Structure and mechanism of a eukaryotic ceramide synthase complex

Ceramide synthases (CerS) catalyze ceramide formation via N-acylation of a sphingoid base with a fatty acyl-CoA and are attractive drug targets for treating numerous metabolic diseases and cancers. Here, we present the cryo-EM structure of a yeast CerS complex, consisting of a catalytic Lac1 subunit and a regulatory Lip1 subunit, in complex with C26-CoA substrate. The CerS holoenzyme exists as a dimer of Lac1-Lip1 heterodimers. Lac1 contains a hydrophilic reaction chamber and a hydrophobic tunnel for binding the CoA moiety and C26-acyl chain of C26-CoA, respectively. Lip1 interacts with both the transmembrane region and the last luminal loop of Lac1 to maintain the proper acyl chain binding tunnel. A lateral opening on Lac1 serves as a potential entrance for the sphingoid base substrate. Our findings provide a template for understanding the working mechanism of eukaryotic ceramide synthases and may facilitate the development of therapeutic CerS modulators. imageIn eukaryotes, ceramide synthase is a multispan enzyme in the endoplasmic reticulum membrane. Here, structural and biochemical studies of the yeast Lac1-Lip1 complex provide new insight into its substrate binding and molecular mechanism.The cryo-EM structure of the yeast Lac1-Lip1 complex bound to C26-CoA substrate reveals that the holoenzyme exists as a dimer of heterodimers.Lac1 contains a hydrophilic reaction chamber and a hydrophobic tunnel that bind the CoA moiety and C26-acyl chain of C26-CoA, respectively.Lip1 interacts with both the transmembrane region and the last luminal loop of Lac1 to maintain the acyl chain binding tunnel.A lateral opening in the membrane-embedded region of Lac1 serves as a potential entry site for the sphingoid base substrate. A structure of the yeast Lac1-Lip1 sphingosine N-acyltransferase complex reveals how a dimer of heterodimers binds its substrate.image