Molecular-Dynamics Simulations of the ATP/apo State of a Multidrug ATP-Binding Cassette Transporter Provide a Structural and Mechanistic Basis for the Asymmetric Occluded State

Abstract

ATP-binding cassette transporters use the energy of ATP hydrolysis to transport substrates across cellular membranes. They have two transmembrane domains and two cytosolic nucleotide-binding domains. Biochemical studies have characterized an occluded state of the transporter in which nucleotide is tenaciously bound in one active site, whereas the opposite active site is empty or binds nucleotide loosely. Here, we report molecular-dynamics simulations of the bacterial multidrug ATP-binding cassette transporter Sav1866. In two simulations of the ATP/apo state, the empty site opened substantially by way of rotation of the nucleotide-binding domain (NBD) core subdomain, whereas the ATP-bound site remained occluded and intact. We correlate our findings with elastic network and molecular-dynamics simulation analyses of the Sav1866 NBD monomer, and with existing experimental data, to argue that the observed transition is physiological, and that the final structure observed in the ATP/apo simulations corresponds to the tight/loose state of the NBD dimer characterized experimentally.

Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

“…MJ0796 is homolog to the NBD of the Gram-negative bacterial Lol complex that transports lipoprotein from the inner to the outer membrane, whereas MsbA is a bacterial lipid flippase homolog to P-glycoprotein (4,5). Our kinetic studies support monomer/dimer modes of operation (6 -14), as opposed to models where the NBDs remain in contact during the catalytic cycle (15)(16)(17)(18). In monomer/dimer models ATP hydrolysis is followed by dissociation of the NBDs, and the energy that drives the conformational changes that lead to substrate transport (power stroke) is provided by NBD association/dissociation.…”

Kinetics of the Association/Dissociation Cycle of an ATP-binding Cassette Nucleotide-binding Domain

“…2) Constant contact model. The NBDs remain in contact during the hydrolysis cycle, and the power stroke results from smaller conformational changes at the NBD-dimer interface (8,(13)(14)(15)17).…”

Homology modelling of human P-glycoprotein

“…The transition pathway of MsbA was investigated with non-equilibrium-driven MD simulations revealing the highly co-operative nature of transmembrane domain (TMD) and NBD movement [18]. Presumably owing to it being one of the first structures solved (and indeed is still considered an excellent template for the outward facing state), the Sav1866 structure has been subjected to many MD studies [19][20][21][22][23][24]. Much can be gleaned from these studies in terms of general exporter function, but in this review, we focus specifically on models of P-gp and then illustrate how such models are typically made and evaluated.…”