Drug resistance to chemotherapeutic agents remains a major challenge in cancer treatment, frequently leading to the clinical failure of otherwise highly effective drugs. One key mechanism underlying this resistance involves multi-drug resistance transporters that efflux drugs from the cytosol into the extracellular environment. P-glycoprotein (P-gp), a transmembrane ATP-binding cassette (ABC) transporter, plays a critical role by reducing intracellular concentrations of a wide variety of xenobiotics that differ greatly in charge, size, and physicochemical properties. P-gp contains two symmetrical nucleotide-binding domains that hydrolyze ATP to drive a conformational switch from an inward-facing to an outward-facing state. This transition is typically triggered by substrate or drug binding, resulting in diminished intracellular levels of anti-cancer compounds. Here, we employ molecular dynamics (MD) simulations to investigate local and global conformational changes of both homodimeric and heterodimeric P-gp within a membrane lipid bilayer. Microsecond-scale simulations derived from high-resolution crystal structures reveal how distinct dimerization states shape the transport mechanism. In addition, we examine the effects of lipid composition and cholesterol concentration on the conformational dynamics of P-gp. Our results demonstrate that lipid composition is an important determinant of the P-gp transport cycle. In particular, cholesterol reduces protein flexibility, suggesting a stabilizing influence on transporter function. Using free-energy perturbation calculations, we quantified the binding affinities of nucleotides and assessed how distinct nucleotide states modulate structural behavior. Collectively, equilibrium MD simulations offer a comprehensive characterization of human P-gp dynamics in eukaryotic membranes, providing deeper insight into its transport behavior.
Shubbar et al. (2026) studied this question.