Long Range Communication between the Drug-Binding Sites and Nucleotide Binding Domains of the Efflux Transporter ABCB1

[Image: see text] The ABC efflux pump P-glycoprotein (P-gp) transports a wide variety of drugs and is inhibited by others. Some drugs stimulate ATP hydrolysis at the nucleotide binding domains (NBDs) and are transported, others uncouple ATP hydrolysis and transport, and others inhibit ATP hydrolysis. The molecular basis for the different behavior of these drugs is not well understood despite the availability of several structural models of P-gp complexes with ligands bound. Hypothetically, ligands differentially alter the conformational dynamics of peptide segments that mediate the coupling between the drug binding sites and the NBDs. Here, we explore by hydrogen-deuterium exchange mass spectrometry the dynamic consequences of a classic substrate and inhibitor, vinblastine and zosuquidar, binding to mouse P-gp (mdr1a) in lipid nanodiscs. The dynamics of P-gp in nucleotide-free, pre-hydrolysis, and post-hydrolysis states in the presence of each drug reveal distinct mechanisms of ATPase stimulation and implications for transport. For both drugs, there are common regions affected in a similar manner, suggesting that particular networks are the key to stimulating ATP hydrolysis. However, drug binding effects diverge in the post-hydrolysis state, particularly in the intracellular helices (ICHs 3 and 4) and neighboring transmembrane helices. The local dynamics and conformational equilibria in this region are critical for the coupling of drug binding and ATP hydrolysis and are differentially modulated in the catalytic cycle.