Association/Dissociation of the Nucleotide-Binding Domains of an ATP-Binding Cassette (ABC) Exporter during the ATP Hydrolysis Cycle

Abstract

ATP-binding cassette (ABC) proteins constitute one of the largest protein families, and most are membrane transport proteins. Eukaryotic exporters in this group, including the multidrug resistance protein P-glycoprotein, have been linked to chemotherapy resistance due to their extrusion of anticancer agents from cells. Like its prokaryotic homolog MsbA, P-glycoprotein has a functional core composed of two transmembrane domains and two nucleotide-binding domains (NBDs). Substrate transport is driven by ATP hydrolysis, but the mechanism of hydrolysis is controversial. The proposed mechanisms can be broadly classified as: 1) monomer-dimer models, where alternating access depends on a large separation of the NBDs (up to 50 angstroms) during the ATP binding/hydrolysis cycle, or 2) constant-contact models, where the NBDs remain associated during the cycle. To address the ABC exporters' mechanism, we used lanthanide-based resonance energy transfer (LRET) to follow the intra-molecular movements of MsbA during the ATP binding/hydrolysis cycle. Although the MsbA NBDs sample three discrete positions during the cycle, the average NBD separation decreases dramatically after the addition of ATP to the Apo (nucleotide-free) protein. ATP binding reduced the predominant NBD separation distance from 50 to 35 angstroms. ATP hydrolysis elicited by MgATP relaxed a fraction of the NBD dimer population towards the Apo state. These steady-state distance measurements are consistent with a monomer-dimer model. The correlation between rates of NBDs dissociation (determined from kinetic LRET measurements) and ATP hydrolysis are consistent with monomer-dimer models, where the NBDs dissociate during each hydrolysis cycle. This work was supported by a grant from CPRIT grant RP101073 and an American Heart Association Pre-doctoral Fellowship 11PRE7360046 to RSC.