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Structures of a phycobilisome in light-harvesting and photoprotected states

Abstract

Phycobilisome (PBS) structures are elaborate antennae in cyanobacteria and red algae1,2. These large protein complexes capture incident sunlight and transfer the energy through a network of embedded pigment molecules called bilins to the photosynthetic reaction centres. However, light harvesting must also be balanced against the risks of photodamage. A known mode of photoprotection is mediated by orange carotenoid protein (OCP), which binds to PBS when light intensities are high to mediate photoprotective, non-photochemical quenching3,4,5,6. Here we use cryogenic electron microscopy to solve four structures of the 6.2 MDa PBS, with and without OCP bound, from the model cyanobacterium Synechocystis sp. PCC 6803. The structures contain a previously undescribed linker protein that binds to the membrane-facing side of PBS. For the unquenched PBS, the structures also reveal three different conformational states of the antenna, two previously unknown. The conformational states result from positional switching of two of the rods and may constitute a new mode of regulation of light harvesting. Only one of the three PBS conformations can bind to OCP, which suggests that not every PBS is equally susceptible to non-photochemical quenching. In the OCP–PBS complex, quenching is achieved through the binding of four 34 kDa OCPs organized as two dimers. The complex reveals the structure of the active form of OCP, in which an approximately 60 Å displacement of its regulatory carboxy terminal domain occurs. Finally, by combining our structure with spectroscopic properties7, we elucidate energy transfer pathways within PBS in both the quenched and light-harvesting states. Collectively, our results provide detailed insights into the biophysical underpinnings of the control of cyanobacterial light harvesting. The data also have implications for bioengineering PBS regulation in natural and artificial light-harvesting systems.

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Fig. 1: Structural overview of PBS and OCP–PBS from Synechocystis sp. PCC 6803.
Fig. 2: Core and rod linkers.
Fig. 3: The newly identified linker ApcG and environment of the terminal emitters.
Fig. 4: Structural basis for conformational switching.
Fig. 5: Structural changes associated with the photoconversion from OCPO to OCPR.
Fig. 6: OCP–PBS interactions.
Fig. 7: Energy flow through PBS.

Data availability

The atomic coordinates have been deposited in the PDB with the accession codes 7SC8 for the rod, 7SC7 for the PBS up-down core, 7SC9 for the core–PBS complex, 7SCB for the PBS–OCP B cylinder, 7SCC for the PBS–OCP T cylinder and 7SCA for the rod of the PBS–OCP sample.

The electron microscopy maps have been deposited in the Electron Microscopy Data Bank with the accession codes 25029 for the PBS rod, 25028 for the PBS up-down core, 25069 for the full map of the PBS up-down conformation and the rod–rod contact, 25070 for the full map of the PBS down-down conformation, 25071 for the full map of the PBS up-up conformation, 25030 for the OCP–PBS core, 25031 for the OCP–PBS rod, 25032 for the B cylinder OCP–PBS, 25033 for the T cylinder OCP PBS and 25068 for the full OCP–PBS. The raw micrographs for all datasets have been deposited in the Electron Microscopy Public Image Archive with the accession code 11133.

Full atomic models for the three PBS conformations are available as Supplementary Data. Because they are based on composite maps and have not been fully refined, they should be used with care. All other data are available from the corresponding authors upon reasonable request.

Code availability

The code used in this study to subtract the streptavidin lattice from the electron micrographs and to calculate the energy transfer within PBS is available on GitHub at https://github.com/pvsauer/StreptavidinLatticeSubtraction and https://github.com/dbina/PBS_2022, respectively.

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Acknowledgements

The authors dedicate this manuscript to the late Nicole Tandeau de Marsac. The authors thank B. Ferlez for taking negative stain electron microscopy images; D. Whitten from the Proteomic Facility at Michigan State University; A. Chintangal and P. Tobias for computational support; D. Toso and J. Remis at the Cal-Cryo facility for support with cryo-EM data collection; R. Glaeser and B.-G. Han for advice about streptavidin grid preparation; and L. Eshun-Wilson for help with data processing. Research in the Kerfeld Lab was supported by the Office of Science of the US Department of Energy (DE-SC0020606). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement no. 795070. D.B. and T.P. thank the Czech Science Foundation (grant no. 19-28323X). D.B. also acknowledges institutional support (RVO:60077344). Molecular graphics and analyses were performed with UCSF ChimeraX with support from NIH R01-GM129325. E.N. is a Howard Hughes Medical Institute Investigator.

