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Abstract
We measured the amplitude of conformational motion in the ATP-binding cassette (ABC) transporter MsbA upon lipopolysaccharide (LPS) binding and following ATP turnover by pulse double electron-electron resonance and fluorescence homotransfer. The distance constraints from both methods reveal large-scale movement of opposite signs in the periplasmic and cytoplasmic part of the transporter upon ATP hydrolysis. LPS induces distinct structural changes that are inhibited by trapping of the transporter in an ATP post-hydrolysis intermediate. The formation of this intermediate involves a 33-Å distance change between the two ABCs, which is consistent with a dimerization-dissociation cycle during transport that leads to their substantial separation in the absence of nucleotides. Our results suggest that ATP-powered transport entails LPS sequestering into the open cytoplasmic chamber prior to its translocation by alternating access of the chamber, made possible by 10–20-Å conformational changes.
Highlights
ATP-binding cassette (ABC) transporters harness the free energy of ATP hydrolysis to power the thermodynamically unfavorable trafficking of a wide spectrum of substrates in and out of the cell [1,2,3]
Clinical multidrug resistance in the treatment of bacterial and fungal infections and cancer chemotherapy can result from the expression of pumps that extrude toxic molecules from the cell
A subclass of these pumps—ATP-binding cassette (ABC) transporters—use energy from ATP to remove a wide range of molecules
Summary
ATP-binding cassette (ABC) transporters harness the free energy of ATP hydrolysis to power the thermodynamically unfavorable trafficking of a wide spectrum of substrates in and out of the cell [1,2,3]. Cooperative ATP binding and hydrolysis occur in a molecular motor composed of two ATPbinding and hydrolysis cassettes (ABCs), referred to as nucleotide-binding domains (NBDs) [4]. In Gram-negative bacteria, the transport of lipid A from its site of synthesis across the inner membrane is critically dependent on the expression of the ABC transporter MsbA. Loss of MsbA activity inhibits growth and is associated with the accumulation of lipid A in the cytoplasmic leaflet of the inner membrane [8,9,10]. Polyspecifity appears to be a common property of ABC efflux systems, whereas importers are substrate specific, often requiring a dedicated high-affinity binding protein for substrate delivery [1]
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