Atomistic View on Substrate Transport in an ABC Exporter

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ATP-binding cassette (ABC) exporters are ATP-driven molecular machines that pump a broad range of transport substrates across biological membranes. Their working mechanism is based on chemomechanical coupling of ATP binding and hydrolysis in the nucleotide-binding domains to large-scale conformational transitions of the transmembrane domains that form the translocation channel for the substrate. However, although substrate translocation is their key biological function, the detailed mechanism by which ABC exporters transport substrate molecules across membranes is still poorly understood. This discrepancy is partly due to the challenges of characterizing this intrinsically dynamic process in atomic detail, which involves coupled changes of transporter conformation and substrate position. Two key aspects stand out as essentially unresolved: (i) The sequence of conformational states through which the transporter cycles and their link to the actual substrate translocation process, and (ii) the free energy profiles and how they compare for substrate-free and -loaded transporters. This project therefore aims at characterizing the conformational dynamics and energetics of substrate translocation in an ABC exporter by all-atom molecular dynamics (MD) simulations. The heterodimeric ABC exporter TM287/288, a bacterial homolog of permeability glycoprotein (Pgp), will be investigated as a prototype. First, multi-microsecond unbiased MD simulations will be carried out to investigate binding of transport substrate verapamil and the associated conformational changes in TM287/288. Second, metadynamics simulations will be carried out to obtain free energy landscapes along collective variables that describe the conformational changes of TM287/288 linked to substrate translocation.This project, which is based on preliminary combined MD simulation/EPR spectroscopy work on TM287/288 in absence of substrate, will reveal the route taken by the substrate through the transporter and the underlying atomic driving forces and mechanical couplings. Furthermore, by comparing the free energy profiles with and without substrate, we aim at understanding the molecular basis for substrate-induced activity enhancement. In addition, we will tackle the open question of what prevents substrate reuptake. Considering the rapidly growing number of X-ray and cryo-EM structures in recent years, this proposed study aims at providing the missing atomic-level picture of the structural dynamics as well as free energy profiles that are necessary to understand – and possibly modify – ABC transporter function.