Probing the allosteric NBD-TMD crosstalk in the ABC transporter MsbA by solid-state NMR.

The ABC transporter MsbA plays a critical role in Gram-negative bacteria in the regulation of the outer membrane by translocating core-LPS across the inner membrane. Additionally, a broad substrate specificity for lipophilic drugs has been shown. The allosteric interplay between substrate binding in the transmembrane domains and ATP binding and turnover in the nucleotide-binding domains must be mediated via the NBD/TMD interface. Previous studies suggested the involvement of two intracellular loops called coupling helix 1 and 2 (CH1, CH2). Here, we demonstrate by solid-state NMR spectroscopy that substantial chemical shift changes within both CH1 and CH2 occur upon substrate binding, in the ATP hydrolysis transition state, and upon inhibitor binding. CH2 is domain-swapped within the MsbA structure, and it is noteworthy that substrate binding induces a larger response in CH2 compared to CH1. Our data demonstrate that CH1 and CH2 undergo structural changes as part of the TMD-NBD cross-talk.

MsbA: an ABC transporter paradigm.

ATP-binding cassette (ABC) transporters play an important role in various cellular processes. They display a similar architecture and share a mechanism which couples ATP hydrolysis to substrate transport. However, in the light of current data and recent experimental progress, this protein superfamily appears as multifaceted as their broad substrate range. Among the prokaryotic ABC transporters, MsbA can serve as a paradigm for research in this field. It is located in the inner membrane of Gram-negative bacteria and functions as a floppase for the lipopolysaccharide (LPS) precursor core-LPS, which is involved in the biogenesis of the bacterial outer membrane. While MsbA shows high similarity to eukaryotic ABC transporters, its expression in Gram-negative bacteria makes it conveniently accessible for many experimental approaches from spectroscopy to 3D structure determination. As an essential protein for bacterial membrane integrity, MsbA has also become an attractive target for the development of novel antibiotics. Furthermore, it serves as a model for multidrug efflux pumps. Here we provide an overview of recent findings and their relevance to the field, highlight the potential of methods such as solid-state NMR and EPR spectroscopy and provide a perspective for future work.