Project Summary/Abstract The goal of this project is to understand at the atomic level how cholesterol modulates integral membrane protein signaling. We seek to address this question in two physiologically important biological pathways, cholesterol homeostasis by the SREBP regulatory machinery and hormone signaling by G protein-coupled receptors (GPCRs). The SREBP machinery centers on Scap, a membrane protein that resides in the endoplasmic reticulum. Scap binds to the SREBP transcription factors and controls their maturation in a cholesterol-dependent manner. There are currently no atomic structures of any vertebrate Scap, and little is known at a detailed biophysical level about Scap's cholesterol binding or conformational changes. Like Scap, GPCRs bind ligands (e.g. hormones, neurotransmitters, and lipids) and undergo conformational changes that trigger intracellular signaling. Multiple GPCR X-ray structures have revealed cholesterol binding interactions, however the functional significance of these interactions is largely unknown. We found that the CB1 cannabinoid receptor can bind allosteric modulators at a membrane-embedded surface that overlaps with its cholesterol binding site. This finding suggests that cholesterol may be an allosteric modulator of CB1 as well as other GPCRs. We are studying the interactions of cholesterol with Scap and GPCRs to better understand how this essential lipid can control diverse functions through these different membrane proteins. We will use cryo-EM to determine the atomic structure of a full-length vertebrate Scap, either bound to its co-regulatory membrane protein Insig or a soluble fragment of SREBP2. These structures will elucidate the architecture of this lipid-sensing machine and provide an atomic basis for cholesterol regulation. In parallel, we will obtain the first NMR spectroscopy data and cryo- EM structural data for GPCRs embedded in native-like phospholipid nanodiscs, with and without cholesterol. These data will reveal whether embedded cholesterol can stabilize particular conformations of a GPCR, and whether cholesterol interactions are maintained in G protein signaling complexes. We propose to use this diverse array of biophysical techniques to obtain unprecedented atomic data on cholesterol regulation of these systems, and our findings may be used to help develop drugs that modulate Scap or different GPCRs through the membrane.