Inward opening (IO) to outward opening (OO) 'catalytic transition' and OO to IO 'relaxation' of P-glycoprotein: to investigate the role of helices in efflux through targeted molecular dynamics simulation.

P-glycoprotein (P-gp) is a membrane protein that effluxes xenobiotics across cell membranes via ATP hydrolysis. It is overexpressed mainly in cancer cells and is responsible for multidrug resistance by effluxing chemotherapeutic molecules. To unearth the coordinated mechanism and function of different domains i.e., nucleotide binding domain (NBD), transmembrane domain (TMD) and transmembrane helices (TMHs) in catalysis, human P-gp was modelled in Inward Opening (IO) and Outward Opening (OO) states and further subjected to targeted molecular dynamics (tMD) simulations. Structural transition frames between IO ⇌ OO were obtained from the clustering of tMD simulation trajectories. Protein model quality scores (ProSA Z-scores) were evaluated for conformational states. The results showed that the IO → OO transition is an energetically uphill process requiring a major structural transition involving 131 distinct conformational states, coupled with ATP hydrolysis. In contrast, the OO → IO relaxation, crucial for resetting the transporter, does not follow the same transition pathway and is an energetically downhill process involving only 90 states, indicating a faster and distinct mechanism. The helix pairs 1&7 and 6&12 are observed to be relatively static, forming the core of the TMD, while pairs 3&9 and 4&10 are moderately dynamic, and pairs 5&11 and 2&8 are highly dynamic, located more peripherally. The static and dynamic nature and position of these helix pairs justify their respective roles in substrate binding and efflux, and these findings may provide insight into the design and development of next-generation P-gp inhibitors. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-025-00389-3.