Ceramide sensing by human SPT-ORMDL complex for establishing sphingolipid homeostasis

Abstract

The ORM/ORMDL family proteins function as regulatory subunits of the serine palmitoyltransferase (SPT) complex, which is the initiating and rate-limiting enzyme in sphingolipid biosynthesis. This complex is tightly regulated by cellular sphingolipid levels, but the sphingolipid sensing mechanism is unknown. Here we show that purified human SPT-ORMDL complexes are inhibited by the central sphingolipid metabolite ceramide. We have solved the cryo-EM structure of the SPT-ORMDL3 complex in a ceramide-bound state. Structure-guided mutational analyses reveal the essential function of this ceramide binding site for the suppression of SPT activity. Structural studies indicate that ceramide can induce and lock the N-terminus of ORMDL3 into an inhibitory conformation. Furthermore, we demonstrate that childhood amyotrophic lateral sclerosis (ALS) variants in the SPTLC1 subunit cause impaired ceramide sensing in the SPT-ORMDL3 mutants. Our work elucidates the molecular basis of ceramide sensing by the SPT-ORMDL complex for establishing sphingolipid homeostasis and indicates an important role of impaired ceramide sensing in disease development.

Fig. 1. Purified human SPT-ORMDL complexes can be inhibited by C6-ceramide.

a Overview of the de novo sphingolipid biosynthetic pathway and the homeostatic regulation of SPT activity by ceramide in an ORMDL-dependent manner. b, c Coomassie blue-stained SDS-PAGE gel (b) and the SPT activity (c) of purified SPT and various SPT-ORMDL3 complexes. ORMDL3* stands for the ORMDL3 construct from our previous study (ref. ), which appears to express a mixture of minor full-length ORMDL3 protein (ORMDL3-FL) and dominant N-terminus truncated ORMDL3 protein. The newly made SPT-ORMDL3 (ORMDL3-FL) complex could be inhibited by C6-ceramide, whereas the SPT, SPT-ORMDL3*, and SPT-ORMDL3 (ORMDL3-ΔN17) complexes had no response to C6-ceramide. Data in (c) are presented as mean values ± SEM of three independent experiments. d SPT activity versus L-serine concentration measured using SPT or SPT-ORMDL3 complex. Data are presented as mean values ± SEM of three independent experiments. The data points were fitted with a Michaelis-Menten equation. e SPT activity versus palmitoyl-CoA concentration for SPT and SPT-ORMDL3 complex. Data are presented as mean values ± SEM of three independent experiments. The data points were fitted with an allosteric sigmoidal equation. f The inhibition curves of human SPT and SPT-ORMDL complexes by C6-ceramide in the in vitro SPT activity assay. Data are presented as mean values ± SEM of three independent experiments. All the estimated IC₅₀ values were summarized in Supplementary Table 1. g Selective inhibition of SPT-ORMDL3 by C6-ceramide among various sphingolipid species or analogs. Data are presented as mean values ± SEM of three independent experiments. The different sphingolipid species or analogs were applied at 10 μM in each assay. Source data are provided as a Source Data file.

Fig. 2. Structure determination of the C6-ceramide-bound SPT-ORMDL3 complex.

a The cryo-EM map and overall structure of the monomeric C6-ceramide-bound SPT-ORMDL3 complex. SPTLC1 and SPTLC2 are shown in light blue and light green, respectively; SPTssa and ORMDL3 are shown in orange and light yellow, respectively; SPTLC1’ is shown in cyan; the lipid-like density is shown in pink (left panel) or blue (right panels). Insets: The lipid-like density, shown in blue meshes, were contoured at 5σ or 3.5σ. C6-ceramide and C24-ceramide are shown as pink sticks; the acyl chains are shown as gray sticks. b, c Comparison of the EM maps (b) and structures (c) of the apo SPT-ORMDL3* complex and C6-ceramide-bound SPT-ORMDL3 complex. Apo* stands for the apo SPT-ORMDL3* complex used in our previous study. The apo SPT-ORMDL3* complex (EMD-30080 and PDB 6M4O) is colored gray. The C6-ceramide-bound SPT-ORMDL3 complex is colored based on the subunits. The ORMDL3-N11 (the 11 residues at the N-terminus) and C6-ceramide in the C6-ceramide-bound SPT-ORMDL3 complex are shown as sticks, and the corresponding maps are colored semi-transparent magenta and pink, respectively (b, zoomed-in view, right). In contrast, the densities for the ORMDL3-N11 and C6-ceramide are not visible in the apo SPT-ORMDL3* complex (b, zoomed-in view, left). Met1 and Pro12, the resolved N-terminus of ORMDL3 and ORMDL3*, are shown as yellow and gray spheres, respectively (c).

Fig. 3. The ceramide binding site in SPT-ORMDL3 complex.

a A close-up view of the density map for the ceramide binding site contoured at 6σ. b Detailed views of the ceramide binding site. Residues involved in the interactions are shown as sticks. All potential polar interactions are indicated by red dashed lines. c Functional characterization of the key residues involved in ceramide binding by SPT activity assay. Data are presented as mean values ± SEM of three independent experiments. d, e Inhibition curves of the SPT-ORMDL3 ceramide-binding variants by C6-ceramide. Curves for the variants mediating the polar interactions and hydrophobic interactions were presented in (d) and (e), respectively. Data are presented as mean values ± SEM of three independent experiments. Source data are provided as a Source Data file.

