Conformational Motion of the ABC Transporter MsbA Induced by ATP Hydrolysis

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. 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. MsbA is a conserved ABC transporter from Gram-negative bacteria with sequence similarity to human multi-drug ABC transporters. MsbA flips the building block of the outer membrane, lipid A, across the inner membrane. The input of ATP energy occurs in two dedicated nucleotide-binding domains (NBDs), whose configuration in intact transporters is controversial. We determined the amplitude of MsbA conformational motion that couples energy expenditure to substrate movement across the membrane. Using molecular probes introduced into the protein sequence, we found that ATP hydrolysis fuels a relative motion of the NBDs close to 30 Å. The movement of the NBDs is coupled to reorientation of the chamber, which binds the lipid substrate from cytoplasmic-facing to extracellular-facing through large amplitude motion on either side of the transporters. In addition to revealing the structural mechanics of transport, these results challenge current models deduced from studies of substrate-specific ABC importers that envisions the two NBDs in contact throughout the ATP hydrolysis cycle.