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Structural basis of the subcortical maternal complex and its implications in reproductive disorders

Nature Structural & Molecular Biology volume 31, pages 115–124 (2024)Cite this article

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Abstract

The subcortical maternal complex (SCMC) plays a crucial role in early embryonic development. Malfunction of SCMC leads to reproductive diseases in women. However, the molecular function and assembly basis for SCMC remain elusive. Here we reconstituted mouse SCMC and solved the structure at atomic resolution using single-particle cryo-electron microscopy. The core complex of SCMC was formed by MATER, TLE6 and FLOPED, and MATER embraced TLE6 and FLOPED via its NACHT and LRR domains. Two core complexes further dimerize through interactions between two LRR domains of MATERs in vitro. FILIA integrates into SCMC by interacting with the carboxyl-terminal region of FLOPED. Zygotes from mice with Floped C-terminus truncation showed delayed development and resembled the phenotype of zygotes from Filia knockout mice. More importantly, the assembly of mouse SCMC was affected by corresponding clinical variants associated with female reproductive diseases and corresponded with a prediction based on the mouse SCMC structure. Our study paves the way for further investigations on SCMC functions during mammalian preimplantation embryonic development and reveals underlying causes of female reproductive diseases related to SCMC mutations, providing a new strategy for the diagnosis of female reproductive disorders.

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Fig. 1: Constitution and overall structure of the SCMC complex.
Fig. 2: Intercomponent interactions of the SCMC core complex.
Fig. 3: FILIA interacts with FLOPED in the SCMC complex.
Fig. 4: Structure mapping of the disease-related mutations and the effects on SCMC formation.

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Data availability

The homology model of MATER, TLE6 and FLOPED was built in a map with SWISS-MODEL using the structures of NLRP3, TLE1 and FILIA (PDB codes: 6NPY, 1GXR and 3V69, respectively) as templates. The structural coordinates and factors were deposited in the Protein Data Bank with ID: 8H96 (SCMCMTO), 8H94 (SCMCMTOK1-124), 8H95 (SCMCMTOK_FL) and 8H93 (dimeric SCMCMTO complex), and the corresponding cryo-EM maps were deposited in Electron Microscopy Data Bank (EMDB) with ID: EMD-34556, EMD-34554, EMD-34555 and EMD-34552, respectively. Source data are provided with this paper.

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Acknowledgements

We thank the SKLB West China Cryo-EM Center of Sichuan University and the Cryo-EM Facility and Supercomputer Center of Westlake University for Cryo-EM support. This work was supported by (1) the National Natural Science Foundation of China (Nos. 31930033 to L.L., 31971132 to D.D., 31871449 to J.C. and 82172634 to H.S.); (2) National Key R&D Program of China (2022YFC2702201) to L.L.; (3) the Key Program of the Science and Technology Bureau of Sichuan (No. 2021YFSY0007) to H.S.; (4) 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (No. ZYYC20013) to H.S.; and (5) Strategic Collaborative Research Program of the Ferring Institute of Reproductive Medicine (180202) to L.L. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Pengliang Chi, Guojin Ou, Dandan Qin, Zhuo Han, Jialu Li.

Authors and Affiliations

  1. Department of Obstetrics, Key Laboratory of Birth Defects and Related Disease of Women and Children of MOE, State Key Laboratory of Biotherapy, West China Second Hospital, Sichuan University, Chengdu, China

    Pengliang Chi, Guojin Ou, Zhuo Han, Jialu Li, Qingting Liu, Sibei Liu, Jinhong Li, Li Guo, Yuechao Lu, Xiang Wang, Hubing Shi & Dong Deng

  2. Clinical Laboratory, West China Second Hospital, Sichuan University, Chengdu, China

    Guojin Ou & Qianqian Qi

  3. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Stem Cell and Regeneration, Beijing Institute of Stem Cell and Regenerative Medicine, Institute of Zoology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing, China

    Dandan Qin, Chengpeng Xu & Lei Li

  4. NHC Key Laboratory of Chronobiology, Sichuan University, Chengdu, China

    Zhuo Han, Jialu Li, Xiang Wang & Dong Deng

  5. Development and Related Diseases of Women and Children Key Laboratory of Sichuan Province, Sichuan University, Chengdu, China

    Zhuo Han, Jialu Li, Xiang Wang & Dong Deng

  6. Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, China

    Qingjie Xiao

  7. Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Guangdong Provincial Key Laboratory of Major Obstetric Diseases, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China

    Zheng Gao

  8. Department of Reproductive Medicine, West China Second Hospital, Sichuan University, Chengdu, China

    Yuechao Lu

  9. Laboratory of Pediatric Surgery, Department of Pediatric Surgery, West China Hospital, Sichuan University, Chengdu, China

    Jing Chen

  10. Laboratory of Integrative Medicine, Clinical Research Center for Breast, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, China

