Cryo-EM structure of ABCG5/G8 in complex with modulating antibodies

Abstract

The heterodimer of ATP-binding cassette transporter ABCG5 and ABCG8 mediates the excretion of sterols from liver and intestine, playing a critical role in cholesterol homeostasis. Here, we present the cryo-EM structure of ABCG5/G8 in complex with the Fab fragments from two monoclonal antibodies at 3.3Å resolution. The high-resolution structure reveals a unique dimer interface between the nucleotide-binding domains (NBD) of opposing transporters, consisting of an ordered network of salt bridges between the conserved NPXDFXXD motif and serving as a pivot point that may be important for the transport cycle. While mAb 11F4 increases the ATPase activity potentially by stabilization of the NBD dimer formation, mAb 2E10 inhibits ATP hydrolysis, likely by restricting the relative movement between the RecA and helical domain of ABCG8 NBD. Our study not only provides insights into the structural elements important for the transport cycle but also reveals novel epitopes for potential therapeutic interventions.

Ins and outs of AlphaFold2 transmembrane protein structure predictions

“…5 a). An ABCG5/ABCG8 structure with a correct registry was also released on 2021-04-07 (PDBID: 7jr7 [ 32 ]), but AF2 template search for building models deposited at EBI used pdb70 downloaded on 2021-02-10 [ 5 ].

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Binding mode analysis of ABCA7 for the prediction of novel Alzheimer's disease therapeutics

“…This was demonstrated for other ABC transporters as well, e.g. , ABCB1 [102] , ABCB2 [103] , ABCB6 [122] , ABCB11 [123] , ABCC7 [124] , ABCC8 [105] , ABCG2 [106] , and ABCG5/G8 [125] , forming either an inward-facing [105] , [106] , [122] , [123] , [124] , [125] or (drug-induced) occluded [102] , [103] conformation. In contrast, most ABC transporters in the ATP-bound state are not able to recognize substrates, as shown for ABCB4 [104] , as these form an outward-facing conformation.…”

Structural analysis of cholesterol binding and sterol selectivity by ABCG5/G8

“…4A/B ). As current apo or ATP-bound structures of ABCG sterol transporters are rather limited to early and end states of catalysis (Lee et al, 2016; Skarda et al, 2021; Sun et al, 2021; Zhang et al, 2021), the sterol poses as observed here may represent the post-efflux conformations as the hydrophobic valve has already been resealed in these ATP-bound structures. For this reason, binding sites within the protein were unable to be detected and subsequently docked.…”

“…Three X-ray diffraction datasets were integrated and scaled with acceptable statistics. The initial model was obtained by molecular replacement using a previously resolved cryo-EM structure (PDB ID: 7JR7) (Zhang et al, 2021), and the motifs involved in ATP binding were adjusted based on the previous crystal structure (PDB ID: 5DO7) (Lee et al, 2016). Two ABCG5/G8 heterodimers are observed in the asymmetric unit.…”

“…The ABC transporter superfamily translocate a broad spectrum of substrates across cellular membranes, with individual members conveying their unique substrate specificities. To determine how ABCG transporters bind sterol ligands, we used previously published apo models ABCG5/G8 (PDB ID: 5DO7 or 7JR7) and ABCG1 (PDB ID: 7R8D) (Lee et al, 2016; Zhang et al, 2021) and carried out molecular docking on the TMD with cholesterol or stigmasterol, a plant sterol previously shown to have the highest accumulation rate in an Abcg5/g8-deficient mouse model (Yu et al, 2004). Using UCSF DOCK6 suite (Allen et al, 2015), we first optimized the docking protocol by carrying out a redocking of the ligands (topotecan, mitoxantrone and imatinib) to reproduce their complex with ABCG2.…”