCP^O (4, 5) and the active NTD (effector domain) form of the protein (9), crystallization of the activated, full-length OCP^R has not been achieved. To identify the protein structural changes that occur after absorption of light by the OCP’s ECN chromophore, we undertook a hybrid approach to structurally characterize OCP^R in solution.In Synechocystis OCP^O, the 4-keto group on the “β1” ring of ECN is H-bonded to two conserved residues, Y201 and W288, in a hydrophobic pocket in the core of the CTD (Fig. S1B) (5). The other end of the carotenoid is positioned between the two four-helix bundles of the NTD. Several conserved residues within 3.9 Å of the carotenoid are known to interact with its extensive conjugation and result in fine tuning of the spectral characteristics of the OCP (Fig. S1B) (4, 5); these residues have been implicated in photochemical function via mutagenesis studies (5). A recent study of the OCP bound to the carotenoid canthaxanthin (OCP-CAN) showed that photoactivation of the OCP results in a substantial translocation (12 Å) of the carotenoid deeper into the NTD (9). Mutational analyses of the full-length OCP and biochemical studies on the constitutively active NTD [commonly known as the red carotenoid protein (RCP)] suggested that the NTD and CTD at least partially separate, resulting in the breakage of an interdomain salt-bridge (R155–E244) upon photoactivation (9–12). Together, the previous studies suggest that large-scale protein structural changes in the OCP accompany carotenoid translocation upon light activation; however, such changes in the context of the full-length protein have yet to be experimentally demonstrated. Here, we report use of X-ray radiolytic labeling with mass spectrometry (XF-MS) and hydrogen/deuterium exchange with mass spectrometry (HDX-MS), which detect residue-specific changes (13–15), to investigate the structural changes that occur during OCP photoactivation. In conjunction with small angle X-ray scattering (SAXS), which enables characterization of global conformational changes in the solution state (16), we show that dissociation of the NTD and CTD is complete in photoactivated OCP. This separation is accompanied by an unfolding of the N-terminal α-helix that is associated with the CTD in the resting state. We also pinpoint changes in specific amino acids and structurally conserved water molecules, providing insight into the signal propagation pathway from carotenoid to protein surface upon photoactivation. Collectively, these data provide a comprehensive view of both global and local intraprotein structural changes in the OCP upon photoactivation that are essential to a mechanistic understanding of cyanobacterial NPQ.