Fig. 1. Details of POPC translocation from the outward-facing transmembrane cavity of ABCA1 through the gateway/annulus complex into the elongated hydrophobic cavity.

The outward-facing transmembrane cavity, TMD-1 and TMD-2 (orange), ECD-1 (peach), and ECD-2 (light blue) and the elongated hydrophobic tunnel (green) were identified in the structure of ABCA1¹³. The elongated hydrophobic tunnel in ECD-1 was demarcated using PyMol cavity algorithm. A Gateway (residues 564–592, magenta) and annulus (residues 69, 71–80, 363, 368–379, cyan). The annulus forms the bottom of the elongated hydrophobic tunnel. Shown is the pathway for diffusion of POPC from the outer leaflet of the membrane bilayer (blue lines) into the outward-facing transmembrane cavity (yellow arrow). B Final frame after 2 µs simulation of the ECDs and TMDs in a POPC bilayer (all-atom model). Five POPC molecules (termed the membrane mound, yellow space-filling) diffused from the outer membrane leaflet into the outward-facing transmembrane cavity (yellow arrow, A) and then displaced ~10–12 Å above the plane of the bilayer into the outward-facing transmembrane cavity. The gateway is the 29-residue charged loop of ECD-1 that binds the membrane mound in CGMD simulations. This loop is contiguous with the outward-facing transmembrane cavity on one side and the annulus of the elongated hydrophobic tunnel. C Location in the gateway of four of the eight Tangier disease point mutations identified in the ECD-1 of human ABCA1. D–F SMD Freeze-frames of the translocation of a single POPC molecule up to and through the annulus and partway into the elongated hydrophobic tunnel. The gateway and annulus are colored magenta and cyan, respectively. D The 1.9 μsec all-atom frame from the 10 μsec CGMD simulation of ABCA1 embedded in a POPC bilayer was the starting structure. The annulus orifice (residues 73-75, 77, 78, 371, 375) is shown in white. E The POPC was translocated by SMD to the annulus orifice. F The POPC then was translocated halfway through the annulus orifice. This process required significant energy in the SMD (Supplementary Fig. 4). To be energetically favorable in vivo, we propose that the outward-facing transmembrane cavity would close, likely from an ATP-dependent process, forcing the PL through a modified orifice.