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Photocatalytic LPOR forms helical lattices that shape membranes for chlorophyll synthesis

Nature Plants volume 7, pages 437–444 (2021)Cite this article

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Abstract

Chlorophyll biosynthesis, crucial to life on Earth, is tightly regulated because its precursors are phototoxic1. In flowering plants, the enzyme light-dependent protochlorophyllide oxidoreductase (LPOR) captures photons to catalyse the penultimate reaction: the reduction of a double bond within protochlorophyllide (Pchlide) to generate chlorophyllide (Chlide)2,3. In darkness, LPOR oligomerizes to facilitate photon energy transfer and catalysis4,5. However, the complete three-dimensional structure of LPOR, the higher-order architecture of LPOR oligomers and the implications of these self-assembled states for catalysis, including how LPOR positions Pchlide and the co-factor NADPH, remain unknown. Here, we report the atomic structure of LPOR assemblies by electron cryo-microscopy. LPOR polymerizes with its substrates into helical filaments around constricted lipid bilayer tubes. Portions of LPOR and Pchlide insert into the outer membrane leaflet, targeting the product, Chlide, to the membrane for the final reaction site of chlorophyll biosynthesis. In addition to its crucial photocatalytic role, we show that in darkness LPOR filaments directly shape membranes into high-curvature tubules with the spectral properties of the prolamellar body, whose light-triggered disassembly provides lipids for thylakoid assembly. Moreover, our structure of the catalytic site challenges previously proposed reaction mechanisms6. Together, our results reveal a new and unexpected synergy between photosynthetic membrane biogenesis and chlorophyll synthesis in plants, orchestrated by LPOR.

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Fig. 1: Plant LPORs form filaments around a membrane bilayer.
Fig. 2: AtPORB oligomerization creates a periodic array of Pchlides.
Fig. 3: Detailed interactions between AtPORB and its substrates suggest a novel catalytic mechanism.
Fig. 4: Reduction of Pchlide to Chlide may introduce conformational changes that lead to filament disassembly.

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Data availability

The atomic model and cryo-EM maps were deposited with PDB (no. 7JK9) and EMDB (no. EMD-22364). In this study the following additional data were used: LPOR structures from T. elongatus (PDB 6L1H) and Synechocystis sp. (PDB 6R48 or 6L1G); LPOR sequence database (Supplementary Data 1; https://doi.org/10.1042/BCJ20200323).

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Acknowledgements

We thank the Frost laboratory for helpful discussions and critical comments; P. Thomas, L. E. Miller-Vedam and M. Sun for computational assistance; N. Poweleit for discussions on helical processing; T. Borowski for generation of Pchlide ligand restraints; M. Braunfeld, D. Bulkley, M. Harrington, A. Myasnikov and Z. Yu of the UCSF Center for Advanced Cryo-EM for microscopy support; and J. Baker-LePain and the QB3 shared cluster (National Institutes of Health (NIH) grant no. 1S10OD021596-01) for computational support. Structural biology applications used in this project were compiled and configured by SBGrid. The Titan X Pascal used for this research was donated by the NVIDIA Corporation. This study was supported by a Bekker scholarship granted by the Polish National Agency for Academic Exchange (no. PPN/BEK/2018/1/00105), a START scholarship granted by the Foundation for Polish Science (no. 024.2018 to M.G) and NIH (no. 1DP2GM110772-01 to A.F). A.F. is further supported by a Faculty Scholar grant from Howard Hughes Medical Institute (HHMI) and is a Chan Zuckerberg Biohub investigator.

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Author notes

  1. Henry C. Nguyen

    Present address: Asher Biotherapeutics, South San Francisco, CA, USA

  2. These authors contributed equally: Henry C. Nguyen, Michal Gabruk.

Authors and Affiliations

  1. Department of Biochemistry & Biophysics, Quantitative Biosciences Institute, University of California, San Francisco, CA, USA

    Henry C. Nguyen, Arthur A. Melo & Adam Frost

  2. Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

    Jerzy Kruk & Michal Gabruk

  3. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Adam Frost

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Contributions

M.G. and A.F. conceived the project. M.G., A.F. and H.C.N. designed experiments and analysed data. M.G. optimized and produced the samples and performed spectroscopy measurements and sequence alignment analysis. H.C.N. assisted in sample optimization and performed cryo-EM experiments and analysis. J.K. and M.G. purified Pchlide. A.A.M. prepared the animation video and assisted with cryo-EM data collection. H.C.N., A.F. and M.G. prepared the manuscript with input from all authors.

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Correspondence to Henry C. Nguyen, Adam Frost or Michal Gabruk.

