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Structural basis of the acyl-transfer mechanism of human GPAT1

Nature Structural & Molecular Biology volume 30, pages 22–30 (2023)Cite this article

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Abstract

Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop–helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.

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Fig. 1: Architecture of human GPAT1.
Fig. 2: Molecular basis of GPAT1 mitochondrial targeting.
Fig. 3: Binding of the palmitoyl-CoA-mimetic inhibitor 2-oxohexadecyl-CoA.
Fig. 4: Binding of the products CoA and palmitoyl-LPA.
Fig. 5: The membrane gate pocket and its role in lipid trafficking.

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Data availability

The sequence of the WT human GPAT1 protein is available in UniProt under the accession code Q9HCL2. The cryo-EM reconstructions for human GPAT1 bound to 2-oxohexadecyl-CoA and CoA–palmitoyl-LPA have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-27898 and EMD-27899, respectively. The coordinates for the models have been deposited in the Protein Data Bank (PDB) under accession codes 8E4Y (2-oxohexadecyl-CoA) and 8E50 (CoA–palmitoyl-LPA). Source data are provided with this paper.

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Acknowledgements

We thank K. Huynh for helpful discussions on electron microscopy data collection and B. Kapinos, E. Ralph, G.S. Walker, J. Adams, L. Zhang, P.R. Verhoest, R. Kurumbail and T.V. Magee for scientific discussions. D.J.W. was supported by a Pfizer Postdoctoral Fellowship. Some molecular graphic images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). The authors received no specific funding for this work.

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Authors and Affiliations

  1. Discovery Sciences, Medicine Design, Pfizer Inc., Groton, CT, USA

    Zachary Lee Johnson, Mark Ammirati, David Jonathan Wasilko, Jeanne S. Chang, Stephen Noell, Timothy L. Foley, Mary A. Piotrowski, Kathleen A. Farley, Lisa M. Aschenbrenner, Kimberly F. Fennell, Mary Xu, Chunyang Guo, Alison E. Varghese, Justin Bellenger, Alandra Quinn, Graham M. West, Matthew C. Griffor, Matthew Calabrese, Claire M. Steppan, Seungil Han & Huixian Wu

  2. Internal Medicine Research Unit, Pfizer Inc., Cambridge, MA, USA

    Hyejin Yoon, Kathleen Smith, Shoh Asano, Katherine Hales, Min Wan & Tamara Gilbert

  3. Internal Medicine, Medicine Design, Pfizer Inc., Cambridge, MA, USA

    Qingyi Yang

  4. Internal Medicine, Medicine Design, Pfizer Inc., Groton, CT, USA

    Jason K. Dutra & Christopher W. am Ende

  5. Biostatistics, Early Clinical Development, Pfizer Inc., Cambridge, MA, USA

    Donald Bennett

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Contributions

Z.L.J. optimized protein purification for cryo-EM, prepared cryo-EM grids, collected and processed cryo-EM data, performed de novo model building, determined the structure of the 2-oxohexadecyl-CoA complex and assisted with writing the manuscript. M.A. collected cryo-EM data. D.J.W. performed cloning, protein expression, purification and thermal shift experiments, collected and processed cryo-EM data, determined the structure of the CoA–palmitoyl-LPA complex and assisted with activity studies. J.S.C. performed protein purification for cryo-EM and LC–MS studies. S.N. designed and performed activity studies. T.L.F. designed and synthesized 2-oxohexadecyl-CoA and 2-oxo-3,3-dimethylbutyl-CoA. H.Y. performed confocal imaging studies. K.S. transfected, fixed and stained cells for confocal imaging studies. S.A. optimized and performed confocal imaging studies. K.H. supervised confocal imaging studies and analyzed data. M.W. supervised cell preparation for confocal imaging studies. Q.Y. performed MD simulations. M.A.P. performed lipid extraction and LC–MS studies. K.A.F. performed NMR characterization of the synthesized acyl-CoA analogs. T.G. assisted with confocal imaging studies. L.M.A. assisted with activity studies. K.F.F. performed protein expression. J.K.D., M.X. and C.G. assisted with LPA-binding confirmation. A.E.V. assisted with protein-purification optimization. J.B. performed purification and QC of the synthesized acyl-CoA analogs. A.Q. performed HR MS characterization of synthesized acyl-CoA analogs. C.W.a.E. supervised synthesis experiments. G.M.W. assisted with MS data analysis. M.C.G. performed cloning. D.B. assisted with confocal imaging data analysis. M.C. assisted with structural data analysis. C.M.S. supervised activity studies. S.H. supervised cryo-EM laboratory operation. H.W. conceived, designed and supervised the project, analyzed the results and wrote the manuscript. All authors analyzed and discussed the results and contributed to the manuscript.

Corresponding author

Correspondence to Huixian Wu.

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The authors were all employees of Pfizer Inc. at the time when the work was performed.

