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. 2023 Jun 13;14:3475. doi: 10.1038/s41467-023-39274-y

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

Fig. 4

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.