Author information

Authors and Affiliations

Authors

Contributions

M.A.D.-M. and P.V.S. designed and performed experiments and interpreted results. C.A.K. designed and supervised the project and interpreted results. H.K. helped with sample preparation and interpreted results. M.S. performed model building, bioinformatics analysis and interpreted results. D.B. and T.P. performed modelling for the energy transfer. B.J.G. performed initial characterization of the specimen for cryo-EM and performed model building. E.N. interpreted results. M.A.D.-M., P.V.S. and C.A.K. wrote the manuscript with help from all authors. M.S. and H.K. are co-second authors.

Corresponding authors

Correspondence to Paul V. Sauer or Cheryl A. Kerfeld.

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The authors declare no competing interests.

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Nature thanks Alexey Amunts, Herbert van Amerongen, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Biochemical and spectroscopic PBS characterization.

a, Representative protein composition analysis of the PBS sample by SDS-PAGE. Identification of the components by MS can be found in Supplementary Table 2. The purification was repeated at least three times, yielding the same results. b, Absorption, and fluorescence spectrum of the isolated PBS in 0.75 M K-phosphate pH 7.5. The PBS shows a maximum absorbance at 620 nm corresponding to phycocyanin (PC), and a shoulder at 650 nm corresponding to allophycocyanin (APC). The fluorescence peak maximum is ~670 nm when PC is preferentially excited (excitation at 580 nm), and a shoulder from 740 nm to 780 nm. c, Sucrose gradient of PBS showing the intense blue band that was used for cryo-EM. d, Fluorescence spectra of PBS before and after in vitro biotinylation. Collectively, these data show that the PBS preparation was structurally and functionally intact.

Extended Data Fig. 2 Cryo-EM data processing of PBS data.

a, Example raw micrograph before and after streptavidin lattice subtraction. b, Fourier transform of a showing Bragg diffraction of streptavidin crystal before subtraction. Tens of thousands of similar micrographs were recorded. c, Processing pipeline of dataset 2, yielding the structures presented in this paper. Blue to red color shadings represent local resolution estimates at FSC = 0.143 ranging from 2 to 6 Å. The three PBS conformations and the structure of the rod are shown at two different thresholds to visualize both overall shape and higher resolution details.

Extended Data Fig. 3 Map and Model quality of PBSup-down.

a, EM density examples of CpcA, ApcE and associated bilins showing map quality of the PBSup-down. b and c, Cropped PBSup-down map, model and corresponding Fourier shell correlation (FSC) curves. Resolution of masked and unmasked models are indicated at FSC = 0.5.

Extended Data Fig. 4 Assembly of the PBS.

The core cylinders consist of four stacked discs; each disc contains three α- and three β-type Apc proteins that form the signature (αβ)6 hexamers. Besides the canonical ApcA and ApcB proteins, B1 and B2 cylinders contain one copy each of ApcD and ApcF, as well as the phycobiliprotein domain of ApcE. These are symmetrically arranged in the two bottom cylinders, oriented towards the membrane. ApcG, ApcD and ApcE protrude out of the core, indicating their likely role in contacting the photosystems. Each rod contains three stacked discs, each consisting of one CpcA/B hexamer. The overall architecture of the PBS core is in agreement with modelling predictions43.

Extended Data Fig. 5 Cryo-EM analysis of the OCPR-PBS complex.

a, Example raw micrograph before and after streptavidin lattice subtraction. b, Fourier transform of (a) showing Bragg diffraction from the streptavidin crystal before subtraction. Tens of thousands of similar micrographs were recorded. c, The workflow for the cryo-EM data processing. Maps of PBS-OCPR and the rod are shown with two different thresholds to show flexible regions and connectivity.

