Structural and Energetic Analysis of the CmABCB1 Substrate Transport

P-Glycoprotein, a member of the ATP-binding cassette (ABC) transporter family (ABCB1), actively exports various hydrophobic compounds from the cell. Crystal structures of the eukaryotic <i>Cyanidioschyzon merolae</i> homologue (CmABCB1) in both inward-facing (IF) and outward-facing (OF) conformations have suggested a transport mechanism involving conformational changes in the transmembrane helices (TMHs) and nucleotide-binding domain (NBD) dimerization. In this study, we employed the string method to compute the minimum free energy path (MFEP) of the conformational transition from IF to OF crystal structure, with a focus on elucidating the structural and energetic basis of a model substrate (rhodamine 6G (R6G)) transport as a most probable path derived from the crystallographic data. ATP binding and subsequent NBD dimerization act as driving forces to overcome the energy barrier associated with frustration occurring in TMH. This process disrupts the aromatic hydrophobic network (AHN), facilitates nonspecific R6G binding, and leads to an intermediate substrate-occluded (Occ) state. R6G interactions weaken the dimer interface and destabilize the Occ state, promoting transition to the stable OF conformation through TMH twisting and squeezing motions. These results highlight the power of MFEP-based free energy landscapes in uncovering the molecular mechanisms of membrane transporters.