Ryan L. Leverenz

A 12 Å carotenoid translocation in a photoswitch associated with cyanobacterial photoprotection

Protection from too much light Photosynthetic organisms protect themselves from too much light using pigment photoswitches that absorb excess energy. Leverenz et al. analyzed the structure of an active, energy-dissipating form of the orange carotenoid protein (OCP) from a cyanobacterium. When activated by excess light, OCP moves its hydrophobic carotenoid pigment 12 Å within the protein to accommodate nonphotochemical quenching by the broader photosynthetic antenna complex. Science, this issue p. 1463 Large-scale pigment movement accompanies photoactivation of the orange carotenoid protein. Pigment-protein and pigment-pigment interactions are of fundamental importance to the light-harvesting and photoprotective functions essential to oxygenic photosynthesis. The orange carotenoid protein (OCP) functions as both a sensor of light and effector of photoprotective energy dissipation in cyanobacteria. We report the atomic-resolution structure of an active form of the OCP consisting of the N-terminal domain and a single noncovalently bound carotenoid pigment. The crystal structure, combined with additional solution-state structural data, reveals that OCP photoactivation is accompanied by a 12 angstrom translocation of the pigment within the protein and a reconfiguration of carotenoid-protein interactions. Our results identify the origin of the photochromic changes in the OCP triggered by light and reveal the structural determinants required for interaction with the light-harvesting antenna during photoprotection.

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

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.

Structural and Functional Modularity of the Orange Carotenoid Protein: Distinct Roles for the N- and C-Terminal Domains in Cyanobacterial Photoprotection

This work reveals key structure-function relationships in the photoprotective orange carotenoid protein. The N-terminal domain is shown to be the effector of energy dissipation; it binds to the phycobilisome and quenches fluorescence without prior photochemical activation. The C-terminal domain dynamically regulates the photoprotective activity of the N-terminal domain in response to light. The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCPO, to an active red form, OCPR. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP’s photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCPR. A comparison of the spectroscopic properties of the RCP with OCPR indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCPR form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain.

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

Significance The orange carotenoid protein (OCP) is critical for the antenna-associated energy-dissipation mechanism of cyanobacteria under high light conditions. We show that light activation causes a global conformation change, the complete separation of the two domains of the OCP. Such a conformational change has been postulated to be a prerequisite for interaction with the antenna. We also identify local structural changes in residue solvent accessibility and roles for structural water molecules in activation of the OCP. By combining small-angle scattering, hydrogen-deuterium exchange, and X-ray hydroxyl radical footprinting studies, we were able to construct a model of the structural changes during the activation of the OCP with an unprecedented level of detail. 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.

Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component

The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

Different Functions of the Paralogs to the N-Terminal Domain of the Orange Carotenoid Protein in the Cyanobacterium Anabaena sp. PCC 7120

The N-terminal domains of the four Orange Carotenoid Protein isoforms defines distinct functions. The photoactive Orange Carotenoid Protein (OCP) is involved in cyanobacterial photoprotection. Its N-terminal domain (NTD) is responsible for interaction with the antenna and induction of excitation energy quenching, while the C-terminal domain is the regulatory domain that senses light and induces photoactivation. In most nitrogen-fixing cyanobacterial strains, there are one to four paralogous genes coding for homologs to the NTD of the OCP. The functions of these proteins are unknown. Here, we study the expression, localization, and function of these genes in Anabaena sp. PCC 7120. We show that the four genes present in the genome are expressed in both vegetative cells and heterocysts but do not seem to have an essential role in heterocyst formation. This study establishes that all four Anabaena NTD-like proteins can bind a carotenoid and the different paralogs have distinct functions. Surprisingly, only one paralog (All4941) was able to interact with the antenna and to induce permanent thermal energy dissipation. Two of the other Anabaena paralogs (All3221 and Alr4783) were shown to be very good singlet oxygen quenchers. The fourth paralog (All1123) does not seem to be involved in photoprotection. Structural homology modeling allowed us to propose specific features responsible for the different functions of these soluble carotenoid-binding proteins.

