High Speed AFM Imaging of Structure and Dynamics of Bacterial ABC Transporter MsbA During Lipid Transport

Abstract

All ATP-binding cassette (ABC) transporters that operate in all species from bacteria to human share a common structural organization, consisting of two distinct domains (i.e., transmembrane domains (TMDs) and cytosolic nucleotide binding domains (NBDs)). MsbA, an ABC transporter, exports lipid A across the cytoplasmic membrane of Gram-negative bacteria. MsbA also exports drugs, conferring multidrug resistance. The crystal and cryo-EM snapshot structures of MsbA bound with different nucleotides have suggested that large conformational changes possibly take place in the TMDs accompanied the ATPase reaction in two NBDs. Therefore, the two NBDs must repeat coupling and uncoupling during multiple ATPase cycles. These dynamic events very likely transmit to the two α-helical bundles connected with the NBDs, so that the TMDs (major parts of the α-helical bundles) undergo different conformations including inward-facing and outward-facing conformations that facilitate substrate entry, translocation and export. However, this structure and dynamics of MsbA during these functional activities have not been verified simultaneously. Using HS-AFM advantage, the structures and dynamics of detergent-solubilized MsbA bound with different nucleotides were successfully real-time imaged to follow their conformational changes. The kinetics of open and closed conformations in NBDs and TMDs was analyzed to evaluate how coupling and uncoupling NBDs could change structural dynamics in TMDs. Next, structural dynamics of TMDs channel and NBDs of MsbA embedded within lipid membrane containing lipid A was also real-time imaged in the presence of different nucleotides and drugs. The translocation of lipid A across TMDs channel was successfully recorded in the controllably oriented MsbA molecules within lipid membrane. Taken together, the dynamic conformational changes in TMDs accompanying ATP hydrolysis in NBDs during lipid A transport unveil a very important operational mechanism in bacterial ABC transporters.