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Structural insights into the molecular mechanisms of OsFBN5-induced OsSPS3 catalysis

Nature Plants volume 12, pages 217–230 (2026)Cite this article

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Abstract

Solanesyl diphosphate synthase (SPS) is crucial for photosynthesis, as it supplies prenyl precursors for the biosynthesis of the photosynthetic electron carrier, plastoquinone-9 (PQ-9). Fibrillin 5 (FBN5) stimulates SPS catalytic activity through direct binding, which is essential for normal plant growth. However, the molecular mechanism of FBN5-mediated SPS catalytic regulation remains unclear. In Oryza sativa (rice), OsSPS3 is an important plastid-localized SPS isoform involved in PQ-9 formation. The Osfbn5 mutant plants display photodamage with exacerbated PQ-9 deficiency when exposed to high light. Here rice serves as a model organism to study SPS and FBN5. We report the crystal structures of the apo and inhibitor-bound forms of OsSPS3, revealing the alternating catalytic mechanism of the asymmetric OsSPS3 dimer. In addition, we report the cryo-electron microscopy structures of the apo and ligand-bound forms of the OsSPS3–FBN5 complex, showing that OsFBN5 binding triggers an open-to-closed conformational transition of a lid-like capping loop within the inactive monomer of OsSPS3, allowing both monomers of dimeric OsSPS3 to be catalytically active. A comparison of the enzymatic activities of the wild-type OsSPS3 homodimer and a recombinant OsSPS3 heterodimer containing one inactive mutant subunit revealed that OsFBN5 enhances the activity of OsSPS3 by inducing a synchronous catalytic mechanism. This work reveals the dynamic catalytic mechanism of OsSPS3 and provides a structural basis for understanding its function and the FBN5-mediated regulation of the PQ-9 biosynthesis pathway.

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Fig. 1: Functional and biochemical characterization of OsSPS3.
Fig. 2: Crystal structures of the asymmetric OsSPS3 dimer.
Fig. 3: The OsSPS3–FBN5 interaction enhances the catalytic activity of OsSPS3.
Fig. 4: Cryo-EM structures of the apo-OsSPS3–FBN5 and GGSPP-bound OsSPS3–FBN5 complexes.
Fig. 5: Comparison of the structures and enzymatic activities of the OsSPS3 dimer and the OsSPS3–FBN5 complex.

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Data availability

The crystal structures have been deposited in the PDB (www.rcsb.org) with accession codes 9JQS (apo-OsSPS3), 9JZF (ZOL/IPP-bound OsSPS3), 9JR6 (aclonifen-bound OsSPS3) and 9JWR (apo-OsFBN5). The atomic coordinates and EM map of apo-OsSPS3–FBN5 and the GGSPP-bound OsSPS3–FBN5 complex have been deposited in the PDB with accession codes 9KBK (apo-OsSPS3–FBN5) and 9VXI (GGSPP-bound OsSPS3–FBN5) and in the Electron Microscopy Data Bank (www.ebi.ac.uk/pdbe/emdb/) with the accession codes EMD-62228 (apo-OsSPS3–FBN5) and EMD-65409 (GGSPP-bound OsSPS3–FBN5). Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (grant no. 2023YFD1700500 to G.-F.Y.), the National Natural Science Foundation of China (grant no. 32241029 to P.Z.) and the China National Postdoctoral Program for Innovative Talent (grant no. BX20240133 to X.-X.S.). We thank the Shanghai Synchrotron Radiation Facility BL02U1 and BL19U1 for providing the facility support.

