Drug-bound P-glycoprotein reveals nucleotide-dependent ligand pockets and ingress channels

Abstract

P-glycoprotein, an ATP-binding cassette exporter, coordinates drug recognition in a central cavity with nucleotide handling at nucleotide binding domains, yet the sequence and coupling of these events remain incompletely defined. Here, we report an integrated computational analysis of inward facing P-glycoprotein ensembles across varying drug locations, nucleotide states, and starting conformations. The sampled inward-facing ensembles of P-glycoprotein display continuous ligand cavity, reshaped by nucleotide occupancy, which enables polyspecific ligand recognition while transmitting allosteric signals to the nucleotide binding domains through intracellular coupling loops. Asymmetry between the two nucleotide binding sites follows from this communication, which alters the nucleotide coordination to potentially affect the catalytic progression. Our simulations reveal dynamic cavity access routes from cytosol and inner membrane, together with transient secondary structure within the interdomain flexible linker. Overall, our study gives insights into how ligand redistribution, asymmetric nucleotide coordination, and linker flexibility may correlate to form a unified view of early transport steps, yielding hypotheses for mutational and kinetic evaluation and informing strategies for small-molecule modulation of transporter function in cellular contexts where multidrug transport affects therapeutic response.