Structural mechanisms of Scap/SREBP signaling in membrane homeostasis

Project Summary/Abstract The goal of this project is to understand at the molecular level how cholesterol exerts negative feedback on its own biosynthesis in mammalian cells. Cholesterol is an essential lipid, both serving as a basic building block of cellular membranes and acting as a precursor for steroid hormones. We seek to understand the atomic structures and mechanism of the membrane proteins Scap and Insig, which control SREBP processing and thereby regulate cholesterol biosynthesis. Scap binds to the SREBP transcription factors and controls their maturation in a cholesterol-dependent manner. Insig binds to Scap when cholesterol levels are high and helps to retain Scap and SREBPs in the endoplasmic reticulum (ER), whereas the COPII adapter proteins Sec23/24 binds to Scap when cholesterol levels are low and promotes trafficking to the Golgi apparatus. We use structural biology and biophysical tools to understand at the atomic level how Scap and Insig respond to membrane cholesterol and regulate SREBPs. Our previous work exploited the chicken orthologs of Scap and Insig, as well as a mutation that locks Scap into a cholesterol bound-like state. In these studies, we elucidated a major conformational rearrangement of Scap's transmembrane architecture and ER luminal loop that is stabilized by Insig binding, and therefore presumed to be promoted by high cholesterol. However there are still no structures of Scap/Insig bound by cholesterol itself, to explain how the lipid influences Scap's conformational equilibrium. We will use single-particle cryo-EM methods to elucidate the high-resolution structure of the mammalian Scap/Insig complex bound to cholesterol. Mutagenesis and SREBP functional studies will be used to probe and validate the importance of different bound cholesterol molecules in SREBP regulation. Further we will carry out purification and cryo-EM structure determination of the Scap/Insig complex in the presence of different small molecules that have been proposed as Scap inhibitors. These structures will show how drugs can interact with this ER membrane protein complex to modulate SREBP signaling, and elucidate whether their binding modes overlap with the natural ligand cholesterol. When membrane cholesterol is low, Scap/SREBP complexes are trafficked to the ER through Scap's interaction with COPII. Determining the structure of the Scap/COPII complex is challenging due to the low affinity of this interaction, and no such cargo/COPII complexes have yet been structurally characterized. We will use yeast display selections to isolate nanobodies that stabilize the Scap/COPII complex, and use these reagents to purify the complex in detergent for single-particle cryo-EM analysis. The structure of this complex will show how COPII recognizes Scap's cytosolic surface under low- cholesterol conditions, and further indicate how the interaction depends on the low-cholesterol conformation of Scap. Overall, our molecular dissection of the Scap/SREBP signaling system will show how cholesterol homeostasis is maintained at the atomic level, and provide a framework for future therapeutic development targeting this pathway in metabolic diseases.