Sphingolipids are essential lipids in eukaryotic membranes. In humans, the first and rate-limiting step of sphingolipid synthesis is catalyzed by the serine palmitoyltransferase holocomplex, which consists of catalytic components (SPTLC1 and SPTLC2) and regulatory components (ssSPTa and ORMDL3). However, the assembly, substrate processing and regulation of the complex are unclear. Here, we present 8 cryo-electron microscopy structures of the human serine palmitoyltransferase holocomplex in various functional states at resolutions of 2.6–3.4 Å. The structures reveal not only how catalytic components recognize the substrate, but also how regulatory components modulate the substrate-binding tunnel to control enzyme activity: ssSPTa engages SPTLC2 and shapes the tunnel to determine substrate specificity. ORMDL3 blocks the tunnel and competes with substrate binding through its amino terminus. These findings provide mechanistic insights into sphingolipid biogenesis governed by the serine palmitoyltransferase complex.
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The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession codes EMD-22598, EMD-22599, EMD-22600, EMD-22601, EMD-22602, EMD-22604, EMD-22605, EMD-22606, and EMD-22608. The corresponding atomic models have been deposited in the Protein Data Bank under accession codes PDB 7K0I, PDB 7K0J, PDB 7K0K, PDB 7K0L, PDB 7K0M, PDB 7K0N, PDB 7K0O, PDB 7K0P, and PDB 7K0Q. Source data are provided with this paper.
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We thank L. Tang and W. Guo at the Cryo-EM Center of St. Jude Children’s Research Hospital for support with data collection and computer infrastructure. We thank C. Kalodimos, S. Blanchard, M. Halic, J. Sun, X. Li, M. Hattori, W. Lü, C. Zhao, J. Lee, and F. Liu for helpful discussions. We thank Z. Luo for assistance with the cartoons. This work was supported by ALSAC.
The authors declare no competing interests.
Peer review information Nature Structural & Molecular Biology thanks Binks Wattenberg, Ming Zhou, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Florian Ullrich and Anke Sparmann were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Summary of image processing procedures of the SPT complex dataset. b, Angular distribution of particles for the final 3D reconstructions. c, Fourier shell correlation (FSC) curves: half map 1 versus half map 2 (black) and model versus summed map (blue). d, Local resolution of cryo-EM maps. In (b), (c), and (d), top panels show the reconstruction of the whole complex and bottom panels show the reconstruction after symmetry expansion and signal subtraction (single protomer). e and f, Cryo-EM map of the SPT complex. In (f), the map is unsharpened and low-pass filtered to show the weaker density of the C-terminal helix of ssSPTa. The identity of the lipids (purple) can not be determined at this resolution.
a, Structure of the cytosolic domains of human SPTLC1 and SPTLC2. For clarity, only one local dimer is shown. b, Structure of the serine palmitoyltransferase from Sphingomonas paucimobilis (SpSPT, PDB 2JG2). c, Overlay of the human SPTLCs and their bacterial homolog. d, Structural comparison of human SPTLCs and their bacterial homolog in the active site.
Extended Data Fig. 3 Analysis of SPTLC2 mutations on key residues involved in the dimeric interface.
a, Representative fluorescence-detection size-exclusion chromatography profiles showing that SPTLC2 Arg302Ala, Arg302Ala-Arg305Ala or Arg302Ala-Arg304Ala-Arg305Ala considerably decreased the dimer population. b, SPT activity measured from cells. d18:0, sphinganine. d18:0 P, sphinganine phosphate. d18:1, sphingosine. Newly synthesized sphingolipids were indicated by deuterium-labeled serine (d2) (mean ± SD; n = 3). c, SPT activity measured from microsomes. (mean ± SD; n = 3). Data for graphs in b and c are available as source data.
Extended Data Fig. 4 Cryo-EM reconstructions and ligand-protein interactions of the SPT complex bound to 3KS or myriocin.
(a to c) SPT-complex bound to 3KS. (d to f) SPT-complex bound to myriocin. a and d, Angular distribution of particles for the final 3D reconstructions. b and e, Fourier shell correlation (FSC) curves: half map 1 versus half map 2 (black) and model versus summed map (blue). c and f, Local resolution of cryo-EM maps. g, Scheme of interactions between 3KS, SPTLC1 (orange), and SPTLC2 (blue). 3KS and PLP are colored black. Dashed lines represent hydrogen bonds and spokes represent hydrophobic interactions. h, Scheme of interactions between myriocin, SPTLC1 (orange), and SPTLC2 (blue). Myriocin and PLP are colored black. i, Densities of 3KS and surrounding residues. j, Densities of myriocin and surrounding residues.
a, Summary of image processing procedures of the SPT-ORM complex dataset. b, Angular distribution of particles for the final 3D reconstruction (class 1). c, Fourier shell correlation (FSC) curves (class 1): half map 1 versus half map 2 (black) and model versus summed map (blue). d, Local resolution of the cryo-EM map (class 1). e, Cryo-EM map of the SPT-ORM complex. f, Cryo-EM structure of ORMDL3. Four transmembrane helices of ORMDL3 are labeled as S1 to S4. The N- and C- terminus of the ORMDL3 are highlighted by spheres. Lipid-like densities were observed around S1 and S2 (lipid 1 and 2), and between S1 and S3 (lipid 3). The identity of the lipids cannot be determined at this resolution. g, Zoomed-in views of densities of lipids and surrounding residues.
SPTLC1 β sheet 1: residues 382–387, 393–398, 443–448. SPTLC1 β sheet 2: 205–209, 184–188, 239–245, 270–274, 302–306, 316–320, 160-164. SPTLC2 β sheet 1: 458–472, 507–511, 493–497. SPTLC2 β sheet 2: 275–281, 253–259, 308–315, 339–344, 372–377, 386–391, 230–235.
Extended Data Fig. 7 Functional analysis of the SPTLC1 mutation disrupting the interface between the SPTLC S1 helix and ORMDL3.
a to e, Sphingolipid contents from cells were measured as an indication of the SPT activity. SPTLC1 ∆S1 mutant is as active as wild type, but the regulation from ORMDL3 is considerably impaired. Representative results are shown (mean ± SD; n = 2). The experiment was repeated multiple times yielding similar results. Data are available as source data.
a to c, SPT-ORM complex (class 2). d to e, SPT-ORM complex (class 3). g to i, SPT-ORM complex (class 4). (a, d, and g) Angular distribution of particles for the final 3D reconstructions. (b, e, and h) Fourier shell correlation (FSC) curves: half map 1 versus half map 2 (black) and model versus summed map (blue). (c, f, and i) Local resolution of cryo-EM maps.
a to c, Two structures of SPT-ORM are overlaid on the left protomer (white) to demonstrate the structural differences of the other protomer (blue or yellow). (a) class 1 versus class 2. (b) class 1 versus class 3. (c) class 1 versus class 3. d, Conformational changes of the membrane dimeric interface among the four structures.
a, Angular distribution of particles for the final 3D reconstruction. b, Fourier shell correlation (FSC) curves: half map 1 versus half map 2 (black) and model versus summed map (blue). c, Local resolution of the cryo-EM map.
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Wang, Y., Niu, Y., Zhang, Z. et al. Structural insights into the regulation of human serine palmitoyltransferase complexes. Nat Struct Mol Biol 28, 240–248 (2021). https://doi.org/10.1038/s41594-020-00551-9
Nature Structural & Molecular Biology (2021)