The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells

Abstract

Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in Escherichia coli cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells.

On the interplay between lipids and asymmetric dynamics of an NBS degenerate ABC transporter

“…g ., for P-gp reconstituted either in detergents or in nanodiscs 11 . However, such assumptions should be carefully considered given the recent resolution of an NBS degenerate ABC transporter adopting wide open IF conformation by means of Cryo-EM using nanobodies 34 . The NBD1 13 amino-acid deletion, such as the absence of the “ball-and-socket” structure for NBD1, might weaken the coupling between NBD1 and TMH10/TMH11.…”

“…Wide-open structures observed in cryo-EM experiments were thus believed to be due to artifacts owing to the use of non-physiological environments for structure resolution 7 , in agreement with structural differences observed e.g., for P-gp reconstituted either in detergents or in nanodiscs 11 . However, such assumptions should be carefully considered given the recent resolution of an NBS degenerate ABC transporter adopting wide open IF conformation by means of Cryo-EM using nanobodies 34 . The NBD1 13 amino-acid deletion, such as the absence of the "ball-and-socket" structure for NBD1, might weaken the coupling between NBD1 and TMH10/TMH11.…”

On the interplay between lipids and asymmetric dynamics of an NBS degenerate ABC transporter

“…This might also explain why IF-to-OF transitions were experimentally observed even in absence of ATP molecules 5 . However, such assumptions should be carefully considered given the recent resolution of an NBS degenerate ABC transporter adopting wide open IF conformation by means of cryo-EM using nanobodies 33 . Our results highlight the importance of NBD and NBS asymmetry as an evolution of ABC transporters which may result from energy saving while keeping transport function by requiring a single ATP hydrolysis.…”

Ultra-Fast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids

“…Consequently, most incell EPR studies to date have relied on the delivery of in vitro purified and spin-labeled proteins back into cells by microinjection, [12][13][14][15][16] electroporation, 6,17,18 osmotic shock, 19 or other permeation techniques. [20][21][22][23] The site-specific incorporation of non-canonical amino acids (ncAAs) by genetic code expansion (GCE) is a powerful method that has been utilized to introduce novel bioorthogonal chemistries into proteins. 24 Previous studies using GCE for spin-labeling have employed ketone condensations, [25][26][27][28][29] 3+2 azide-alkyne cycloadditions, 17,30,31 Suzuki-Miyaura couplings, 32 quinone methide Michael additions, 33 and inverse electron demand Diels-Alder (IEDDA) cycloadditions.…”