Structural Characterization of the Sterol-Sensing Domain in the NPC1 and SCAP Membrane Proteins

Abstract

Sterols are essential biomolecules in animals that aid in hormone and bile acid synthesis, and serve as key structural components of the cell membrane. Cholesterol homeostasis heavily regulates cellular levels of sterols, but this biochemical process remains unclear. It is thought to be controlled by several membrane proteins, including Niemann-Pick disease type C-1 (NPC1) protein and sterol regulatory element-binding protein (SREBP)-cleavage-activating protein (SCAP). These proteins’ functions are dependent on a highly-conserved sterol-sensing domain (SSD) that is thought to regulate interactions with sterols. However, there is no direct evidence of the mechanisms of the SSD in the aforementioned proteins. To overcome the inherent challenges of studying membrane proteins, cholesterol, and their exact interactions, single-particle electron microscopy (cryo-EM) was used to provide structural insights on both cholesterol homeostasis and other processes involving SSDs. The goal of the investigation is to capture high-resolution images of NPC1’s SSD binding interface with cholesterol, as well as the sterol-dependent interactions between Insulin induced gene-1 (INSIG1) and SCAP, which also occurs through the SSD. We present several constructs of human SCAP that indicate detectable protein expression levels and stability. Furthermore, a 6.9 angstrom (Å) structure of NPC1 in nanodisc has been resolved with no sterol density indicated in the predicted binding pocket of the SSD. These results provide deeper insights on the complexity and dynamics of proteins involved in cholesterol metabolism and signaling. Optimization of the construct designs and cryo-samples will be necessary in future experiments to ultimately image these proteins and their interactions at the SSD.