    Hubing Shi

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  1. Pengliang Chi
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Contributions

P.C. performed the expression constructs design, protein purification, EM sample preparation and data curation. G.O. and Z.H. performed immunoprecipitation, immunoblotting and immunofluorescence. Jialu L. performed EM data collection, computation and model building. D.Q., Z.G. and C.X. performed the experiments on animals and oocytes/zygotes. Q.X. participated in the model building. Q.Q., Q.L., S.L. and Jinhong L. participated in data analysis and assisted with immunoprecipitation. L.G., Y.L., J.C., X.W., H.S., L.L. and D.D. participated in structural and biochemical data analysis, experiment design and fundings. P.C., G.O., D.Q., H.Z., Jialu L., L.L. and D.D. wrote and edited the manuscript. L.L. and D.D. supervised the overall project.

Corresponding authors

Correspondence to Lei Li or Dong Deng.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Dimitris Typas was the Primary Editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Sequence alignment of mouse MATER and human MATER.

Mouse MATER lacks the N terminal PYD domain lacks the N terminal repeat (black box outlined). The PYD domain of human MATER was outlined with the green box. The β-strand 7 was essential for binding with FLOPED. The residues of CTL (W1046, Y1047, D1053) in contact with LRR (R843, D930, S985) were conserved in human MATER.

Extended Data Fig. 2 Sequence alignment of TEL6 and FLOPED.

a. Sequence alignment of mouse TEL6 and human TLE6. The green box highlights the N-terminal region of TLE6, which contains an MIH for binding with MATER. The blue box highlights the conserved WDR domain. b. Sequence alignment of mouse FLOPED and human FLOPED. The β-strand for binding with MATER is outlined with the blue box.

Extended Data Fig. 3 Flowchart of cryo-EM data processing and analysis of the map density for the SCMCMTO complex.

a. Representative micrograph of the cryo-EM and workflow of cryo-EM data processing. Details are described in the Methods. The green circles represented the single particles. The classes outlined with red boxes in 2D classification averages represent the dimeric SCMC complex. b. Gold-standard Fourier shell correlation (GSFSC) curve for the refined map of the SCMCMTO complex. c. The particle angular distribution for the map of the SCMCMTO complex. d. The local resolution of the map of the SCMCMTO complex.

Extended Data Fig. 4 Flowchart of cryo-EM data processing and analysis of the map density for the SCMCMTOK1-124 complex.

a. Representative micrograph of the cryo-EM and workflow of cryo-EM data processing. Details are described in the Methods. The green circles represented the single particles. The classes outlined with red boxes in 2D classification averages represent the dimeric SCMC complex. b. Gold-standard Fourier shell correlation (GSFSC) curve for the refined map of the SCMCMTOK1-124 complex. c. Particle angular distribution for the map of the SCMCMTOK1-124 complex. d. Superposition of the cryo-EM maps of SCMCMTO and SCMCMTOK1-124. e. Structure alignment of the models built with SCMCMTO and SCMCMTOK1-124, RMSD = 0.706 Å.

Extended Data Fig. 5 Cryo-EM densities of the SCMCMTO complex.

The atomic model was fitted in the cryo-EM map of SCMCMTO. The domains were grouped in dotted line boxes and the peptide regions were indicated by the range of numbers.

Extended Data Fig. 6 Flowchart of cryo-EM data processing and analysis of the map density for the SCMCMTO dimer of the trimer complex.

a. Representative micrograph of the cryo-EM and workflow of cryo-EM data processing. Details were described in the Methods. The green circles represented the single particles. b. Gold-standard Fourier shell correlation (GSFSC) curve for the refined map of the SCMCMTO dimer of the trimer complex. c. Particle angular distribution for the map of the SCMCMTO dimer of the trimer complex. d. Back-to-back dimerization interface between LRR domains of the dimeric SCMCMTO complex. e. Immunoprecipitation was performed to analyze the back-to-back dimerization interface. Strep-TLE6 and Strep-FLOPED were mixed with vector, Flag-MATER (WT), or Flag-MATER variant (F858D/M861D/L886D) and transfected into 293 F cells. The cells were collected, lysed with 1% Triton X-100 and incubated with SCMC protein purified from Sf9. The proteins were purified with anti-Flag affinity gel and analyzed by WB. Three biologically repeated experiments were independently performed. f. Determination of the molecular mass by multi-angle light scattering coupled with size exclusion chromatography. A 200 µL volume of SCMCMTO (approximately 400 µg) in PBS buffer was applied to a preequilibrated Superose 6 increase 10/300 column. The UV280 absorbance is shown as the red line, and the molecular mass is shown as the blue line.

Source data

Extended Data Fig. 7 Intercomponent interactions of SCMC and the sequence alignment of the MIH motif.

a. Overall view of the interfaces among the subunits of the SCMC complex. The six interfaces are indicated with dotted line boxes and labeled with Roman numerals. b. Sequence alignment of the MIH motif of TLE6 among species.

