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Structural basis of energy transfer in Porphyridium purpureum phycobilisome

Nature volume 579, pages 146–151 (2020)Cite this article

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Abstract

Photosynthetic organisms have developed various light-harvesting systems to adapt to their environments1. Phycobilisomes are large light-harvesting protein complexes found in cyanobacteria and red algae2,3,4, although how the energies of the chromophores within these complexes are modulated by their environment is unclear. Here we report the cryo-electron microscopy structure of a 14.7-megadalton phycobilisome with a hemiellipsoidal shape from the red alga Porphyridium purpureum. Within this complex we determine the structures of 706 protein subunits, including 528 phycoerythrin, 72 phycocyanin, 46 allophycocyanin and 60 linker proteins. In addition, 1,598 chromophores are resolved comprising 1,430 phycoerythrobilin, 48 phycourobilin and 120 phycocyanobilin molecules. The markedly improved resolution of our structure compared with that of the phycobilisome of Griffithsia pacifica5 enabled us to build an accurate atomic model of the P. purpureum phycobilisome system. The model reveals how the linker proteins affect the microenvironment of the chromophores, and suggests that interactions of the aromatic amino acids of the linker proteins with the chromophores may be a key factor in fine-tuning the energy states of the chromophores to ensure the efficient unidirectional transfer of energy.

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Fig. 1: Overall architecture of the PBS from P. purpureum.
Fig. 2: Interactions of the linker proteins LRγs and LRCs with chromophores in the rod Rc.
Fig. 3: The bilins of ApcD and ApcF and their surrounding residues.
Fig. 4: The conformation of PCB in LCM.
Fig. 5: Key bilins in the core.

Data availability

The atomic coordinates have been deposited in the Protein Data Bank with the accession code 6KGX. The electron microscopy maps have been deposited in the Electron Microscopy Data Bank with accession codes EMD-9976 for the overall map and EMD-9977 through to EMD-9988 for the 12 local maps. The raw electron microscopy images used to build the 3D structure are available from the corresponding authors upon request.

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Acknowledgements

We thank the staff at the Tsinghua University Branch of the National Protein Science Facility (Beijing) for technical support on the Cryo-EM and High-Performance Computation platforms; J. Wang for model validation; D. Liu for model building and structure refinement; H. Lin and X. Pan for discussion; and X. Li and H.-W. Wang for recommendations for the computation. This work was supported by the National Basic Research Program (grants 2016YFA0501101 and 2017YFA0504600 to S.-F.S.) and the National Natural Science Foundation of China (grants 31670745 and 31861143048 to S.-F.S.).

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Author notes

  1. These authors contributed equally: Jianfei Ma, Xin You, Shan Sun

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Jianfei Ma, Xin You, Shan Sun & Sen-Fang Sui

  2. Yantai Institute of Coast Zone Research, Chinese Academy of Sciences, Yantai, China

    Xiaoxiao Wang & Song Qin

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  1. Jianfei Ma
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  6. Sen-Fang Sui
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Contributions

S.-F.S. supervised the project; J.M. prepared the samples, collected and analysed the electron microscopy data, performed the initial model building and the biochemical and biophysical analyses; X.Y. performed the model building and the structure refinement; J.M., X.Y., S.S. and S.-F.S. analysed the structure; X.W. helped with the electron microscopy data collection and the biochemical and biophysical analyses; S.Q. contributed to the sample selection; J.M. and X.Y. wrote the initial draft; and S.S. and S.-F.S. edited the manuscript.

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Correspondence to Sen-Fang Sui.

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Extended data figures and tables

Extended Data Fig. 1 Preparation and characterization of the PBS from P. purpureum.

a, Isolation of PBSs using sucrose density gradient centrifugation. Three visible bands were observed. Band 1 is the sample of PBSs used for single-particle analysis in this study. The purification of PBS was repeated independently at least three times with similar results. b, Analysis of the protein composition of band 1 by SDS–PAGE stained with ZnSO4 to enable the detection of bilin-containing proteins with ultraviolet light by Zn-enhanced fluorescence. The bands of LRγ4, 5, 7, 8 and PBPs identified by mass spectrometric analysis are indicated. For gel source data, see Supplementary Fig. 1. The purification and characterization of the protein composition was repeated independently at least three times with similar results. c, Absorption spectrum of band 1 and the PBS from G. pacifica. The peaks at 498 nm, 620 nm and 650 nm are from phycourobilins, PCBs of phycocyanins and PCBs of allophycocyanins, respectively. The peaks at 540 nm and 565 nm are from PEBs. The reduced absorption of the P. purpureum PBS compared with the G. pacifica PBS at 498 nm indicates that the phycourobilin content of P. purpureum is much lower than that of G. pacifica. d, Fluorescence emission spectra of the three bands. Emission maxima at 580 nm and 676 nm represent the disassembled phycoerythrin hexamer and the terminal emitter in the intact PBS, respectively. Band 1 has an emission peak at 676 nm, band 2 at 580 nm and band 3 has two emission peaks at 676 nm and 580 nm, indicating that band 1 contains intact PBSs, band 2 contains free PBPs and band 3 contains partially disassembled PBSs. e, Results of the mass spectrometric analysis of purified PBSs. Two batches of sample were analysed. The similar results confirmed the consistency of our purification method.

