Probing the ATP Hydrolysis Cycle of the ABC Multidrug Transporter LmrA by Pulsed EPR Spectroscopy

Abstract

Members of the ATP binding cassette (ABC) transporter superfamily translocate various types of molecules across the membrane at the expense of ATP. This requires cycling through a number of catalytic states. Here, we report conformational changes throughout the catalytic cycle of LmrA, a homodimeric multidrug ABC transporter from L. lactis. Using site-directed spin labeling and pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy, we have probed the reorientation of the nucleotide binding domains and transmembrane helix 6 which is of particular relevance to drug binding and part of the dimerization interface. Our data show that LmrA samples a very large conformational space in its apo state, which is significantly reduced upon nucleotide binding. ATP binding but not hydrolysis is required to trigger this conformational change, which results in a relatively fixed orientation of both the nucleotide binding domains and transmembrane helices 6. This orientation is maintained throughout the ATP hydrolysis cycle until the protein cycles back to its apo state. Our data present strong evidence that switching between two dynamically and structurally distinct states is required for substrate translocation.

Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA

“…However, ATP binding and ATP hydrolysis were also reported to cause MD separation in MsbA and homologs (8,9,11,12,29). To investigate the effects of substrate binding on the conformation of MsbA at the MDs, we used our previously described cysteine reporter at the extracellular end of the MDs, A281C in MsbA-cl.…”

“…ADP⅐V i (hydrolysis intermediate)-trapped MsbA was also observed in the outwardfacing conformation, similar to the AMP-PNP-bound structure (7). Biochemical verifications of these conformations include electron paramagnetic resonance (EPR) studies on MsbA (8 -10) and LmrA (11), hydrogen/deuterium exchange-mass spectrometry on BmrA (12), inter-molecular cysteine crosslinking on MsbA in membrane vesicles (13,14), and most recently, by luminescence resonance energy transfer on MsbA (15).…”

Conformational cycle and ion-coupling mechanism of the Na + /hydantoin transporter Mhp1

“…This methodology has been successfully applied to define coupled conformational cycles for a number of transporter classes (13,(26)(27)(28)(29)(30)(31)(32). We find that patterns of distance distributions between pairs of spin labels monitoring the intra-and extracellular sides of Mhp1 are consistent with isomerization between the crystallographic inward-and outward-facing conformations.…”

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain

“…Furthermore, the inward-facing conformation has been biochemically validated in bacterial ABC exporters, using electron paramagnetic resonance on multicopy suppressor of Htrb mutations (MsbA) (13) and LmrA (14), cysteine cross-linking of MsbA (15), and hydrogen/deuterium exchange coupled to mass spectrometry of BmrA (16). The inward-facing conformation is a key intermediate in the alternating access mechanism (7), as it allows the transporter to scan the inner leaflet of the membrane for substrates.…”