Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle

Abstract

This paper is dedicated to the memory of our friend and colleague Andreas Schmidlin P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an~10 A Ê resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding af®nity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P i . Biochemical data suggest that these rearrangements may involve rotation of transmembrane a-helices. A mechanism for transport is suggested.

Structure and function of ABC transporters: the ATP switch provides flexible control

“…Spectroscopic studies of LmrA lead to a similar conclusion [45]. The nature of these conformational changes remains to be elucidated, although there is clearly a substantial repacking of the TMDs throughout the depth of the membrane [5,14]. Experimental evidence suggests that threading of α-helices into and out of the lipid bilayer is unlikely [46].…”

“…Although it has long been predicted from primary sequence data that the TMDs each consist of multiple membrane-spanning α-helices, it was only with the first X-ray structure, for a bacterial lipid transporter MsbA, that this was formally confirmed [7]. Low-to-medium resolution structures of the mammalian multidrug resistance P-glycoprotein (P-gp; ABCB1), obtained by electron microscopy of single particles [13] and electron cryomicroscopy of 2-D crystals [5,14], showed that the TMDs form an enclosed aqueous chamber/pore in the membrane which, in the basal state, appears open at the extracellular face but closed intracellularly, with the two NBDs exposed as cytoplasmic lobes (Fig. 1).…”

“…These conformational changes have been visualised directly for P-gp by 2-D crystallography [5,14]. Comparison of 2-D crystal structures of ATP (AMP-PNP)-bound P-gp with those of P-gp trapped in the posthydrolytic transition state (with vanadate; see below) showed that the major conformational change in the TMDs occurs upon ATP binding and that subsequent ATP hydrolysis and ADP/Pi release introduce more limited changes [5]. Spectroscopic studies of LmrA lead to a similar conclusion [45].…”

“…However, direct measurement of the binding affinity of the drug vinblastine to P-gp has been possible. The use of non-hydrolysable ATP analogues (both AMP-PNP and ATP-γ-S) showed that ATP binding, in the absence of hydrolysis, is sufficient to reduce substratebinding affinity [5,47,48] with little effect on binding capacity or affinity for a modulator which binds to an allosteric site but is not transported, arguing against nonspecific disruption of protein structure. For the cystic fibrosis transmembrane regulator, although initial studies suggested that ATP hydrolysis drives channel opening, a more detailed analysis has revealed that channel opening can be mediated by ATP binding to the NBDs in the absence of hydrolysis [49].…”

“…Vanadate is able to stabilise the NBD dimer with one ATP and one ADP.Vi in a transition state. Biochemical evidence suggests that this is an activated state [52] with a distinct conformation of the TMDs of P-gp, which retains a low affinity for vinblastine [5,53]. Comparison of the high-resolution structures of ATP-and ADP-bound HlyB-NBD [40] suggests that some of the energy of hydrolysis be used to distort a helix (number 6; Fig.…”