Specificity of the Cyanobacterial Orange Carotenoid Protein: Influences of Orange Carotenoid Protein and Phycobilisome Structures

In cyanobacteria, the binding properties of a carotenoid energy quencher and the structure of the core of phycobilisome antenna determine the amplitude of photoprotection and the specificity of the mechanism. Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome (PB), the extramembranous light-harvesting antenna. This mechanism is triggered by the photoactive Orange Carotenoid Protein (OCP), which acts both as the photosensor and the energy quencher. The OCP binds the core of the PB. The structure of this core differs in diverse cyanobacterial strains. Here, using two isolated OCPs and four classes of PBs, we demonstrated that differences exist between OCPs related to PB binding, photoactivity, and carotenoid binding. Synechocystis PCC 6803 (hereafter Synechocystis) OCP, but not Arthrospira platensis PCC 7345 (hereafter Arthrospira) OCP, can attach echinenone in addition to hydroxyechinenone. Arthrospira OCP binds more strongly than Synechocystis OCP to all types of PBs. Synechocystis OCP can strongly bind only its own PB in 0.8 m potassium phosphate. However, if the Synechocystis OCP binds to the PB at very high phosphate concentrations (approximately 1.4 m), it is able to quench the fluorescence of any type of PB, even those isolated from strains that lack the OCP-mediated photoprotective mechanism. Thus, the determining step for the induction of photoprotection is the binding of the OCP to PBs. Our results also indicated that the structure of PBs, at least in vitro, significantly influences OCP binding and the stabilization of OCP-PB complexes. Finally, the fact that the OCP induced large fluorescence quenching even in the two-cylinder core of Synechococcus elongatus PBs strongly suggested that OCP binds to one of the basal allophycocyanin cylinders.

Structure, Diversity, and Evolution of a New Family of Soluble Carotenoid-Binding Proteins in Cyanobacteria

Using a phylogenomic approach, we have identified and subclassified a new family of carotenoid-binding proteins. These proteins have sequence homology to the N-terminal domain (NTD) of the Orange Carotenoid Protein (OCP), and are referred to as Helical Carotenoid Proteins (HCPs). These proteins comprise at least nine distinct clades and are found in diverse organisms, frequently as multiple paralogs representing the distinct clades. These seem to be out-paralogs maintained from ancient duplications associated with subfunctionalization. All of the HCPs share conservation of the residues for carotenoid binding, and we confirm that carotenoid binding is a fundamental property of HCPs. We solved two crystal structures of the Nostoc sp. PCC 7120 HCP1 protein, each binding a different carotenoid, suggesting that the proteins flexibly bind a range of carotenoids. Based on a comprehensive phylogenetic analysis, we propose that one of the HCP subtypes is likely the evolutionary ancestor of the NTD of the OCP, which arose following a domain fusion event. However, we predict that the majority of HCPs have functions distinct from the NTD of the OCP. Our results demonstrate that the HCPs are a new family of functionally diverse carotenoid-binding proteins found among ecophysiologically diverse cyanobacteria.

Insights into the structural changes occurring upon photoconversion in the orange carotenoid protein from broadband two-dimensional electronic spectroscopy.

Carotenoids play an essential role in photoprotection, interacting with other pigments to safely dissipate excess absorbed energy as heat. In cyanobacteria, the short time scale photoprotective mechanisms involve the photoactive orange carotenoid protein (OCP), which binds a single carbonyl carotenoid. Blue-green light induces the photoswitching of OCP from its ground state form (OCPO) to a metastable photoproduct (OCPR). OCPR can bind to the phycobilisome antenna and induce fluorescence quenching. The photoswitching is accompanied by structural and functional changes at the level of the protein and of the bound carotenoid. Here, we use broadband two-dimensional electronic spectroscopy to study the differences in excited state dynamics of the carotenoid in the two forms of OCP. Our results provide insight into the origin of the pronounced vibrational lineshape and oscillatory dynamics observed in linear absorption and 2D electronic spectroscopy of OCPO and the large inhomogeneous broadening in OCPR, with consequences for the chemical function of the two forms.

Raman Optical Activity Reveals Carotenoid Photoactivation Events in the Orange Carotenoid Protein in Solution

The orange carotenoid protein (OCP) plays an important role in photoprotection in cyanobacteria, which is achieved by the photoconversion from the orange dark state (OCPO) to the red active state (OCPR). Using Raman optical activity (ROA), we studied the conformations of the carotenoid chromophore in the active sites of OCPO and OCPR. This ROA measurement directly observed the chromophore conformation of native OCP in solution, and the measurement of OCPR first demonstrated the ROA spectroscopy for the transient species. For OCPO, the spectral features of ROA were mostly reproduced by the quantum chemical calculation based on the crystal structure of the OCP. Within the spatial resolution (∼2 Å), a slight modification of the polyene-chain distortion improved the agreement between the observed and calculated ROA spectra. While the crystal structure of OCPR is not available, the ROA spectrum of OCPR was reproduced by using the crystal structure of red carotenoid protein (RCP), an OCPR proxy. The present results showed that the chromophore conformations in the crystal structures of OCP and RCP hold true for OCPO and OCPR in solution. Particularly, ROA spectroscopy of the native OCPR provides a direct support for the 12 Å translocation of chromophore in the photoactivation, which was proposed by X-ray crystallography using RCP [R. L. Leverenz, M. Sutter, et al. Science 2015, 348, 1463-1466].