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Peer review information Nature Plants thanks Alexey Amunts, Benjamin Engel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Plant LPOR can form filaments of different sizes.

a, Negative stain electron microscopy (EM) micrographs of AtPORB filaments with different diameters formed from liposomes composed of 2.5 mol% MGDG / 50 mol% DGDG / 47.5 mol% PG (orange frame) or 50 mol% MGDG / 25 mol% DGDG / 25 mol% PG (black frames). Scale bar: 25 nm. b, Negative stain EM micrographs of AtPORB filaments produced from liposomes mimicking the lipid composition of PLBs (52 mol% MGDG / 32 mol% DGDG / 8 mol% PG, 8 mol% SQDG; orange frame, chloroplast inner membranes (50 mol% MGDG / 30 mol% DGDG / 8 mol% PG, 5 mol% SQDG, 7 mol% PC, cyan frame) and our optimized membrane composition (50 mol% MGDG / 35 mol% DGDG / 15 mol% PG, black frame). Scale bar: 25 nm. c, Low-temperature fluorescence emission spectra before (left panel) and after illumination (right panel) of the filaments produced with the liposomes mimicking the lipids of PLBs (orange), chloroplast inner membrane (cyan) and for our optimized membrane composition (black). Grey vertical lines indicate the emission maxima of: free Pchlide (632 nm), Pchlide within PLBs or the membrane-bound filaments (655 nm), and reduced Chlide (690 nm).

Extended Data Fig. 2 CryoEM validation of the AtPORB filament.

a, Representative fit of the cryoEM density to the atomic model. b, Angular distribution of the membrane-bound AtPORB cryoEM 3D reconstruction. c, Independent half-map and map-model Fourier shell correlation (FSC) of the reconstruction. d, Local resolution estimates.

Extended Data Fig. 3 A 4-start helix of plant LPOR.

Exterior and top-down views of the 4-start PORB helix. Each start comprises a strand of antiparallel dimers, colored in orange, green, cyan, or magenta.

Extended Data Fig. 4 Comparison of plant and cyanobacterial LPORs.

a, Secondary structure elements of AtPORB annotated onto the tertiary structure. b, The comparison of the sequences of AtPORB, T. elongatus LPOR (Thermo), Synechocystis LPOR (Synech), and the consensus sequences of LPORs originating from plants and cyanobacteria (187 and 301 sequences, respectively). The size of the letter indicates the frequency of the given amino acid at the given position, while x indicates multiple amino acids with low frequency at a given position. Transit peptides of plant LPORs are excluded. Residues conserved in all sequences are grey. The secondary structure elements and Pchlide loop of AtPORB are marked. AtPORB residues known to play a role in oligomerization and substrate binding are highlighted with a pale red background. Orange/cyan/grey polygons indicate residues of NADPH/Pchlide/MGDG binding sites, respectively. Edges of the polygon highlighted in green indicate the oligomerization interfaces the given residue is involved in: bottom edge – antiparallel dimer, top edge – intra-strand interface, side edges – inter-strand interface. The previously proposed catalytic YxxxK motif is marked in red. Cysteine and serine residues that we suggest may be catalytically involved in the reaction are marked in green. c, Schematic representation of the LPOR interfaces for the polygons in b. d, Negative stain EM micrographs of AtPORB (left) and Synechocystis LPOR (right) filaments produced with our optimized membrane composition. Scale bar: 25 nm.

Extended Data Fig. 5 Comparison of the NADPH binding site across plants and cyanobacteria.

a, NADPH binding pocket of AtPORB. b, NADPH binding pocket of LPOR form T. elongatus (PDB: 6L1H). c, NADPH binding pocket of LPOR form Synechocystis (PDB: 6L1G). Hydrophobic interactions are shown as orange circles and hydrogen bonds are shown as dashed lines. Green dashed lines indicate the identical bonds between homologous residues. Resides in red indicate non-conserved residues. Waters are shown as small light blue circles.

Extended Data Fig. 6 Structural comparison of Pchlide binding motifs between Pchlide-bound plant and Pchlide-free cyanobacterial LPOR.

Superposition of Pchlide-bound AtPORB (dark green) with Pchlide-free LPOR from Synechocystis LPOR (a, dark purple, PDB: 6R48 or 6L1G), T. elongatus (b, PDB: 6RNW, orange), and T. elongatus (c, PDB: 6L1H, slate blue).

Extended Data Fig. 7 AtPORB S228A is less active upon illumination than WT, C309 mutants or Y276F mutant.

a, the spectra of the reactions before (blue) and after (orange) the illumination of AtPORB WT and selected mutants. b, proposed proton-relay path (green dashed lines) and hydride-relay path (yellow dashed line).

Supplementary information

Supplementary Information

Supplementary Table 1.

Reporting Summary

Supplementary Video 1

Structure of the membrane-bound LPOR filament with substrates bound.

Supplementary Video 2

Conformational changes following Pchlide binding.

Supplementary Video 3

Mechanistic model of substrate binding, polymerization and membrane remodelling by plant LPOR.

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Nguyen, H.C., Melo, A.A., Kruk, J. et al. Photocatalytic LPOR forms helical lattices that shape membranes for chlorophyll synthesis. Nat. Plants 7, 437–444 (2021). https://doi.org/10.1038/s41477-021-00885-2

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