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Extended data

Extended Data Fig. 1 Purification and characterization of GPAT1 protein for functional and structural studies.

a, Representative SEC profiles for GPAT1 WT (blue) and the cryo-EM construct GPAT1(Δ1−79) (green) and SDS-PAGE of SEC fractions for WT GPAT1. Purification of the constructs was repeated more than 5 times independently with similar results. b, Specific activity of purified GPAT1. The G3P-free control was subtracted to calculate specific GPAT1 activity in all subsequent experiments. Data in mean ± s.d. derived from technical duplicates from one representative experiment are shown. The experiment was repeated at least 3 times independently with similar results. c, Comparison of specific activities of WT and GPAT1(Δ1−79) at a protein concentration of 12.5 nM. Data are mean ± s.d. derived from 4 independent repeats. d, Initial rate of reaction versus G3P concentrations using purified WT GPAT1. The initial rates in various concentrations of G3P can be fit with the Michaelis-Menten equation, producing a Vmax of 3.0 ± 1.5 (nM sec−1 nM−1) and a Michaelis constant (Km) of 24 ± 10 mM for G3P derived from n = 6 independent experiments. e, Initial rate of reaction versus palmitoyl-CoA concentrations using purified WT GPAT1. The initial rates in various concentrations of palmitoyl-CoA would not fit well with the traditional Michaelis-Menten equation but could be fit with the Michaelis-Menten substrate inhibition equation, producing a Vmax of 4.8 ± 1.0 (nM sec−1 nM−1), a Michaelis constant (Km) of 18 ± 7 μM and a Ki of 48 ± 40 μM for palmitoyl-CoA derived from n = 6 independent experiments. Data in mean ± s.d. derived from technical duplicates from one representative experiment are shown in d and e. f, Inflection temperatures (Ti) of WT GPAT1 by measuring the tryptophan fluorescence ratio at 350 nm/330 nm in the presence of palmitoyl-CoA at specified concentrations. Data are mean ± s.d. derived from 4 independent repeats. g, Chemical structures of palmitoyl-CoA and the non-hydrolysable acyl-CoA analogs used in the study. The uncropped gel image for a and numerical data for graphs in b−f are available as source data.

Source data

Extended Data Fig. 2 Cryo-EM structure determination for the 2-oxohexadecyl-CoA complex.

a, Flow chart of cryo-EM data processing, including a representative micrograph and representative 2D class averages showing distinct views and features of the complex. b, Final cryo-EM reconstruction colored by local resolution. c, Fourier shell correlation (FSC) curve indicates an overall nominal resolution of 3.40 Å using the gold-standard FSC=0.143 criterion. Numerical data for c are available as source data.

Source data

Extended Data Fig. 3 Cryo-EM structure determination for the CoA/palmitoyl-LPA complex.

a, Flow chart of cryo-EM data processing, including a representative micrograph and representative 2D class averages showing distinct views and features of the complex. b, Final cryo-EM reconstruction colored by local resolution. c, Fourier shell correlation (FSC) curve indicates an overall nominal resolution of 3.67 Å using the gold-standard FSC=0.143 criterion. Numerical data for c are available as source data.

Source data

Extended Data Fig. 4 Quality of the cryo-EM maps.

a-b, Models and corresponding cryo-EM density for representative regions of the 2-oxohexadecyl-CoA (a) and the CoA/palmitoyl-LPA (b) complex structures. Figures were prepared using UCSF Chimera.

Extended Data Fig. 5 Secondary structure diagram and sequence alignment of human GPAT1 with mammalian and bacterial GPAT orthologs.

The protein sequences were extracted from UniProt accession numbers Q9HCL2 from Homo sapiens (GPAT1_HUMAN); Q61586 from Mus musculus (GPAT1_MOUSE); P97564 from Rattus norvegicus (GPAT1_RAT); P0A7A7 from Escherichia coli (PLSB_ECOLI, amino acids 50–805); and P44857 from Haemophilus influenzae (PLSB_HAEIN, amino acids 30–804). Protein sequences are denoted as conserved sequences (*), conservative mutations (:), semi-conservative mutations (.), and non-conservative mutations (no symbol). Secondary structural elements corresponding to the 3D structure of human GPAT1 are shown above the alignment, with structural domains colored in the same scheme as Fig. 1. Black dashed lines are residues that were not included in the cryo-EM construct, whereas the colored dashed lines are regions that were included in the construct but not modeled in the structure. Cylinders and arrows represent α-helices and β-strands, respectively. Membrane-associating sequences are highlighted in gray. Catalytic amino acids are in red text. The four conserved sequence blocks are enclosed in black boxes.

Extended Data Fig. 6 Comparison of the human GPAT1 structure with published acyltransferase structures.

a-d, Structures of the acyltransferase domain (NTD) of human GPAT1 (a), squash GPAT (PDB ID 1K30) (b), phosphatidyl-myo-inositol mannosides acyltransferase A from M. smegmatis (PatA, PDB ID 5F34) (c), and 1-acyl-sn-glycerol-3-phosphate acyltransferase from Thermotoga maritima (PlsC, PDB ID 5KYM) (d). Side chains of the catalytic dyad residues are shown as sticks.

