Niemann-Pick disease type C (NPC) is associated with mutations in NPC1 and NPC2, whose gene products are key players in the endosomal/lysosomal egress of low-density lipoprotein-derived cholesterol. NPC1 is also the intracellular receptor for Ebola virus (EBOV). Here, we present a 4.4 Å structure of full-length human NPC1 and a low-resolution reconstruction of NPC1 in complex with the cleaved glycoprotein (GPcl) of EBOV, both determined by single-particle electron cryomicroscopy. NPC1 contains 13 transmembrane segments (TMs) and three distinct lumenal domains A (also designated NTD), C, and I. TMs 2-13 exhibit a typical resistance-nodulation-cell division fold, among which TMs 3-7 constitute the sterol-sensing domain conserved in several proteins involved in cholesterol metabolism and signaling. A trimeric EBOV-GPcl binds to one NPC1 monomer through the domain C. Our structural and biochemical characterizations provide an important framework for mechanistic understanding of NPC1-mediated intracellular cholesterol trafficking and Ebola virus infection.

ABCA1, an ATP-binding cassette (ABC) subfamily A exporter, mediates the cellular efflux of phospholipids and cholesterol to the extracellular acceptor apolipoprotein A-I (apoA-I) for generation of nascent high-density lipoprotein (HDL). Mutations of human ABCA1 are associated with Tangier disease and familial HDL deficiency. Here, we report the cryo-EM structure of human ABCA1 with nominal resolutions of 4.1 Å for the overall structure and 3.9 Å for the massive extracellular domain. The nucleotide-binding domains (NBDs) display a nucleotide-free state, while the two transmembrane domains (TMDs) contact each other through a narrow interface in the intracellular leaflet of the membrane. In addition to TMDs and NBDs, two extracellular domains of ABCA1 enclose an elongated hydrophobic tunnel. Structural mapping of dozens of disease-related mutations allows potential interpretation of their diverse pathogenic mechanisms. Structural-based analysis suggests a plausible "lateral access" mechanism for ABCA1-mediated lipid export that may be distinct from the conventional alternating-access paradigm.

The Hedgehog (Hh) pathway involved in development and regeneration is activated by the extracellular binding of Hh to the membrane receptor Patched (Ptch). We report the structures of human Ptch1 alone and in complex with the N-terminal domain of human Sonic hedgehog (ShhN) at resolutions of 3.9 and 3.6 angstroms, respectively, as determined by cryo-electron microscopy. Ptch1 comprises two interacting extracellular domains, ECD1 and ECD2, and 12 transmembrane segments (TMs), with TMs 2 to 6 constituting the sterol-sensing domain (SSD). Two steroid-shaped densities are resolved in both structures, one enclosed by ECD1/2 and the other in the membrane-facing cavity of the SSD. Structure-guided mutational analysis shows that interaction between ShhN and Ptch1 is steroid-dependent. The structure of a steroid binding-deficient Ptch1 mutant displays pronounced conformational rearrangements.

The Hedgehog (Hh) pathway controls embryonic development and postnatal tissue maintenance and regeneration. Inhibition of Hh receptor Patched (Ptch) by the Hh ligands relieves suppression of signaling cascades. Here, we report the cryo-EM structure of tetrameric Ptch1 in complex with the palmitoylated N-terminal signaling domain of human Sonic hedgehog (ShhN p ) at a 4:2 stoichiometric ratio. The structure shows that four Ptch1 protomers are organized as a loose dimer of dimers. Each dimer binds to one ShhN p through two distinct inhibitory interfaces, one mainly through the N-terminal peptide and the palmitoyl moiety of ShhN p and the other through the Ca 2+ -mediated interface on ShhN p . Map comparison reveals that the cholesteryl moiety of native ShhN occupies a recently identified extracellular steroid binding pocket in Ptch1. Our structure elucidates the tetrameric assembly of Ptch1 and suggests an asymmetric mode of action of the Hh ligands for inhibiting the potential cholesterol transport activity of Ptch1.

The SREBP pathway controls cellular homeostasis of sterols. The key players in this pathway, Scap and Insig-1/2, are membrane-embedded sterol sensors. 25-hydroxycholesterol (25HC)-dependent association of Scap and Insigs acts as the master switch for the SREBP pathway. Here, we present cryo-EM analysis of the human Scap and Insig-2 complex in the presence of 25HC, with the transmembrane (TM) domains determined at an average resolution of 3.7 Å. The sterol sensing domain (SSD) in Scap and all six TMs in Insig-2 were resolved. A 25HC molecule is sandwiched between the S4-S6 segments in Scap and TMs 3/4 in Insig-2 in the luminal leaflet of the membrane. Unwinding of the middle of the Scap-S4 segment is crucial for 25HC binding and Insig association.

Highlights d Cryo-EM structure of human Scap/Insig-2 complex in the presence of digitonin d The luminal L1 and L7 domains of Scap interact in the complex structure d Scap(D428A) exhibits the same conformation as WT in the complex with Insig-2 d Scap(D428A) suppresses SREBP activation even in the absence of Insig binding

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The sterol regulatory element-binding protein (SREBP) pathway senses the cellular cholesterol level through sterol regulated association between Scap and Insig. Despite the recent structural determination of the transmembrane domains of human Scap and Insig-2 bound to 25-hydroxycholesterol (25HC), the structure and regulatory mechanism of the luminal domains of Scap by cholesterol remains elusive. Here, combining cryo-EM analysis and artificial intelligence-facilitated structural prediction, we report the structure of the human Scap/Insig-2 complex in the presence of digitonin instead of 25HC. Despite the lack of sequence similarity, the structure of the luminal domain Loop 1 and a co-folded segment in Loop 7 of Scap resembles that of the luminal/extracellular domain in NPC1 and related proteins. Comparison of the sterol-loaded structures of these proteins provides clues of the regulation of Loop 1/7 interaction by cholesterol. We also show that the structure of Scap(D428A), which suppresses SREBP activation under sterol depletion, is identical to WT when complexed with Insig-2, although the gain of function may also involve a later step in protein trafficking.