Abstract
Lysosomal transmembrane acetylation of heparan sulfates (HS) is catalyzed by HS acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT), whose dysfunction leads to lysosomal storage diseases. The mechanism by which HGSNAT, the sole non-hydrolase enzyme in HS degradation, brings cytosolic acetyl-coenzyme A (Ac-CoA) and lysosomal HS together for N-acyltransferase reactions remains unclear. Here, we present cryogenic-electron microscopy structures of HGSNAT alone, complexed with Ac-CoA and with acetylated products. These structures explain that Ac-CoA binding from the cytosolic side causes dimeric HGSNAT to form a transmembrane tunnel. Within this tunnel, catalytic histidine and asparagine approach the lumen and instigate the transfer of the acetyl group from Ac-CoA to the glucosamine group of HS. Our study unveils a transmembrane acetylation mechanism that may help advance therapeutic strategies targeting lysosomal storage diseases.
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Data availability
The sequence of human HGSNAT is available at the following link: https://www.uniprot.org/uniprotkb/Q68CP4/entry#sequences. The cryo-EM maps and coordinates of apo-HGSNAT, Ac-CoA-HGSNAT, CoA/NAG-HGSNAT and CoA/4MU-NAG-HGSNAT complexes have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-36376, EMD-36386, EMD-36387 and EMD-37264 and in the Protein Data Bank (PDB) under accession codes 8JKV, 8JL1, 8JL3 and 8W4A, respectively. Source data are provided with this paper.
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Acknowledgements
We thank the Cryo-Electron Microscopy Center at the Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry for the help with data collection. We thank ShanghaiTech Cryo-EM Imaging Facility for its assistance in data collection. We thank the Molecular and Cell Biology Core Facility at the School of Life Science and Technology, ShanghaiTech University, for providing technical support. This work was supported by Shanghai Lingang Laboratory (grant nos. LG-QS-202203-03 to J.Y. and LG-QS-202203-05 to J.G.) and Shanghai Pujiang Program (grant no. 22PJ1410300 to J.G.).
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J.Y. and J.G. designed the project. R.X. and Y.N. performed the sample preparation for cryo-EM. R.X., Y.N. and C.G. performed the biochemistry studies. R.X. performed the fluorescence-based acetylation assay. F.R. and Z.Z. carried out MS analysis. J.Y. and J.G. performed the cryo-EM data collection, data analysis and model building. J.Y., J.G., A.V.P. and X.P. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Biochemical characterization and cryo-EM analysis of human HGSNAT.
a. Topology of HGSNAT. Unresolved regions are marked with dashed lines. b. Representative size exclusion chromatography (SEC) profile (left) of HGSNAT and SDS-PAGE gel (right) of HGSNAT in the presence or absence of β-ME. The trace and gel image are representative of 4 experimental replicates. c. A representative cryo-EM micrograph of HGSNAT. (out of 30,545 similar micrographs). d. Selected representative 2D class averages.
Extended Data Fig. 2 A representative flow chart of data processing and representative densities of the HGSNAT map.
a. Flow chart of data processing for Ac-CoA-bound HGSNAT complexes. Details may be found in Methods. b. Cryo-EM densities of the transmembrane helixes in the Ac-CoA-bound HGSNAT complexes.
Extended Data Fig. 3 3D reconstructions of the apo, Ac-CoA-bound, CoA/NAG-bound, and CoA/4MU-NAG-bound HGSNAT complexes.
a, e, j, o. Local resolution estimates of the apo (a), Ac-CoA-bound (e), CoA/NAG-bound (j), and CoA/4MU-NAG-bound (o) HGSNAT maps. b, f, k, p. FSC curves before and after masking and between the model and the final maps of the apo (b), Ac-CoA-bound (f), CoA/NAG-bound (k), and CoA/4MU-NAG-bound (p) HGSNAT. c, g, l, q. Projection orientation distribution of apo (c), Ac-CoA-bound (g), CoA/NAG-bound (l), and CoA/4MU-NAG-bound (q) HGSNAT maps, generated from cryoSPARC. d, h, m, r. The 3DFSC sphericity of the apo (d), Ac-CoA-bound (h), CoA/NAG-bound (m), and CoA/4MU-NAG-bound (r) HGSNAT maps, analyzed with 3DFSC in cryoSPARC. i, n, s. Densities of the substrates determined in the Ac-CoA-bound (i), CoA/NAG-bound (n), and CoA/4MU-NAG-bound (s) HGSNAT complexes. t. Densities of the Met282, Phe313, Lys341, and Arg345 in the apo and Ac-CoA-bound HGSNAT structures to illustrate the repositioning of these four residues upon the binding of Ac-CoA.
Extended Data Fig. 4 Effects of the indicated mutants on the activity of HGSNAT.
a. The relative enzyme activities of the indicated mutants. Data are shown as mean ± standard deviations, n = 3 biologically independent samples. b–s. Size exclusion chromatography profiles of the HGSNAT mutants.
Extended Data Fig. 5 Thermostability of wild type HGSNAT and HGSNAT mutants.
a–d. Melting curves of wild type HGSNAT, indicated HGSNAT mutants and CFP. The CFP tag, located at the C-terminal of the wild type HGSNAT and mutants, was used for protein detection. Data are shown as mean ± standard deviations, n = 3 biologically independent samples. e. The interactions in the NTD-TMD interface. Possible hydrogen bonds are indicated with dashed lines.
Extended Data Fig. 6 LC-MS identification of the endogenous Ac-CoA in HGSNAT.
a. LC profiles of commercial standard Ac-CoA (top) and purified HGSNAT enzyme from the solubilization of the whole cells without any additional dialysis (bottom). b. MS/MS spectrum of commercial standard Ac-CoA (top) and purified HGSNAT enzyme from the solubilization of the whole cells without any additional dialysis (bottom).
Extended Data Fig. 7 Sequence alignment of the HGSNAT across various species.
Secondary elements are labeled above the alignment. The catalytic residues Asn286 and His297 are highlighted and indicated with red stars.
Extended Data Fig. 8 The comparisons of the overall structure and the positions of catalytic site between HGSNAT with lipid-modifying MBOATs.
a–e. The overall structure of HGSNAT (a) in comparison with HHAT (b), Porcupine (c), ACAT1 (d), and DGAT1 (e). The acyl-CoA and hallmark catalytic histidine in each enzyme are indicated and shown in ball and stick style.
Supplementary information
Supplementary Video
Illustrating the transmembrane acetylation reaction catalyzed by HGSNAT.
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Xu, R., Ning, Y., Ren, F. et al. Structure and mechanism of lysosome transmembrane acetylation by HGSNAT. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01315-5
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DOI: https://doi.org/10.1038/s41594-024-01315-5