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Cryo-EM structures of human GMPPA–GMPPB complex reveal how cells maintain GDP-mannose homeostasis

Nature Structural & Molecular Biology volume 28, pages 1–12 (2021)Cite this article

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Abstract

GDP-mannose (GDP-Man) is a key metabolite essential for protein glycosylation and glycophosphatidylinositol anchor synthesis, and aberrant cellular GDP-Man levels have been associated with multiple human diseases. How cells maintain homeostasis of GDP-Man is unknown. Here, we report the cryo-EM structures of human GMPPA–GMPPB complex, the protein machinery responsible for GDP-Man synthesis, in complex with GDP-Man or GTP. Unexpectedly, we find that the catalytically inactive subunit GMPPA displays a much higher affinity to GDP-Man than the active subunit GMPPB and, subsequently, inhibits the catalytic activity of GMPPB through a unique C-terminal loop of GMPPA. Importantly, disruption of the interactions between GMPPA and GMPPB or the binding of GDP-Man to GMPPA in zebrafish leads to abnormal brain development and muscle abnormality, analogous to phenotypes observed in individuals carrying GMPPA or GMPPB mutations. We conclude that GMPPA acts as a cellular sensor to maintain mannose homeostasis through allosterically regulating GMPPB.

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Fig. 1: Structures of GMPPA–GMPPB–GDP-Man and GMPPA–GMPPB–GTP complexes.
Fig. 2: Overall structures and catalytic centers of GMPPA and GMPPB.
Fig. 3: Subunit arrangement of the GMPPA–GMPPB complex and three types of dimeric interface.
Fig. 4: GMPPA regulates the catalytic activity of GMPPB.
Fig. 5: GMPPA inhibits the catalytic activity of GMPPB by sensing GDP-Man concentration.
Fig. 6: GMPPA and GMPBB disease mutants disrupt complex assembly or alter enzymatic activity.
Fig. 7: GMPPA and GMPPB regulate neuronal and muscle development in zebrafish by controlling GDP-Man concentration.

Data availability

Structural data of GMPPA/GMPPB bound to GDP-Man and GTP are deposited in the Protein Data Bank (PDB) under accession nos. 7D72 (GDP-Man), 7D73 (state I) and 7D74 (state II), respectively. The cryo-EM maps are deposited in the Electron Microscopy Data Bank with accession codes 30599 (GDP-Man), 30600 (state I) and 30601 (state II), respectively. All other data are available from the authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the Core Facilities of Peking University School of Life Sciences for assistance with negative-staining electron microscopy; the Cryo-EM Platform of Peking University and the Electron Microscopy Laboratory of Peking University for cryo-EM data collection; and the High-performance Computing Platform of Peking University for help with computation. The studies were supported by the National Science Foundation of China (grants 31725007 and 31630087 to N.G., and 91854121 and 31871429 to D.J.), the National Key Research and Development Program of China (grants 2019YFA0508904 to N.G. and 2018YFC1005004 to D.J.), Sichuan Science and Technology Program (grant 2018RZ0128 to D.J.) and the Qidong-SLS Innovation Fund (to N.G.).

Author information

Author notes

  1. These authors contributed equally: Lvqin Zheng, Zhe Liu, Yan Wang.

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Peking-Tsinghua Joint Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, School of Life Sciences, Peking University, Beijing, China

    Lvqin Zheng & Ning Gao

  2. Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy, Sichuan University, Chengdu, China

    Zhe Liu, Yan Wang, Fan Yang, Jinrui Wang, Wenjie Huang, Jiao Qin, Min Tian, Xiaotang Cai & Da Jia

  3. School of Life Sciences, Tsinghua University, Beijing, China

    Xiaohui Liu

  4. Department of Pediatric Surgery and Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China

    Xianming Mo

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Contributions

Y.W. generated protein samples for EM analysis and performed biochemical work with assistance from W.H., M.T. and J.Q. F.Y. and J.W. performed cellular studies. L.Z. performed cryo-EM data acquisition and data processing. L.Z. and N.G. built and refined the model. Z.L. carried out zebrafish studies, X.C. determined GDP-Man concentration and X.L. and X.M. provided technical assistance. L.Z., N.G. and D.J. wrote the manuscript. N.G. and D.J. supervised the project.

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Correspondence to Ning Gao or Da Jia.

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The authors declare no competing interests.