Author information

Author notes

  1. These authors contributed equally: Han Xiao, Xing-Xing Shi, Min Li, You-Wang Wang.

Authors and Affiliations

  1. State Key Laboratory of Green Pesticide, Central China Normal University, Wuhan, People’s Republic of China

    Han Xiao, Xing-Xing Shi, Min Li, Da-Wei Wang, Long-Can Mei, Hong-Yan Lin & Guang-Fu Yang

  2. International Joint Research Center for Intelligent Biosensor Technology and Health, Central China Normal University, Wuhan, People’s Republic of China

    Han Xiao, Xing-Xing Shi, Min Li, Da-Wei Wang, Long-Can Mei, Hong-Yan Lin & Guang-Fu Yang

  3. Key Laboratory of Epigenetic Regulation and Intervention, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s Republic of China

    You-Wang Wang & Ping Zhu

  4. University of Chinese Academy of Sciences, Beijing, People’s Republic of China

    You-Wang Wang & Ping Zhu

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Contributions

G.-F.Y. and P.Z. conceived and designed this project. H.X. and M.L. performed the crystal structure data collection and determination. H.X. and Y.-W.W. prepared the samples, screened the cryo grids and performed the data acquisition, image processing and structure determination. H.X. and X.-X.S. analysed the structures and biochemical data. X.-X.S. performed the computational research. D.-W.W., L.-C.M. and H.-Y.L. performed the compound screening and provided technical advice. G.-F.Y., P.Z., X.-X.S. and H.X. finalized the manuscript.

Corresponding authors

Correspondence to Ping Zhu or Guang-Fu Yang.

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The authors declare no competing interests.

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Nature Plants thanks Hyun Uk Kim, Peng Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phenotypes of OsSPS double-knockout rice mutants and the identification of OsSPS3 inhibitors.

a, Representative images of T0 generation plants showing phenotypic differences between the wild-type control and selected double-knockout (Ossps2/3, Ossps1/3, and Ossps1/2) mutants. Scale bar, 1 cm. b, Size exclusion chromatography (SEC) profile (insert, left) and SDS‒PAGE analysis (insert, right) of the purified recombinant OsSPS3 protein (left). Molecular weight estimation of WT-OsSPS3 by gel filtration chromatography (right). The log molecular weight (Da) of three molecular standards (Aldolase, 158 kDa; Conalbumin, 75 kDa; Ovalbumin, 44 kDa) was plotted against their retention volume (mL). c, Catalytic activity of OsSPS3 in the presence of the screened compounds. The data were calculated from three independent experiments (n = 3, shown as the mean ± s.d.). d, 3D surface plots showing the dependence of reaction velocity on substrate concentration (GGPP) and inhibitor concentration (aclonifen and ZOL) to OsSPS3. The mesh represents the nonlinear analysis.

Source data

Extended Data Fig. 2 Structural characterization of OsSPS3 and its ligand-binding interactions.

a, Cartoon representation of apo-OsSPS3 with α-helices shown as cylinders in a spectrum from blue to red (N- to C-terminus). Helices are labelled A-P. b, Representative 2Fo-Fc electron density maps (contoured at 1.0 σ, mesh surface) of the N-terminal regions in OsSPS3α (green) and OsSPS3β (blue). c, Representative 2Fo-Fc electron density maps (contoured at 1.0 σ, mesh surface) of loops I and loop II of OsSPS3α (green) and OsSPS3β (blue). d, Molecular dynamics (MD) simulations (100 ns) of the docked GGPP-IPP-bound OsSPS3 dimer structure. The original structure (light blue) and the stable structure (light green) after the MD simulation are shown as cylinders. The bound substrates are depicted as spheres and colored differently, with IPP-1 in magenta, IPP-2 in orange, GGPP-1 in yellow, and GGPP-2 in purple. e, Molecular formulas and electron density maps (gray mesh, contours at 1.0 σ) of ZOL and IPP (carbon: cyan; oxygen: red; nitrogen: blue; phosphorus: orange) in OsSPS3α. ZOL and IPP are shown as sticks. Magnesium ions and water molecules are shown as green–yellow and red spheres, respectively. f, Structural superposition of the apo (dark gray) and ZOL/IPP-bound (blue) OsSPS3 dimer. g, Electron density map (gray mesh, contoured at 1.0 σ) and molecular structure of aclonifen (carbon: gray; oxygen: red; nitrogen: blue; chlorine: green) bound to the OsSPS3 dimer interface. The ligand is shown in stick representation.

