Cholesterol homeostasis is controlled by the sterol regulatory element–binding protein (SREBP) pathway. SREBPs (SREBP-1a, -1c, and -2) are transcription factors with an N-terminal transcription factor domain and a C-terminal regulatory domain (CTD) connected by a transmembrane (TM) hairpin. Among the key players in this pathway, two membrane proteins—Scap and Insig (Insig-1 or -2)—together monitor the sterol level in the endoplasmic reticulum (ER) membrane.
SREBP-CTD constitutively interacts with the C-terminal WD40 domain of Scap. The N terminus of Scap comprises eight TM segments, among which S2 to S6 constitute the sterol-sensing domain (SSD). The SSD is also found in several membrane proteins that are involved in cholesterol metabolism or transport. When the cellular cholesterol level surpasses a certain threshold, the SREBP-2–Scap complex is anchored to the ER membrane through a sterol-dependent interaction between the Scap-SSD and Insig. The interaction is more sensitive to some cholesterol derivatives, like 25-hydroxycholesterol (25HC), than to cholesterol. When cholesterol levels drop, Scap dissociates from Insig. The SREBP-2–Scap complex is subsequently translocated by the COPII vesicles to the Golgi, where the transcription factor domain of SREBP-2 is liberated after two sequential proteolytic cleavages and is transported into the nucleus to activate gene expression for cholesterol synthesis and uptake. Despite rigorous characterizations, the molecular basis for sterol-regulated interaction between Scap and Insig remains elusive.
The structure of the Scap-Insig-25HC ternary complex will not only reveal the basis for sterol sensing by Scap and Insig but also facilitate the development of potential therapeutics against viral infections and for cancer treatment. Modern methods of single-particle cryo–electron microscopy (cryo-EM) provide a powerful tool to elucidate the structure of this relatively small and highly dynamic membrane protein complex.
WD40-deleted human Scap (residues 1 to 752) and full-length human Insig-2 were transiently coexpressed in HEK293F cells. Supplementation of 25HC during protein expression and isolation was necessary to maintain an intact complex. For cryo-EM analysis, a guided multireference three-dimensional classification method was combined with Relion and CryoSparc. The TM region was determined at resolutions of 3.3 to 3.9 Å, whereas the luminal domains were of lower resolutions, insufficient for model building. Seven TMs—including the entire SSD—in Scap and all six TMs in Insig-2 were resolved.
TMs 1, 2, 3, and 6 of Insig-2 enclose a hydrophobic pocket in which there is no density corresponding to a sterol. A stretch of density that can perfectly fit 25HC is sandwiched by the two proteins in the luminal leaflet of the membrane. Whereas the binding site is mainly constituted by hydrophobic residues on TMs 3 and 4 of Insig-2 and S4, S5, and S6 of Scap, the 25-OH group at the end of the iso-octanol tail of 25HC is exposed to the cytosolic milieu through a hydrophilic cavity enclosed by Scap and Insig-2, which affords a potential explanation for the preference of 25HC over cholesterol in promoting the interaction between Scap and Insig.
S4 in Scap is broken in the middle, resulting in two half helices, S4a and S4b. Compared with the structures of SSD-containing proteins NPC1 and patched 1, in which S4 is straight, the tilting of S4a toward the interior of the SSD creates the space for 25HC accommodation and for the displacement of S2, which constitutes a major interface with Insig. The sandwiched 25HC functions as more than a molecular glue. Its presence stabilizes the unwound conformation of Scap-S4 that is crucial for Insig association. Scap mutations, such as D428A (Asp428 → Ala) and Q432A (Gln432 → Ala), that may lower the energy penalty for S4 unwinding, allow for complex formation with Insig in the absence of sterols. Therefore, the interdependence of S4 unwinding, 25HC accommodation, and Insig binding establishes the molecular basis for sterol sensing.
The cryo-EM structure of the human Scap and Insig-2 complex bound to 25HC, together with biochemical analyses, shed light on the mechanistic understanding of sterol sensing in the SREBP pathway. Unwinding of Scap-S4 serves as the switch for 25HC binding and Insig association. Our studies also provide structural interpretations for two well-characterized Scap mutations, the gain-of-function D428A and loss-of-function Y298C (Tyr298 → Cys).