Extended Data Fig. 2 Cryo-EM analysis of the PBS from P. purpureum.

a, A representative motion-corrected electron micrograph of PBSs. Scale bar, 50 nm. Tens of thousands of micrographs were collected with similar results. b, Fourier power spectrum of the micrograph showing the Thon ring extending to 2.25 Å. Tens of thousands of micrographs were collected with similar results. c, Typical good, reference-free 2D class averages from single-particle PBS images. Scale bar, 20 nm. More than three rounds of 2D class average were performed with similar results. d, Gold-standard Fourier shell correlation (FSC) curves for the 3D electron microscopy reconstructions of the PBS. Blue curve, FSC curve for the overall structure; green curve, FSC curve for the core region that was masked during refinement. e, FSC curves for the cross-validation of the atomic model. The small difference between work and free FSC curves suggested that the model was not overfitted. f, The workflow for the 2D and 3D classifications for cryo-EM data processing. The masking strategy for dealing with sub-regions of PBS is enclosed within dashed lines. For details, see ‘Cryo-EM data analysis’ in Methods.

Extended Data Fig. 3 Characterization of different types of chromophore.

a, Cryo-EM densities (mesh) of bilins (stick) bound to LRγ4 in the rod Rb, LRγ5 in rod Rd, LRγ7 in the hexamer Ha and LRγ8 in the rod Rd. b, The densities (mesh) of some PCB and PEB bilins (stick) in R-phycocyanins and phycoerythrins from rods Ra and Rb to show their different coplanarities. All of the density maps of PCB bilins showed a very flat conformation of rings B, C and D, consistent with the carbon–carbon double bond between rings C and D in PCB that constraints the movement of ring D, so that ring D is coplanar with the B–C plane. Conversely, most of the density maps of PEB displayed a curved conformation of rings B, C and D owing to the single carbon–carbon bond between rings C and D in PEB that allows the rotation of ring D, so that ring D deviates from the B–C plane. However, some PEBs in R-phycocyanin also showed a planar conformation—such as \({}^{{\rm{Ra1I}}}{{\rm{\beta }}}_{3}^{153}\) and \({}^{{\rm{Rb1I}}}{{\rm{\beta }}}_{1}^{153}\)—although to a lesser extent than that for a typical PCB molecule. c, Dihedral angles of three kinds of chromophore. The dihedral angles Φ1, Ψ1, Φ2 . . . are defined by the atoms NA–C(4)–C(5)–C(6), C(4)–C(5)–C(6)–NB, NB–C(9)–C(10)–C(11) . . . etc.

Extended Data Fig. 4 Overall structure of the PBS from P. purpureum and comparison with that from G. pacifica.

a, Schematic diagram showing the organization of the rods and the core from two perpendicular views. The colouring scheme is the same as in Fig. 1e. b, Structure of the core from two perpendicular views shows the assembly and arrangement of the core layers. c, Overall structure of the PBS overlapped with the G. pacifica PBS displayed in surface representation from three perpendicular views. The additional hexamers in the G. pacifica PBS are coloured white and labelled. d, Schematic model of the PBS architecture. The connections between PBS components are shown. Dark and light colours show C2 symmetric parts of rods. Dark and light salmon, phycoerythrin hexamers in rod; dark and light brown, extra phycoerythrin hexamers; dark and light forest green, phycocyanin hexamers; blue, allophycocyanin trimer; large rectangular box, Pfam00427 domains; small rectangular box, Pfam01383 domains; square box, CBDγ. e, Comparison of linker proteins from P. purpureum with those from G. pacifica. Structures of the 19 well-resolved linker proteins (magenta) are superimposed with those from the G. pacifica PBS (cyan). The linker proteins share very high structural conservation—such as the Pfam00427 domain in the rod–core linker (LRC)1–3/LRC1′–3′, the rod linker (LR)1–3/LR1′–3′ and LCM/LCM′, the Pfam01383 domain in the core linker (LC)/LC′ and LR1/ LR1′, the FAS1 domain in LRC6/LRC6′ and LR9/LR9′, the CBDγ domain in LRγ4–5/LRγ4′–5′ and LRγ7–8/ LRγ7′–8′, the coiled-coil motif at the C termini of LRC2–3/LRC2′–3′, and the long α-helix in the middle of the LRC4–5/LRC4′–5′. Note that LR6 from the P. purpureum PBS is different from LRγ6 from the G. pacifica PBS, therefore they are not aligned. Domains of \({{\rm{\alpha }}}^{{{\rm{L}}}_{{\rm{CM}}}}\), Pfam00427 (00427), Pfam01383 (01383), CBDγ, and FAS1 are labelled.