Extended Data Fig. 6 Map and Model quality of PBS-OCPR.

a, examples of map density quality from different regions of the PBS-OCPR. b-e, Map and Model quality of PBS-OCPR. Local resolution maps, models and corresponding Fourier shell correlation (FSC) curves. Resolution of masked and unmasked models are indicated at FSC = 0.5. Maps are colored by local resolution from 2 to 4 Å at FSC = 0.143.

Extended Data Fig. 7 Synechocystis sp. PCC 6803 OCP Purification and OCP-PBS interaction.

a, Representative coomassie blue-stained SDS-PAGE. The purification was repeated at least three times with similar results. b, UV-Vis absorption spectra of the OCPO (inactive form) and OCPR (active form). c, Fluorescence quenching of 1:20 (PBS:OCP). The PBS is biotinylated. d, Titration PBS:OCP fluorescence quenching at various ratios indicated. e–g, Close-up view of the interaction between OCP and the PBS with the supporting densities.

Extended Data Fig. 8 Structural comparison between light-harvesting PBS and quenched PBS.

a, Superimposition of the PBS-OCPR (colored as in Fig. 1) and PBS up-up (grey). b, Superimposition of the PBS-OCPR and the PBS down-down conformation (transparent surface). Both shown in surface representation. c, Model of OCPR (red) from Synechocystis PCC sp. 6803 structure superposed into the comparable position of the PBS structure from Anabaena sp. strain PCC 7120 PBS (PDB:7EYD) d, Model of OCPR (red) from Synechocystis PCC sp. 6803 structure superposed into the comparable position of the PBS structure from Synechococcus sp. PCC 7002 PBS (PDB:7EXT13). The rods are omitted for clarity.

Extended Data Fig. 9 Spectroscopic parameters and energy flow in quenched PBS.

a, Effect of the transition dipole moment (TDM) of canthaxanthin in OCP on the lifetime of excitation in PBS; the horizontal dashed line indicates the lifetime of unquenched PBS (TDM = 0) b, Distribution of TDM values of echinenone in OCP, adapted from68. In panels (a) and (b) the red vertical dashed line indicates the mean TDM value, 2.3 D, used in our calculations; the range marked by grey lines is used for simulations shown in panel (e). Reproduced from Ref. 68 with permission from the PCCP Owner Societies. c, Normalized spectra of APC fluorescence (red) and estimated S0-S1 absorption of CAN; d, Dependence of the bilin-to-CAN energy transfer time on the CAN S1 energy for the two closes bilin-CAN pairs. The dashed vertical line in panels (c) and (d) indicates the S1 energy 14000 cm−1 used in all calculations; e, Decay associated spectra obtained from a simulation based on the OCP-PBS structure. It represents the excitation energy flow in the fully quenched OCP-PBS after excitation into the far end of the rod. The value of the green component, corresponding to the quenching, depends on the TDM value shown in panel (a), 400 ps is obtained for the mean TDM value of 2.3 D, 200 ps corresponds to TDM of 4 D. f, The same spectra obtained from fitting the time-resolved fluorescence data (adapted with permission from7). g, PBS array with OCP bound. OCP biding sites are still accessible. h, Simulation of excitation energy flow in a phycobilisome pair (PBS1, black; PBS2, red) after excitation into the outer tip of the PBS1 bottom rod. Cyan (single PBS) and blue (PBS pair) kinetics monitors population in the rod. Grey, black and red lines represent excitation summed over all bilins in the PBS. Grey line shows the total excitation in a single PBS without OCP; In the PBS pair, the excitation is distributed from the initially excited PBS1 (black) to PBS2 (red). Solid line, PBS pair without OCP; short dash, each PBS binds 4 OCPs; long-dash PBS2 binds 4 OCPs, PBS1 has no OCP. This simulation shows that the effect of OCP is shared between PBS in the pair. Analysis of transfer times suggested less than 20 inter-PBS bilin pairs with transfer times shorter than 20 ps. Majority of those were in the top cylinder of the core. Several close contacts were also identified between rods, however, the comparison of the blue and cyan traces shows that arranging PBS into a pair has little effect on the excitation dynamics within a rod.