Extended Data Fig. 3 Structural and functional analysis of OsFBN5.

a, Yeast two-hybrid assay confirming the interaction between OsSPS3 and OsFBN5. SD-LW, synthetic defined medium lacking Leu and Trp; SD-LWH, synthetic defined medium lacking Leu, Trp, and His (supplemented with X-α-Gal); and SD-LWAH, synthetic defined medium lacking Leu, Trp, Ade, and His (supplemented with X-α-Gal). b, GST pull-down assay demonstrating a direct interaction between OsSPS3 and OsFBN5. c, Gel filtration binding shift assay results of apo-OsSPS1 (purple) and OsSPS1 combined with OsFBN5 (green); and apo-OsSPS2 (dark khaki) and OsSPS2 combined with OsFBN5 (light steel blue). d, Representative T0 of the wild-type (WT) and Osfbn5 knockout (KO) mutant plants. Scale bar, 1 cm. e, Transcript analysis of OsSPS family genes in Osfbn5-KO mutants. Relative expression levels of Osfbn5, Ossps1, Ossps2, and Ossps3 in four independent Osfbn5-KO T0 lines. Data are presented as the mean ± s.d. f, Trimeric OsFBN5 (PDB code: 9JWR) shown in ribbon representation, with each monomer colored differently (salmon, dark salmon, light salmon). g, Size exclusion chromatography profile and SDS‒PAGE analysis of purified OsFBN5. h, Enzyme kinetic parameters (kcat/Km) of OsSPS3 in the presence of OsFBN5 and three different allylic substrates with IPP as the counter substrate applied at a fixed concentration. i, Dose‒response effects and kinetic inhibition of OsSPS3 by ZOL in the presence of OsFBN5. The data were calculated from three independent experiments (n = 3, shown as the mean ± s.d.). The lines represent linear fits. The Ki values were determined from nonlinear analysis.

Source data

Extended Data Fig. 4 Cryo-EM data analysis of the apo-OsSPS3–FBN5 complex.

a, Anion exchange chromatography and SDS‒PAGE analysis of 3 peaks. Peak 1 corresponds to the apo-OsFBN5 (black), peak 2 represents the OsSPS3–FBN5 complex (blue), and peak 3 contains apo-OsSPS3 (light brown). Each corresponding band is labelled. This experiment was independently repeated three times with similar results. b, Representative negative staining images. The black arrows indicate the complex particles. Scale bar, 100 nm. This experiment was independently repeated three times with similar results. c, Flow chart for cryo-EM data analysis of the apo-OsSPS3–FBN5 complex. d, FSC curves for the apo-OsSPS3–FBN5 complex. e, Map of the apo-OsSPS3–FBN5 complex is colored according to estimated local resolution. f, Sample maps of N-terminal subdomains (helices A, B and C), dimerization interface (helices G and H), loops I, II, and III and the interaction interface of the apo-OsSPS3–FBN5 complex.

Source data

Extended Data Fig. 5 Cryo-EM data analysis of the GGSPP-bound OsSPS3–FBN5 complex.

a, Flow chart for cryo-EM data analysis of GGSPP-bound OsSPS3–FBN5 complex. b, Orientation distribution of the aligned particles in the final 3D reconstruction output from CryoSPARC. c, Gold-standard Fourier shell correlation (GSFSC) curves of GGSPP-bound OsSPS3–FBN5 complex d, Local resolution calculated using CryoSPARC. The map is colored according to the local resolution. e, Sample maps of N-terminal subdomains (helices A, B and C), loops Ⅰ, II, and III and the dimerization interface (helices G and H) of the GGSPP-bound OsSPS3–FBN5 complex. f, EM density of GGSPP/Co2+.