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Peer review information Nature Structural & Molecular Biology thanks Jon Agirre, Hudson Freeze and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Purification and biochemical analysis of the GMPPA/GMPPB complex purified from sf9 cells.

a, Left: Elution profile of the GMPPA/GMPPB complex from gel filtration chromatography. Proteins were concentrated to 1 ml, and separated by a Superdex 200 gel filtration column (GE Healthcare), with an elution buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM DTT, 10% (v/v) glycerol, 0.02 % DDM). The size of GMPPA/GMPPB in solution was determined by fitting the calibration curve on the right. Right: standardization of the same size exclusion column. Thyroglobulin (670 KDa), γ-globulin (158 KDa), Ovalbumin (44 KDa), and Myoglobin (17 KDa) were eluted under the exact same conditions as GMPPA/GMPPB. b, Coomassie blue stained SDS-PAGE gels of samples after gel filtration chromatography in (a). A representative gel obtained from one out of three independent experiments was shown. Numbers representing elution volume. c, Formation of GDP-Man using GTP and man-1-p as substrates at different time points, in the presence of GMPPB or GMPPA/GMPPB. d, Formation of GTP using GDP-Man and PPi as substrates at different time points, in the presence of GMPPB or GMPPA/GMPPB. e, Formation of GDP-Man using GTP and man-1-p as substrates under different conditions, in the presence of GMPPA/GMPPB. Data shown in c, d, and e represent mean ± s.d. of 3 biologically independent experiments.

Extended Data Fig. 2 Cryo-EM image processing of GMPPA/GMPPB complex bound to GDP-Man.

a, A representative raw cryo-EM image. b, Representative two-dimensional class averages of the GMPPA/GMPPB particles bound to GDP-Man. c, Image-processing workflow, including 3D classification, structural refinement, masked-based refinement, CTF refinement and Bayesian polishing. d, Gold-standard FSC curve of the cryo-EM map. e, Final local resolution estimation of the density map.

Extended Data Fig. 3 Cryo-EM image processing of GMPPA/GMPPB complex bound to GTP.

a, A representative raw cryo-EM image. b, Representative two-dimensional class averages of the GMPPA/GMPPB particles bound to GTP. c, Image-processing workflow of the GMPPA/GMPPB particles bound to GTP. d, Gold-standard FSC curve of the final density maps of both state I (orange) and state II (sky blue). e, Final local resolution estimation of the cryo-EM maps of state I (left) and state II (right).

Extended Data Fig. 4 Sequence alignment of GMPPA and GMPPB subunits from different species.

Alignment of GMPPA and GMPPB sequences from Homo sapiens, Danio rerio and Schizosaccharomyces pombe. The sequence is numbered according to the GMPPA sequence of Homo sapiens.

Extended Data Fig. 5 Structural comparison GMPPA, GMPPB and their bacterial homologs.

a, Comparison between different conformations of GMPPA subunits. The left panel shows the structures of A1 and A2. A1 and A2 are coloured blue and blown, respectively. The right panel is the superimposition of A1 and A2 and the difference between two structures are highlighted red in A2. b, Magnified view of the boxed region in panel a for the A1 conformation. c, Magnified view of the boxed region in panel a for the A2 conformation. d, Superimposition of four GMPPB subunits (B1, B2, B3, B4). The loop between β7 and β8 display up to 5 Å shift. In the conformation of B1, this loop interacts with the C-loop of GMPPA1. e, Structural comparison of GMPPA/GMPPB heterodimer and T. maritima GMP homodimer (PDB: 2X5Z). f, Structures of GMPPB (left upper); ST0452N-Acetylglucosamine-1-Phosphate Uridyltransferase (PDB: 5Z0A) from Sulfurisphaera tokodaii (right upper); glucose-1-phosphate thymidylyltransferase (PDB: 2GGO) from Sulfurisphaera tokodaii (left bottom). Structural comparison of the three proteins (right bottom). g, Diagrams of domain organization of GMPPA and GMPPB. The β strands and α helices are represented as arrows and cylinders, respectively.

Extended Data Fig. 6 Detail interactions of type I and type III interfaces.

a, Detail interactions of type I interface between GMPPB subunits. Detail interactions of type I interface between A1 and A2, which is largely mediated by hydrophobic and hydrophobic interactions (left). b, Detail interactions of type III interface. The N-loops are shown in red dash line. c,d, The indicated GST-tagged GMPPA (c) or GMPPB (d) constructs and HA-GMPPA-WT (left) or HA-GMPPB-WT (right) were co-transfected in HEK293T cells. Total cell lysates were precipitated with glutathione-Sepharose beads. Bound samples were analyzed by immunoblotting using antibodies against GST and HA. Shown are representative gels from three biologically independent experiments.