Extended Data Fig. 8 Structure of MATER and comparisons with homologs.

a. Structure of MATER, FISNA (97-138 aa), NBD domain (139-292 aa), HD1 domain (293-353 aa), WHD domain (354-463 aa), HD2 domain (464-564 aa), LRR domain (565-1037 aa), and CTL (1038-1059 aa). b. Density of the ATP/ADP binding pocket (highlighted with the red circle). Walker A and Walker B motifs are shown in magenta. c. ATP hydrolysis assay for MATER, MATER Walker A mutant, SCMCMTO, NLRP3, and BSA. Then, 0.6 µM proteins and 1 mM ATP were incubated in reaction buffer, and the reactions were incubated in a time course over 240 min at 25°C. The Reagent buffer was added to the reaction mixture and further incubated for 30 min and absorbance at 620 nm was determined by BioTek Synergy H1. d. ATPase activities of MATER, MATER Walker A mutant, SCMCMTO, NLRP3, and BSA were determined by incubating the samples with 1 mM ATP for 240 min at 25°C. The reactions were further incubated with Reagent buffer for 30 min, and the absorbance at 620 nm was determined. The data points indicated n = 3 biological repeats and the error bars present the means ± SD; two-tailed unpaired Student’s t-test; n.s., no significance; ****, p < 0.0001. p (MATER vs MATER-GKS-3A) = 0.6481, p (MATER vs SCMC) = 0.2102, p (MATER vs NLRP3) = 1.56 × 10−5, p (MATER vs BSA) = 0.5164. e. Comparison of NACHT domain of MATER (SCMCMTO) with that of NLRP3 (7pzc) by superimposing NBD domain, RMSD = 0.959 Å. The first helix of HD1 of MATER rotates 65° away from NBD than that of NLRP3. f. Surface display of the ATP/ADP binding pocket in MATER (SCMCMTO), which is highlighted with a red circle. g. Surface display of the ATP/ADP binding pocket in NLRP3 (6npy, inactive), ADP was present as the sphere. h. Surface display of the ATP/ADP binding pocket in NLRP3 (7pzc, decamer). i. Surface display of the ATP/ADP binding pocket in NLRP3 (8ej4, active). j. Surface display of the ATP/ADP binding pocket in NLRC4 (4kxf). k. Surface display of the ATP/ADP binding pocket in CED-4 (2a5y). l. Sequence alignment of the Walker A motif and Sensor 2 motif of MATER (SCMCMTO) with those of NLRP3. m. Distances of key residues of NLRP3 with ADP(7pzc). n. Distances of the corresponding key residues of MATER with ADP. ADP in MATER was superimposed by the NBD domain of NLRP3 (7pzc) and that of MATER (SCMCMTO), RMSD = 1.003 Å. o. Hydrogen bonds between CTL and LRR.

Source data

Extended Data Fig. 9 FLOPED directly binds and stabilizes FILIA through its C-terminus in the SCMC.

a. Representative micrograph of the cryo-EM and workflow of cryo-EM data processing. Details are described in the Methods. The green circles represented the single particles. b. Gold-standard Fourier shell correlation (GSFSC) curve for the refined map of the SCMCMTOK-FL complex. c. Particle angular distribution for the map of the SCMCMTOK-FL complex. d. Alignment of FILIA (white, PDB:3v69) and FLOPED (green), RMSD = 1.237 Å. e. Cell lysis of GST pull-down corresponding to Fig. 3c. Two biologically repeated experiments were performed. f. Real-time quantitative PCR of the SCMC components in GV oocytes. 30 GV oocytes from FlopedΔC/ΔC and 30 GV oocytes from WT mice were used for RNA extraction and qPCR.

Source data

Extended Data Fig. 10 Cryo-EM densities of locations of the disease-associated mutations in the interface (related to Fig. 4b, 4c).

a. Cryo-EM densities related to the mutation T305R of TLE6 and the interface with FLOPED. b. Cryo-EM densities related to R32W/P/G mutations of FLOPED and the interface with MATER.

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Supplementary Tables

Supplenentary Table 1. The summary of the clinical variants, mouse counterparts and the symptom of patients. Supplementary Table 2. The mCSM prediction of the protein-protein interaction stability.

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Source Data Fig. 1

Unprocessed western blots and gels.

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Unprocessed western blots.

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Unprocessed western blots and gels.

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Statistical source data.

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Unprocessed western blots.

Source Data Extended Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

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Chi, P., Ou, G., Qin, D. et al. Structural basis of the subcortical maternal complex and its implications in reproductive disorders. Nat Struct Mol Biol 31, 115–124 (2024). https://doi.org/10.1038/s41594-023-01153-x

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