Extended Data Fig. 5 Interactions between LRC proteins and the core.

a, Organization of LRC proteins LRC1–3/LRC1′–3′ and the core. The grooves on the α subunits that contact the linker helices are shown in red. b, Structural similarity and differences among LRC1a, LRC1b and LRC1c. These rod–core linkers are superimposed relative to the Pfam00427 domain. The helices that interact with the core are boxed. c, Structural similarity of LRC2 and LRC3, as demonstrated by superimposition of the Pfam00427 domain at the N termini and the coiled-coil motif at the C termini. The helices interacting with the core are boxed. d–f, Interactions between the αAPC subunit and the helices of LRC1b (d), LRC2 (e) and LRC3 (f). The residues involved in the interaction of LRC proteins are coloured green and shown in stick representation. The αAPC are shown in surface representation, and the residues involved in the interaction are red.

Extended Data Fig. 6 Interactions of the linker proteins LRγs and LRCs with chromophores in the rod Rd.

a, Bottom, overall structure of the rod Rd with the hexamers shown in surface representation and the linker proteins shown in cartoon representation. Top, structure of the layer Rd3I. Proteins and bilins are shown in cartoon and sphere representations, respectively. Three β subunits are coloured differently and the β82 PEBs are boxed and analysed in detail in b–d. b, The interactions between the residue Y63 and the bilin \({{\rm{\gamma }}}_{{{\rm{L}}}_{{\rm{R}}}{\rm{\gamma }}5}^{135}\) from LRγ5 with the bilin \({}^{{\rm{Rd3I}}}{{\rm{\beta }}}_{2}^{82}\). c, The interaction between F122 from LRγ5 and the bilin \({}^{{\rm{Rd3I}}}{{\rm{\beta }}}_{1}^{82}\). d, The interaction between F107 from LRγ5 and the bilin \({}^{{\rm{Rd3I}}}{{\rm{\beta }}}_{3}^{82}\). e, A focused view of the structure of the layer Rd1I showing the central triangle area. PBPs, the linker protein, bilins and residues are shown in surface, cartoon, ball-and-stick and stick representations, respectively. Three β82 PCBs are boxed and analysed in detail in f–h. f, The interactions between Y201 and F207 from LRC2 and the bilin \({}^{{\rm{Rd1I}}}{{\rm{\beta }}}_{2}^{82}\). g, The interaction between Y90 from LRC2 and the bilin \({}^{{\rm{Rd1I}}}{{\rm{\beta }}}_{1}^{82}\). h, The interaction between Y137 from LRC2 and the bilin \({}^{{\rm{Rd1I}}}{{\rm{\beta }}}_{3}^{82}\).

Extended Data Fig. 7 Comparisons of linker proteins from both P. purpureum and G. pacifica.