Extended Data Fig. 6 Structural and functional analysis of the OsSPS3–FBN5 complex.

a, Structural superposition of the apo-OsFBN5 (salmon) with the GGSPP-bound OsSPS3–FBN5 complex, with one of the OsFBN5 units colored khaki. b, Structural alignment of the OsFBN5 β-barrel channel with the OsSPS3 catalytic pockets. c, Comparison of the apo-OsSPS3–FBN5 (gray) and GGSPP-bound OsSPS3–FBN5 (blue) cryo-EM structures reveals a flip in the side chain conformation of Phe237. d, Michaelis–Menten curves of OsSPS3(R201A) in the absence (triangles) and presence (circles) of OsFBN5 with GGPP as the allylic substrate and IPP as the counter substrate applied at a fixed concentration. The data were calculated from three independent experiments (n = 3, shown as the mean ± s.d.). e, Structural superposition of the ZOL/IPP bound OsSPS3 (pink) with the GGSPP-bound OsSPS3–FBN5 complex (sky blue). f, Superposition of ZOL/IPP-bound OsSPS3 with the GGSPP-bound OsSPS3–FBN5 complex showing conformational changes in the dimerization helices G and H. The arrows indicate the deflection angle. g, Structural alignment of active and inactive monomers of ZOL/IPP-bound OsSPS3 with the OsSPS3 monomer in GGSPP-bound OsSPS3–FBN5. h, Molecular dynamics (MD) simulations reveal that the stable binding of the substrates GGPP/IPP is mediated by critical residues (Arg188 and Arg189) in both OsSPS3 monomers of the OsSPS3–FBN5 complex.

Source data

Extended Data Fig. 7 Biochemical characterization of the recombinant OsSPS3 heterodimer.

a, Microscale thermophoresis assay results of OsSPS3(WT/AA) binding to OsFBN5. Western blotting demonstrated successful purification of the OsSPS3 heterodimer (insert, left). The data were calculated from three independent experiments (n = 3, shown as the mean ± s.d.). b, The Michaelis–Menten curve of OsSPS3(WT/AA) in the absence (triangles) of OsFBN5 with GGPP as the allylic substrate and IPP as the counter substrate at a fixed concentration. The data were calculated from three independent experiments (n = 3, shown as the mean ± s.d.). c, The Michaelis–Menten curve of OsSPS3(R201A) in the presence (circles) of OsFBN5 with GGPP as the allylic substrate and IPP as the counter substrate at a fixed concentration. The data were calculated from three independent experiments (n = 3, shown as the mean ± s.d.).

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Extended Data Fig. 8 AlphaFold3 (AF3)-predicted structure of the OsSPS3–FBN5 complex and a proposed model of OsFBN5 acting as an OsSPS3 substrate transporter.

a, Two clusters of AF3-predicted structures of the OsSPS3–FBN5 complex on the basis of the ipTM scores. b, Superposition of the AF3-cluster2 (AF3-4) structure and the cryo-EM structure of the OsSPS3–FBN5 complex. The black box shows the different conformations of loop III in the two structures. c, Two substrate-binding pockets of the predicted AF3-cluster-2 structures. d, Microscale thermophoresis assay results of apo-OsFBN5 binding to three different allylic substrates (C10-GPP, C15-FPP and C20-GGPP). The dissociation constants were calculated from three independent experiments (n = 3, shown as the mean ± s.d.). e, Mutational analysis of the OsFBN5 hydrophobic channel. The data were calculated from three independent experiments (n = 3, shown as the mean ± s.d.). f, Schematic model of FBN5 acting as a substrate transporter for SPS catalysis and the catalytic product being released from the SPS dimer interface. g, Structural alignment of the predicted C45-SPP-bound structure with the experimental GGSPP-bound structure. The structures are depicted as cylinders. In the experimental GGSPP-bound structure, the two OsSPS3 chains are shown in green and blue cylinders, respectively. The ligands are shown as sticks. The proposed elongation path of the C45-SPP product is indicated by a red dashed arrow. h, Microscale thermophoresis assay results and the Michaelis–Menten curve of OsSPS3(E352D) in the presence of OsFBN5. The data were calculated from three independent experiments (n = 3, shown as the mean ± s.d.).

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Xiao, H., Shi, XX., Li, M. et al. Structural insights into the molecular mechanisms of OsFBN5-induced OsSPS3 catalysis. Nat. Plants 12, 217–230 (2026). https://doi.org/10.1038/s41477-025-02184-6

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