Extended Data Fig. 7 SEC profiles of purified GPAT1 mutant proteins.

a, Analytical SEC profiles of WT and GPAT1(Δ1–79) in comparison with different C-terminal truncation constructs. b-f, SEC profiles of various GPAT1 constructs with different N-terminal truncations (b), or mutations at positions indicated in each graph (c-f). Monomeric GPAT1 protein is indicated by an asterisk (*) in each figure.

Extended Data Fig. 8 Mapping of the previously proposed transmembrane regions onto the GPAT1 structure.

a-b, Regions corresponding to previously proposed transmembrane segments TM1 (a) and TM2 (b) are mapped onto the human GPAT1 structure and shown in white surface representation. As revealed by the structure, these two regions are not embedded in the membrane; instead, they contain residues mostly from the CTD (α15, α16, α19, and α20) and are about 12–22 Å away from the membrane.

Extended Data Fig. 9 The membrane gate pocket for palmitoyl-LPA binding and egress.

a, Root mean square fluctuation (RMSF) of the membrane-associating helices α6, α9, and α12 during two independent MD simulation runs. To calculate the RMSF, MD trajectories were first superposed based on GPAT1 protein backbone heavy atoms. Positions of the heavy atoms of amino acids from α6 (residues 183–200), α9 (residues 264–271), and α12 (residues 379–390) obtained in all MD trajectories were used for RMSF calculations. b-c, Lipid-like cryo-EM density peaks in the membrane gate pocket in the 2-oxohexadecyl-CoA structure (b) and the CoA/palmitoyl-LPA structure (c). d, LC profiles of selected ion chromatograms of palmitoyl-LPA (top) and palmitoyl-d9-LPA (internal standard, bottom) extracted from the GPAT1–2-oxohexadecyl-CoA sample. e, MS spectra of LC fraction at the retention time of 1.65 ± 0.1 min in d (top) and the corresponding fraction from blank buffer run (bottom). The m/z peak corresponding to the endogenous palmitoyl-LPA is highlighted in red. f-g, Tandem mass spectrometry (MS/MS) spectra of ions with m/z 409.236 (f) and m/z 418.290 (g) identified from LC–MS. Fragmentation profiles of the ions further confirmed the selected m/z ions are palmitoyl-LPA and palmitoyl-d9-LPA, respectively. Chemical structures of palmitoyl-LPA and palmitoyl-d9-LPA are shown in f and g, respectively. Only ions corresponding to palmitoyl-LPA or palmitoyl-d9-LPA fragmentation are labeled for clarity. LC–MS analyses were performed in two independent experiments with different extraction methods using either methanol or 1-butanol (see Methods). Similar LC–MS profiles and MS spectra were observed using both methods. Only liquid chromatography–tandem mass spectrometry (LC–MS/MS) data from the 1-butanol extraction method are shown. h, Palmitoyl-LPA binds to the membrane gate pocket with a short residence time. The coordinates of GPAT1 with a palmitoyl-LPA molecule bound to the membrane gate pocket as shown in Fig. 5a was used as the starting model for simulations. Distances between the mass center of the LPA aliphatic chain and the center of the membrane gate along the axis perpendicular to the membrane plane observed in MD simulations are plotted against simulation time. Data from two independent simulations are shown. In both runs, the palmitoyl-LPA dropped out from the membrane gate at around 250 ns. Data for graphs in a and h are available as source data.

Source data

Extended Data Fig. 10 Comparison of the GPAT1 cryo-EM structure with the AlphaFold model.

a-d, Overlays of the GPAT1–2-oxohexadecyl-CoA cryo-EM structure (teal) with the AlphaFold model (UniProt ID Q9HCL2, firebrick red) in Cα ribbon representation from four different views. The unmodeled regions of the cryo-EM structure are also hidden in the AlphaFold model for clarity. e, Differences observed between the cryo-EM structure and the predicted AlphaFold model at the membrane gate helices, α12 and α9, as well as at selected side chains in the catalytic site and the membrane gate pocket. The highlighted residues, R278, R279, R328, and H363 are shown in sticks. Polar interactions between R278 and R328 observed in the cryo-EM structure are indicated by dashed lines. The small molecules, 2-oxohexadecyl-CoA in the catalytic site and palmitoyl-LPA in the membrane gate pocket resolved in the cryo-EM structure are shown in green and light gray sticks, respectively.

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Reporting Summary

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Supplementary Table 1

Mutagenesis primer sequences.

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Johnson, Z.L., Ammirati, M., Wasilko, D.J. et al. Structural basis of the acyl-transfer mechanism of human GPAT1. Nat Struct Mol Biol 30, 22–30 (2023). https://doi.org/10.1038/s41594-022-00884-7

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