a, b, Structural alignment of LRγ linker proteins in the outmost hexamers of various rods from the P. purpureum PBS (a) and the G. pacifica PBS (b). β82 PEBs and residues of LRγ linker proteins are shown in ball-and-stick and stick representations, respectively. Note that an aromatic residue from the LRγ linker is present near to each β82 PEB to form π–π interactions, and one bilin from the LRγ linker \(({{\rm{\gamma }}}_{{{\rm{L}}}_{{\rm{R}}}{\rm{\gamma }}})\) always provides additional π electrons to the conjugation system of the \({{\rm{\beta }}}_{2}^{82}\) PEB. These aromatic residues and the bilins from LRγ linker proteins are conserved in both P. purpureum and G. pacifica. c, Sequence alignment of LRγ4–5 from P. purpureum and other red algae. Three aromatic residues interacting with the β82 PEBs and the cysteine residues used to link the bilins close to the β82 PEBs are marked by stars. LRgamma4_GP and LRgamma5_GP, LRγ4–5 from G. pacifica; PXF41621.1, γ-subunit from Gracilariopsis chorda; XP_005715244.1, γ-subunit from Chondrus crispus; OSX79262, γ-subunit from Porphyra umbilicalis; AAN39000.1, γ-subunit from Griffithsia japonica; AXQ05179.1, γ-subunit from Agarophyton chilense. d, Structural alignment of LRC1 linker proteins from P. purpureum and G. pacifica in the phycocyanin hexamer showing the bilin \({{\rm{\beta }}}_{2}^{82}\) and the surroundings. The key histidine residue close to the \({{\rm{\beta }}}_{2}^{82}\) PCB is conserved. e, Sequence alignment of LRC1 from P. purpureum and other red algal and cyanobacterial species. The key histidine residue close to the β82 PCBs is marked with a star. LRC1_GP, LRC1 from G. pacifica; YP_009294673.1, LRC1 from red algal G. chorda; YP_007627464.1, LRC1 from red algal C. crispus; YP_009413376.1, LRC1 from red algal P. umbilicalis; YP_009244497.1, LRC1 from red algal A. chilense; WP_006617749.1, LRC1 from cyanobacteria Arthrospira platensis; WP_009783358.1, LRC1 from cyanobacteria Lyngbya sp. PCC 8106; WP_017720249.1, LRC1 from cyanobacteria Oscillatoria sp. PCC 10802; WP_071516454.1, LRC1 from cyanobacteria Geitlerinema sp. PCC 9228. f, Structural alignment of the LRC2 and LRC3 linker proteins from P. purpureum and G. pacifica in the phycoerythrin hexamer proximal to the core showing the bilin \({{\rm{\beta }}}_{2}^{82}\) and the surroundings. Two aromatic residues near to the \({{\rm{\beta }}}_{2}^{82}\) PEB are conserved in both P. purpureum and G. pacifica. g, Sequence alignment of LRC2–3 from P. purpureum and other red algae. Two aromatic residues close to the β82 PEBs are marked with stars. LRC2_GP and LRC3_GP are from G. pacifica. PXF39827.1, XP_005715536.1 and OSX69059.1 are from G. chorda, C. crispus and P. umbilicalis, respectively.

Extended Data Fig. 8 Characterization of ApcD, ApcF and the α subunit domain of LCM.

a, Magnified view of the superimposition of ApcD proteins from P. purpureum, G. pacifica (GP_ApcD), Synechocystis PCC 6803 (4PO5_ApcD) and the α subunit of the core layer A3 (α_CoreA3). Bilins and residues are shown in ball-and-stick and stick representations, respectively. Three aromatic residues near to the PCB are conserved in all ApcD proteins, but not in the α subunit of the core layer A3. b, Magnified view of the superimposition of ApcF proteins from P. purpureum and G. pacifica (GP_ApcF), and the β subunit of the core A2 (β_CoreA2). \({}^{{\rm{A2}}}{{\rm{\beta }}}_{{\rm{ApcF}}}^{87}\) is shown in ball-and-stick representation in sand. c, A schematic of interactions between \({}^{{\rm{A2}}}{{\rm{\beta }}}_{{\rm{ApcF}}}^{87}\) and the hydrophobic cap. d, Magnified view of the PCB pocket of ApcF (left), \({}^{{\rm{A2}}}{{\rm{\beta }}}_{1}^{81}\) (middle) and \({}^{{\rm{A2}}}{{\rm{\beta }}}_{2}^{81}\) (right). The protein is shown in surface representation and coloured on the basis of amino acid hydrophobicity (see colour bar). The side chains of hydrophobic residues within 5 Å of the PCB are shown in stick representation. e, Magnified view of the structural alignment of the hydrophobic caps formed by LCM proteins from P. purpureum and G. pacifica. f, Schematic of the steric hindrance experienced by Y140/LCM and the ZZZasa configuration of \({}^{{\rm{A2}}}{{\rm{\alpha }}}_{{{\rm{L}}}_{{\rm{CM}}}}^{186}\). g, Structural alignment of \({{\rm{\alpha }}}^{{{\rm{L}}}_{{\rm{CM}}}}\), ApcD, ApcF, the α subunit (ApcA_A2) and the β subunit (ApcB_A2) in the core. The PCB pockets are indicated in the magnified view on the right.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table
Extended Data Table 2 Summary of proteins, chromophores and model validation
Full size table

Supplementary information

41586_2020_2020_MOESM1_ESM.pdf

Supplementary Figure 1 | Uncropped SDS-PAGE gels for Extended Data Fig. 1b a, The SDS-PAGE gel stained by the coomassie brilliant blue to show the protein marker. b, The SDS-PAGE gel stained by ZnSO4 to show the bilin-containing proteins.

Reporting Summary

Supplementary Table 1 | 25 PBS protein sequences.

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Ma, J., You, X., Sun, S. et al. Structural basis of energy transfer in Porphyridium purpureum phycobilisome. Nature 579, 146–151 (2020). https://doi.org/10.1038/s41586-020-2020-7

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