Distinct Conformational Spectrum of Homologous Multidrug ABC Transporters

ATP-binding cassette (ABC) exporters are ubiquitously found in all kingdoms of life and their members play significant roles in mediating drug pharmacokinetics and multidrug resistance in the clinic. Significant questions and controversies remain regarding the relevance of their conformations observed in X-ray structures, their structural dynamics, and mechanism of transport. Here, we used single particle electron microscopy (EM) to delineate the entire conformational spectrum of two homologous ABC exporters (bacterial MsbA and mammalian P-glycoprotein) and the influence of nucleotide and substrate binding. Newly developed amphiphiles in complex with lipids that support high protein stability and activity enabled EM visualization of individual complexes in a membrane-mimicking environment. The data provide a comprehensive view of the conformational flexibility of these ABC exporters under various states and demonstrate not only similarities but striking differences between their mechanistic and energetic regulation of conformational changes. [Display omitted] •New tools enabled EM visualization of dynamic transporter conformations in lipids•Distinct conformational spectra of two homologous ABC transporters were revealed•P-gp prevails in inward-facing conformations under ATP hydrolysis conditions•MsbA with ATP displays both outward and a continuum of inward-facing conformations Moeller et al. used single particle electron microscopy to delineate the entire conformational spectrum of two homologous ATP-binding cassette exporters and the influence of nucleotide and substrate binding. The data provide new insights into mechanistic and energetic regulation of conformational changes for the transport function.