Fig. 4. The N-terminus of ORMDL3 is essential for SPT inhibition.

a The resolved N-terminus of ORMDL3 in the C6-ceramide-bound SPT-ORMDL3 map. The ORMDL3-N11 (the 11 residues at the N-terminus) are highlighted in magenta; all the other parts are shown in the same coloring scheme as above. The maps are shown as semi-transparent. b Close-up views of the density map and coordination of ORMDL3-N11. The electron densities for ORMDL3-N11, shown as blue mesh, were contoured at 6σ. The residues that form polar interactions with ORMDL3-N11 in SPTLC1, SPTLC2, and ORMDL3 are shown as sticks, and the potential polar interactions are indicated by red dashed lines. c Functional characterization of ORMDL3 N-terminal deletion variants and N2A variant by SPT activity assay. Data are presented as mean values ± SEM of three independent experiments. d The loss of C6-ceramide-mediated inhibition of SPT activity for the ORMDL3 N-terminal deletion variants and N2A variant. Data are presented as mean values ± SEM of three independent experiments. e Comparison of the EM maps for ORMDL3-N11 (upper) and ceramide (lower) in the apo wild-type (WT) SPT-ORMDL3 complex and the ORMDL3-ΔN2 mutant. The ORMDL3-N11 is no longer visible in the ORMDL3-ΔN2 mutant. The ceramide-like density largely disappeared in the EM map of the ORMDL3-ΔN2 mutant. f Release of the N-terminus of ORMDL3 for substrate binding in the ORMDL3-ΔN2 mutant. The ORMDL3-ΔN2 mutant structure was superimposed with the C6-ceramide-bound WT SPT-ORMDL3 structure and S-CoA-bound SPT-ORMDL3* structure (PDB 7CQK). S-CoA, short for S-(2-oxoheptadecyl)-CoA and a nonreactive analog of palmitoyl-CoA, is displayed as black spheres. The C6-ceramide-bound SPT-ORMDL3 structure is colored gray, and the ORMDL3-ΔN2 mutant structure is colored based on the subunits as above. Asn11, the resolved N-terminus of ORMDL3-ΔN2, is shown as a yellow sphere. g Conformational changes of the ceramide-binding site in the ORMDL3-ΔN2 mutant. The ORMDL3-ΔN2 mutant structure (colored based on the subunits) was superimposed with the C6-ceramide-bound WT SPT-ORMDL3 structure (gray). Source data are provided as a Source Data file.

Fig. 5. Ceramide induces and locks the N-terminus of ORMDL3 into an inhibition conformation and the enzymatic kinetics of two SPT-ORMDL3 variants.

a Superposition of the apo WT SPT-ORMDL3 structure (gray) and the ORMDL3-N13A mutant structure (colored based on the subunits). Met1 and Asn11, the resolved N-terminus of WT ORMDL3 and ORMDL3-N13A, respectively, are shown as spheres. Insets: The EM maps for ORMDL3-N11 (upper) and ceramide-like density (lower) in the apo WT SPT-ORMDL3 and ORMDL3-N13A mutant structures. The ORMDL3-N11 is no longer visible in the ORMDL3-N13A mutant. The ceramide-like density largely disappeared in the EM map of the ORMDL3-N13A mutant. b Conformational changes of the ceramide-binding site and the ORMDL3-N11 coordinating residues in the ORMDL3-N13A mutant structure. The ORMDL3-N13A mutant structure (colored based on the subunits) was superimposed with the C6-ceramide-bound WT SPT-ORMDL3 structure (gray). The residues involved in ORMDL3-N11 coordination are underscored. c Enzymatic kinetics of the ORMDL3-N13A and ORMDL3-ΔN17 variants compared to those of the WT SPT and SPT-ORMDL3 complexes. Data are presented as mean values ± SEM of three independent experiments. Source data are provided as a Source Data file.

Fig. 6. The impaired ceramide sensing by ALS-associated SPTLC1 variants.

a A close-up view of the ALS-associated SPTLC1 variants. b The SPTLC1 ALS variants exhibit little effect on ORMDL3 binding. The SPT-ORMDL3 complexes bearing ALS variants, purified by size exclusion chromatography, were visualized by Coomassie-blue stained SDS-PAGE gel. c Functional characterization of the ALS variants by SPT activity assay. Data are presented as mean values ± SEM of three independent experiments. d The impaired C6-ceramide-mediated SPT-ORMDL3 activity inhibition for the ALS variants. Data are presented as mean values ± SEM of three independent experiments. Source data are provided as a Source Data file.

Fig. 7. A working model for the homeostatic regulation of SPT-ORMDL complex by ceramide and biological implications of the ALS-associated SPTLC1 variants.

At low ceramide levels, the ORMDL N-terminus remains flexible, thus allowing the entry of substrate for SPT catalysis. The constant reaction of SPT-ORMDL would lead to the accumulation of ceramide, which would inhibit SPT-ORMDL by locking the N-terminus of ORMDL3 into an inhibition conformation. The fixed ORMDL N-terminus would block the entry of substrate for further catalysis. The Asn2 in ORMDL is important for maintaining the inactive conformation of the SPT-ORMDL complex. The Asn13 in ORMDL is critical for ceramide binding. The ALS-associated SPTLC1 variants, located near the transmembrane interface between SPTLC1 and ORMDL, impair the ceramide sensing of the SPT-ORMDL mutants. The consequently unregulated SPT activity of ALS variants would result in unrestrained sphingolipid biosynthesis.