Nucleotide asymmetry and flexible linker dynamics modulate drug efflux cycle of P-glycoprotein, A computational study.

Despite advancements in oncology, multidrug resistance (MDR) mediated by P-glycoprotein (P-gp/ABCB1) remains a major barrier to chemotherapy. P-gp is an ATP-binding cassette transporter that undergoes nucleotide-driven structural rearrangements to efflux chemotherapeutics, but the mechanistic details of the substrate transport remain poorly resolved. Here, we performed high-throughput multi-replica molecular dynamics to simulate P-gp in a lipid bilayer (totaling ∼110 µs) to dissect nucleotide-dependent conformational changes across the transport cycle. Our adaptive sampling strategy reveals asymmetric nucleotide coordination at nucleotide-binding sites (NBS), which correlates with transmembrane domain (TMD) restructuring for substrate efflux. The experimentally unresolved flexible linker transiently forms up to five turns of α-helix that affects the nucleotide binding domain (NBD) dimerization process. We identified conformation-dependent substrate/allocrite pathways including nucleotide-specific access routes, while TMD-linker interaction facilitates substrate access tunnel formation. Together, these pathways reveal that the concerted interplay of nucleotide occupancy, linker dynamics, and overall protein conformation governs the structural plasticity and broad substrate promiscuity of the substrate binding cavity in P-gp. By integrating these findings, this work bridges static structural data with dynamic functional insights to further our understanding of the P-gp substrate translocation cycle.