The Essential Role of the N-Terminal Domain of the Orange Carotenoid Protein in Cyanobacterial Photoprotection: Importance of a Positive Charge for Phycobilisome Binding

Wilson, Adjélé; Gwizdala, Michal; Mezzetti, Alberto; Alexandre, Maxime; Kerfeld, Cheryl A.; Kirilovsky, Diana

doi:10.1105/tpc.112.096909

Cited by 87 publications

(107 citation statements)

References 27 publications

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“…4B). Recently, the OCP has been proposed to interact with the phycobilisome through its N-terminal domain, specifically through the surface surrounding Arg155, which is typically buried in interaction with the C-terminal domain in the OCP o (18). We have confirmed the interaction of the FRP with the C-terminal domain of the OCP by coimmunoprecipitation.…”

Section: Discussionsupporting

confidence: 68%

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Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria

Sutter

Wilson

Leverenz

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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128

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Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. To prevent high-light induced damage, cyanobacteria have developed photoprotective mechanisms. One involves a photoactive carotenoid protein that decreases the transfer of excess energy to the reaction centers. This protein, the orange carotenoid protein (OCP), is present in most cyanobacterial strains; it is activated by high light conditions and able to dissipate excess energy at the site of the light-harvesting antennae, the phycobilisomes. Restoration of normal antenna capacity involves the fluorescence recovery protein (FRP). The FRP acts to dissociate the OCP from the phycobilisomes by accelerating the conversion of the active red OCP to the inactive orange form. We have determined the 3D crystal structure of the FRP at 2.5 Å resolution. Remarkably, the FRP is found in two very different conformational and oligomeric states in the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that the dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity. nonphotochemical quenching | Synechocystis L ight is vital for the survival and growth of photosynthetic organisms. In natural environments, these organisms are exposed to varying light conditions in addition to the day/night cycle. Too much exposure to light causes the formation of reactive oxygen species that damage the sensitive photochemical reaction centers, and thus, a careful regulation of energy flow is critical. Under low light conditions, an efficient energy collection by the antennae complexes is achieved, whereas under high light conditions, the excess energy has to be diverted from photosynthesis (1).Plants and cyanobacteria have evolved different ways to deal with excess energy arriving at the reaction centers. Higher plants and green algae contain antenna complexes consisting of transmembrane proteins that sense the acidification of the thylakoid lumen and react by switching from efficient energy collection to heat dissipation (1, 2). Cyanobacteria, however, contain antenna complexes called phycobilisomes, which are membrane-anchored and consist of phycobilin proteins. Instead of sensing the effect of high light through pH changes, cyanobacterial phycobilisomes are quenched by a protein capable of directly sensing high light conditions, the orange carotenoid protein (OCP). The 35 kDa OCP consists of two distinct domains that encompass a ketocarotenoid (3′-hydroxyechinenone) in an all trans conformation (3-6). Irradiance with high light changes the OCP from an inactive orange (OCP o ) to an active red form (OCP r ) that is capable of binding to the phycobilisomes to prevent excess energy from flowing to the reaction centers. ...

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Section: Discussionsupporting

confidence: 68%

“…In the strains overexpressing the C-terminal domain, the original WT OCP was suppressed by partial deletion of the slr1963 gene as described in ref. 18. The His-tagged C-terminal domain was isolated by Nickel affinity as described in ref.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria

Sutter

Wilson

Leverenz

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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128

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“…S1, and Table S6). R155, which participates in a salt bridge between the NTD and CTD, and which plays an important role in interaction with the phycobilisome during energy dissipation (12), is associated with water cluster 1, which includes HOH1020 (Fig. 4 and Fig.…”

Section: Hdx-msmentioning

confidence: 99%

“…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)(10)(11)(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.…”

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confidence: 99%

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

Gupta

Guttman

Leverenz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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.orange carotenoid protein | photoprotection | X-ray footprinting | hydrogen deuterium exchange | SAXS P hotosynthetic 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 pHinduced 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 ).The NTD and CTD of the OCP have discrete functions. The isolated NTD acts as an effector domain that binds to the antenna whereas the CTD has been proposed to play a sensory/regulatory role in controlling the OCP's photoprotective function (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 cy...

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“…The two domains are joined by a long (;25 amino acids) and flexible loop linker (Kerfeld et al, 2003;Wilson et al, 2010). OCP o has a closed conformation, and OCP r has an open conformation, allowing the interaction between the carotenoid and the bilin in the core of phycobilisome (Wilson et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher

et al. 2014

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Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.

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The Essential Role of the N-Terminal Domain of the Orange Carotenoid Protein in Cyanobacterial Photoprotection: Importance of a Positive Charge for Phycobilisome Binding

Cited by 87 publications

References 27 publications

Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria

Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria

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

The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher

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