Structure of the human lipid exporter ABCB4 in a lipid environment

Abstract

ABCB4 is an ATP-binding cassette transporter that extrudes phosphatidylcholine into the bile canaliculi of the liver. Its dysfunction or inhibition by drugs can cause severe, chronic liver disease or drug-induced liver injury. We determined the cryo-EM structure of nanodisc-reconstituted human ABCB4 trapped in an ATP-bound state at a resolution of 3.2 Å. The nucleotide binding domains form a closed conformation containing two bound ATP molecules, but only one of the ATPase sites contains bound Mg2+. The transmembrane domains adopt a collapsed conformation at the level of the lipid bilayer, but we observed a large, hydrophilic and fully occluded cavity at the level of the cytoplasmic membrane boundary, with no ligand bound. This indicates a state following substrate release but prior to ATP hydrolysis. Our results rationalize disease-causing mutations in human ABCB4 and suggest an 'alternating access' mechanism of lipid extrusion, distinct from the 'credit card swipe' model of other lipid transporters.

Structural and functional diversity calls for a new classification of ABC transporters

“…The directions of substrate transport are indicated by solid and dashed red arrows. The structures have the following Protein Data Bank (PDB) accession codes: MalFGK 2 ‐MalE: 2R6G [12]; BtuC 2 D 2 ‐BtuF: 4FI3 [50]; EcfTAA’‐FolT: 4HUQ [14]; Sav1866: 2HYD [15]; TmrAB: 5MKK [51]; TM287/288: 4Q4H [52]; McjD: 4PL0 [53]; PCAT1: 6V9Z [54]; Atm1: 4MYH [55]; MRP1: 5UJA [56]; PrtD: 5L22 [57]; P‐gp: 4M1M [58]; TAP1/2: 5U1D [59]; ABCB4: 6S7P [60]; ABCB8: 5OCH; ABCB10: 3ZDQ [61]; ABCB11: 6LR0 [62]; MsbA: 5TV4 [63]; PglK: 6HRC [64]; YbtPQ: 6P6J [31]; IrtAB: 6TEJ [32]; Rv1819c: 6TQF [33]; ABCD4: 6JBJ [30]; CFTR: 5UAK [65]; SUR1: 6BAA [66]; Wzm‐WztN: 6OIH [25]; TarGH: 6JBH [26]; ABCG5/8: 5DO7 [16]; ABCG2: 6HCO [67]; ABCA1: 5XJY [23]; LptB 2 FG: 5X5Y [17]; MacB: 5LJ7 [21]. ABC, ATP‐binding cassette; β‐jr, β‐jellyroll‐like domain; C, C terminus; CH, coupling helix; CoH, connecting helix; EH, elbow helix; N, N terminus; NBD, nucleotide‐binding domain; P2, extracytoplasmic loop; PG, periplasmic gate helix; PLD, periplasmic domain; TMD, transmembrane domain.…”

Cryo‐EM of ABC transporters: an ice‐cold solution to everything?

“…Therefore, the ability to image membrane proteins, embedded in lipid nanodisks [98], seems to be very appealing. This system provides a lipid environment, which is considered to be close to native, while still allowing for high-resolution structure determination, as demonstrated by several examples in the ABC-transporter field [7,8,25,56] (Fig. 2).…”

“…A range for ABC transporters are now becoming common even without such fiducials [19,22,23,26,42,[44][45][46][47]49,51,56,91,92]. Therefore, it is preferable to avoid antibodies whenever possible to minimize the risk of locking the transporter in a nonphysiological conformation.…”

“…Such technology has been shown to enable high‐resolution characterization of as small membrane proteins as, for example, 50‐kDa malaria parasite transporter [18] and has also been successfully applied to several ABC transporters [8,24,54]. On the other hand, resolutions in the 3.1–3.5 Å range for ABC transporters are now becoming common even without such fiducials [19,22,23,26,42,44–47,49,51,56,91,92]. Therefore, it is preferable to avoid antibodies whenever possible to minimize the risk of locking the transporter in a nonphysiological conformation.…”

“…As such, cryo-EM enabled the analysis of ABC transporters that defied crystallography, such as the cystic fibrosis transmembrane conductance regulator (CFTR) [6,[19][20][21][22][23] and ABCG2 [7,[24][25][26], also known as breast cancer resistance protein (BCRP). Moreover, the low sample amount requirements in cryo-EM provide the opportunity to study low-abundant, native, fragile, or flexible complexes [27][28][29][30][31][32][33][34], while the high tolerance to various hydrophobic environments [35][36][37][38][39][40][41] enables structure determination of ABC transporters not only in different detergents [5,6,28,29,[42][43][44][45][46][47][48][49][50][51][52], but also within a lipid environment [8,24,[53][54][55][56]. Additionally, cryo-EM can dissect confo...…”