Extended Data Fig. 10 Molecular details of the PBS.

a, Alignment of ApcE with red algae homologs (PDB:5Y6P Griffithsia pacifica, PDB:6KGX Porphyridium purpureum). RMSD is 1.9 Å, differences are circled. b, Structural models of ApcC, ApcG and CpcD. CpcD and ApcC consist of a single pf01383 domain. c, Alignment of CpcG1 with red algae homologs. Circle in CpcG1 shows C-terminal domain. CpcG1 is related to algal LRC1 linker with RMSD of 0.7 Å in the pf00427 domain. The C-terminal domain of CpcG1 has homology to LRC1a with RMSD of 0.6 Å. d, Comparison of CpcC1 and CpcC2. eg, interaction between CpcD, CpcC2, CpcC1 and CpcG1. Interacting residues are highlighted. h, Alignment of ApcD and ApcF with red algae homologs. ApcD is related to the red algal homolog with RMSD of 0.6 Å while ApcF aligns with an RMSD of 0.6 Å with its homolog. The only major differences exist in loop regions. i, Comparison of densities of ApcG between our study and13. Reconstructions of the Synechococcus sp. strain PCC 7002 PBS and Anabaena sp. strain PCC 7120 PBS show ApcG density after map smoothening (Gaussian filter σ = 1 Å).

Extended Data Fig. 11 PBS rod conformation.

a, Side view of AcpAB disc showing the groove network that organizes CpcG1 linker attachment to the PBS core. Selected residues are labeled. b, Salt bridge formed between two neighboring rods when the mobile rod adopts the ‘down’ conformation. EM density is transparent, dark grey and shows the conformation of R150 when the mobile rod is ‘up’. A bilin molecule in very close proximity of R150 of CpcB is also shown.

Extended Data Fig. 12 OCPR features. a and b, electrostatics of the protein environment of the carotenoid tunnel mapped on the OCPR bound to the PBS.

a, Carotenoid tunnel with the β1 and β2 indicated and carotenoid shown as sticks. b, Electrostatic surfaces of the proteins around the carotenoid tunnel and the nearby bilins; carotenoid and bilins shown as sticks. c, CTD-CTD List of residues that interact within 4 Å. d, Close-up view of the P276-W277-F278 loop covering the opening to the carotenoid tunnel in the CTD of OCPO. e, Sequence conservation logo for the CTD (beginning at residue 199) of the OCP. Residues involved in the dimerization are highlighted with a yellow dot.

Extended Data Fig. 13 Decay associated spectra (DAS) and PBS arrays.

DAS spectra obtained either a, from simulation or b, from fitting the data obtained from time-resolved fluorescence (right, adapted with permission from ref. 7) monitoring the excitation energy flow in the PBS. In simulations, PBS was excited into the far end of the rod. The ~100 ps component (green) in the experimental data was interpreted as due to excitation annihilation83, hence it is not present in the simulation. c, Absorption (black) and emission (red) spectra of bilins used to model the excitation energy transfer in PBS. d, PBS arrays were modeled based on cryo-electron tomography data from ref. 25. Only the up-up conformation is compatible with PBS arrays. e, The up-down conformation would result in array termination. f, A PBS switching to the down-down conformation could result in array dispersion.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics of PBS
Extended Data Table 2 Cryo-EM data collection, refinement and validation statistics of OCP–PBS

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1, 2, 4, 5 and 6, and Supplementary Fig. 1.

Reporting Summary

Supplementary Table 3

ApcG UniProt.

Supplementary Data

Atomic model coordinate .cif files for holo-PBS in the up-up conformation, the up-down conformation and the down-down conformation. These atomic models are consensus models assembled from the individual models as described in the Methods. These models have not been refined and therefore only serve for easier visualization of the entire PBS.

Supplementary Video 1

Display of the CTD displacement and carotenoid translocation in OCPR. The carotenoid appears in sphere representation. β1 and β2 surfaces are also labelled.

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Domínguez-Martín, M.A., Sauer, P.V., Kirst, H. et al. Structures of a phycobilisome in light-harvesting and photoprotected states. Nature 609, 835–845 (2022). https://doi.org/10.1038/s41586-022-05156-4

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