A structural framework for unidirectional transport by a bacterial ABC exporter

Abstract

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

Molecular insights into the human ABCB6 transporter

“…Previously Atm1/ABCB7/HMT1/ABCB6 subfamily model built by NaAtm shows that apo-form contains only one cavity for substrate binding 31 , 32 . In the apo-state, hABCB6 adopts an inward-facing conformation and TMDs of hABCB6 contain the substrate-binding cavity which is divided into two parts: a closed cavity 2 and an open-form cavity 1 (Fig.…”

Does the ATP‐bound EQ mutant reflect the pre‐ or post‐ATP hydrolysis state in the catalytic cycle of human P‐glycoprotein (ABCB1)?

“…The sequence of events in the transmembrane region resulting in the efflux of the substrate remains to be clearly elucidated. Structural studies of the bacterial transporters Atm1 and TmrAB have shown that the conformational landscape of these transporters is rather complex [3,4]. Figure 5A is a schematic representation of a possible mechanism showing conformational changes that occur in WT and EQ mutant P-gp during the ATP hydrolysis cycle and the Table in Fig.…”

“…In contrast, in the IC conformation the NBDs dimerize, and the transmembrane region is rearranged, expelling the substrate from the cell [1]. It is clear that this is an oversimplified model and that transporters actually undergo many different conformational changes during the ATP‐coupled transport cycle, as recently shown for the bacterial transporters Atm1 and TmrAB, for which at least eight different conformations were solved using X‐ray crystallography and cryo‐electron microscopy (cryo‐EM), respectively [3,4]. For instance, it has been proposed that the ADP and vanadate (V i )‐trapped conformation of P‐gp resembles the transition state during the hydrolysis of ATP [5], yet such a conformation has yet to be solved.…”

Molecular basis of cholesterol efflux via ABCG subfamily transporters

“…G1 assumes a closed conformation when ATP binds the NBS, which collapses site 1 while simultaneously pushing the neutral sterol substrate to the extracellular cavity, where it binds extracellular lipid acceptors or enters the outer hemileaflet of the bilayer. Upon extracellular cholesterol release and ATP hydrolysis, the transporter returns to the resting state, ready to transport another cholesterol molecule ( 35 , 42 – 44 ). Further structural analysis of G5G8 will be required to determine how ATP hydrolysis can rearrange the TMHs so that it flips from an inward- to an outward-facing state, as has been found to occur in other ABC transporters.…”