Structural basis of the mechanism and inhibition of a human ceramide synthase

nature structural & molecular biology Article https://doi.org/10.1038/s41594-024-01414-3 Structural basis of the mechanism and inhibition of a human ceramide synthase Received: 22 November 2023 Accepted: 1 October 2024 Published online: xx xx xxxx Tomas C. Pascoa 1 , Ashley C. W. Pike 1, Christofer S. Tautermann Gamma Chi 1, Michael Traub2, Andrew Quigley 1,4, Rod Chalk1, Saša Štefanić 3,5, Sven Thamm2, Alexander Pautsch2, Elisabeth P. Carpenter 1 , Gisela Schnapp 2 & David B. Sauer 1 , 2 Check for updates Ceramides are bioactive sphingolipids crucial for regulating cellular metabolism. Ceramides and dihydroceramides are synthesized by six ceramide synthase (CerS) enzymes, each with specificity for different acyl-CoA substrates. Ceramide with a 16-carbon acyl chain (C16 ceramide) has been implicated in obesity, insulin resistance and liver disease and the C16 ceramide-synthesizing CerS6 is regarded as an attractive drug target for obesity-associated disease. Despite their importance, the molecular mechanism underlying ceramide synthesis by CerS enzymes remains poorly understood. Here we report cryo-electron microscopy structures of human CerS6, capturing covalent intermediate and product-bound states. These structures, along with biochemical characterization, reveal that CerS catalysis proceeds through a ping-pong reaction mechanism involving a covalent acyl–enzyme intermediate. Notably, the product-bound structure was obtained upon reaction with the mycotoxin fumonisin B1, yielding insights into its inhibition of CerS. These results provide a framework for understanding CerS function, selectivity and inhibition and open routes for future drug discovery. Ceramides are the precursors for the synthesis of complex sphingolipids and are bioactive signaling lipids. In particular, ceramides have been proposed as key metabolic sensors to promote fatty acid use and storage during excessive fatty acid availability1. Abnormal ceramide accumulation is associated with metabolic dysfunction and elevated levels of ceramides have been observed in obesity-related metabolic disorders such as diabetes, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH)2–4. Ceramides are composed of a sphingosine (d18:1) long-chain base with an N-linked acyl chain, the length of which is critical to the lipids’ biological functions and roles in pathophysiology5. For example, C16:0 ceramide is the most common ceramide in adipose tissue and its levels are elevated in this tissue of obese humans2. In addition, insulin resistance is correlated with plasma C16:0 and C18:0 ceramides, subcutaneous adipose tissue C16:0 ceramides and hepatic C16:0 and C18:0 ceramides6–8. Moreover, total ceramides and C16:0, C22:0 and C24:1 dihydroceramides were found to be elevated in the liver of insulin-resistant patients with NASH3. In mammals, de novo synthesis of ceramides is preceded by synthesis of dihydroceramides through the N-acylation of the sphingoid long-chain base sphinganine (dihydrosphingosine; d18:0) by one of six ceramide synthases (CerS1–CerS6)9. Alternatively, CerS can directly reacylate recycled sphingosine in the salvage pathway10. Of these enzymes, recent observations from human and mouse studies Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, UK. 2Boehringer Ingelheim Pharma, GmbH & Co. KG, Biberach, Germany. 3Institute of Parasitology, Vetsuisse and Medical Faculty, University of Zürich, Zürich, Switzerland. 4Present address: Membrane Protein Laboratory, Research Complex at Harwell, Diamond Light Source, Ltd., Harwell Science and Innovation Campus, Didcot, UK. 5Present address: Nanobody Service Facility, University of Zürich, AgroVet-Strickhof, Lindau, Switzerland. e-mail: ; ; ; 1 Nature Structural & Molecular Biology Article https://doi.org/10.1038/s41594-024-01414-3 b a N LH ER Nb22 6c 6c 3 1a 3 6b 4 2 90° 1a 7 CerS6 1b Cyto 5b 6a 2 7 4 75 Å 6a 1b 5b C CH3 CH3 CH1 LH CH1 CH2 CH2 Hox-like domains 70 Å c N LH 2 1a 3 4 5a 6b LH 7 His211 5b TM1a 6a TM4 TM3 Cyto 1b CH1 N TM6c TM6b His211 TM6a C CH3 1.5 CerS6 CerS6 + Palm +238.45 Da 43,738.56 1.0 43,500.11 0.5 0 43,450 43,600 43,750 43,900 Deconvoluted mass (AMU) TM5b TM7 CH2 Intensity (counts) (×104) ER e d 6c C Fig. 1 | Cryo-EM structure of human CerS6. a, Cryo-EM map of the CerS6 dimer (blue) with one copy of Nb22 (gray) bound to each CerS6 monomer. b, Overall cartoon cylinder representation of the CerS6 dimer structure. One of the monomers is rainbow-colored from purple (N terminus) to red (C terminus). LH, luminal helix. c, Schematic representation of the CerS6 seven-TM helix topology. d, Cartoon representation of the transmembrane domain of a CerS6 monomer. The covalent acyl–imidazole species is shown in stick representation (acyl chain, pink carbon atoms) and the Coulombic potential map for this covalent species is shown as a pink transparent surface. The Hox-like domain was omitted for clarity. e, Denaturing intact protein MS analysis of purified CerS6 protein, revealing the presence of a covalent modification (+238.45 Da), which matches the expected mass shift corresponding to covalent attachment of a palmitoyl (C16:0) chain. This adduct peak was present in all purifications tested (n = 6). AMU, atomic mass units. Cyto, cytoplasm. highlighted CerS6 as a target for treating obesity-associated metabolic disease, including type 2 diabetes and NASH2,5,11–13. Deletion of CerS6 in mice granted protection against diet-induced obesity, steatohepatitis and insulin resistance2,13 and liver-specific deletion improved glucose tolerance and mitochondrial morphology13. Strikingly, this occurs in CerS6Δ/Δ mice but not in CerS5Δ/Δ mice, although both show a preference for C16-CoA and, therefore, primarily produce C16 ceramides14, and hepatic C16:0 ceramide levels were reduced in both knockouts13. Resulting from the specific interaction of CerS6-derived C16:0 sphingolipids in mitochondria with the mitochondrial fission factor13, this revealed that the subcellular localization of ceramide production can lead to drastically different physiological outcomes13. In further support of the therapeutic potential of inhibiting CerS6, ablation of the protein’s expression in an obese insulin resistance mouse model led to improved body fat, oral glucose tolerance and insulin sensitivity11. Central to their enzymatic function, CerSs contain a TRAM– Lag1–CLN8 (TLC) homology domain15, including a 52-residue Lag1p motif with conserved histidines and aspartates required for activity16,17. CerS2–CerS6 also contain a nonessential Hox-like domain between transmembrane (TM) domains 1 and 2 (ref. 18), flanked by two essential and conserved positively charged residues19. Regulators of CerS activity include phosphorylation20, glycosylation21, dimerization22 and other protein–protein interactions23,24. The six mammalian CerSs also have (...truncated)