Source data

Extended Data Fig. 7 Determination of GMPPB and GMPPA/GMPPB complex kinetic constants.

a, Lineweaver Burk double reciprocal plots 1/V = f(1/[GTP]) of GMPPB and GMPPA/GMPPB complex. The forward reactions were performed at 37 °C for 4 minutes. The enzyme activities were analyzed with a range of GTP concentrations (7.5~150 μM) and a fixed Man-1-P concentration (150 μM). b, Lineweaver Burk double reciprocal plots 1/V = f(1/[man-1-P]) of GMPPB and GMPPA/GMPPB complex. Reactions were performed at 37 °C for 4 minutes, similar to a. The enzyme activities were analyzed with a range of Man-1-P concentrations (7.5~150 μM) and a fixed GTP concentration (150 μM). c, Lineweaver Burk double reciprocal plots 1/V = f(1/[GDP-man]) of GMPPB and GMPPA/GMPPB complex. The reverse reactions were performed at 37 °C for 5 minutes. The enzyme activities were analyzed with a range of GDP-Man concentrations (7.5~150 μM) and a fixed PPi concentration (150 μM). d, Kinetic constants (Km, Kcat, Kcat /Km) of GMPPB and GMPPA/B complex for Man-1-P, GTP, and GDP-Man. e, Effect of GDP-Man on the initial velocity (V0) of GMPPB and GMPPA/GMPPB. Reactions containing 2 μM GMPPB or 10 μM GMPPA/GMPPB, 150 μM GTP, 150 μM man-1-P, and a range of GDP-Man concentrations (0~320 μM), were carried out at 37 °C for 4 minutes. The reactions were then terminated, and the amount of PPi produced was determined. The V0 at each GDP-Man concentration was normalized against V0, 0 (V0 in the absence of GDP-Man). Data shown in a, b, c, and e represent mean ± s.d. of 3 biologically independent experiments.

Extended Data Fig. 8 Depletion of GMPPA in HEK293T cells increased GDP-mannose level.

a, HEK293T cells were lentiviral transduced with a vector encoding shRNA sequence against GMPPA (shPPA) or an control vector (shControl). Two cell sublines with different knock-down effects were selected, shPPA1 and shPPA2. The levels of GMPPA and GADPH were analyzed by immunoblotting. b, Chromatographic separation and measurement of GDP-hexose by HPLC-MS/MS. Pure GDP-mannose and GDP-fucose were used as standards. c, GDP-mannose amount in 1 × 106 HEK293T shControl and shPPA1/2 cells. d, GDP-mannose concentrations calculated from data in (c). Data shown in c and d represent mean ± s.d. of 3 biologically independent experiments.

Source data

Extended Data Fig. 9 Depletion of GMPPA and GMPBB in zebrafish embryos.

a, Gmppb (left) and gmppa (right) protein levels in control and morphant embryos at 1 dpf. Embryos that were injected with control mo (Control, 5 ng), gmppb mo (5 ng) + p53 mo (5 ng) (PPB-MO), or gmppa mo1 (2.5 ng) + mo2 (2.5 ng) + p53 mo (5 ng) (PPA-MO) at the stage of one cell. GAPDH was used as a control for protein amount. b-e, Embryos were injected with control mo (Control, 5 ng), gmppb mo (5 ng) + p53 mo (5 ng) (PPB-MO), gmppb mo (5 ng) + p53 mo (5 ng)+ GDP-Man (1 fmol/embryo) (PPB-MO + GDP-Man), and then analyzed for GDP-Man amount (b), glycosylation pattern (c), and Sox2 expression (d), at 3 hpf. For experiments in b and c, yolk was removed before analysis. c. Glycosylation was determined by biotinylated Lens culinaris lectin (LCA), followed by detection with streptavidin-conjugated horseradish peroxidase. d. SOX2 expression was determined by real-time PCR. Data in b and d represents mean ± s.d. of 3 biologically independent experiments. Results were evaluated by two-tailed unpaired t tests (**P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant). e, Different doses of GDP-Man from 0 fmol to 20,000 fmol was injected into zebrafish embryos, and number of live and dead embryos at 1 dpf were recorded. ‘n’ represents the number of zebrafish embryos per group. f, Tg [hb9: GFP]ml2 transgenic zebrafish was treated as in (e), and the length of CaP motor neurons was analyzed. Representative blots obtained from one out of three independent experiments were shown in a and c.

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Zheng, L., Liu, Z., Wang, Y. et al. Cryo-EM structures of human GMPPA–GMPPB complex reveal how cells maintain GDP-mannose homeostasis. Nat Struct Mol Biol 28, 1–12 (2021). https://doi.org/10.1038/s41594-021-00591-9

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