Atomistic insight into the mechanism of substrate recognition and transport by the bile salt export pump (BSEP) using computational microscopy

ABCB11, also known as the Bile Salt Export Pump (BSEP), is an essential member of the ABC transporter family that primarily exports bile salts across the bile canaliculi. ABCB11 undergoes extensive conformational transitions during its transport cycle. Although the recent cryo-EM structures provide a brief insight into the catalytic process, the detailed molecular mechanism of the substrate translocation and the role of cofactors in facilitating the transport cycle remain elusive. Given its structural homology with other ABC transporter proteins, ABCB11 is presumed to adopt an alternative access mechanism for its transport activity. However, the proposed mechanism is not characterized thoroughly. In this study, we have employed well-tempered metadynamics and all-atom molecular dynamics simulations to decipher the overall transport mechanism of ABCB11 within a membrane environment. Our workflow incorporates enhanced sampling and systematic docking of substrates and cofactor molecules to map the full conformational landscape during the substrate transport of the protein. From the free energy surface of the Apo state, the ABCB11 structure in the minima has been identified, which subsequently served as a basis for analyzing substrate-bound, prehydrolysis, and post-hydrolysis states. Initially, the Apo form of ABCB11 adopts an “inwardfacing (IF)” conformation. Substrate binding induces conformational flexibility, allowing the protein to access occluded and distinct “IF” conformations with varying degrees of NBD separations. Furthermore, the nucleotide binding drives conformational transitions in the ABCB11. From the free energy surface, we have identified a catalytically potent, collapsed conformation primed for hydrolysis. Hydrolysis drives the receptor to adopt a more collapsed conformation, thereby facilitating the “inward-facing” to “outward-facing” conformational switch, which enables substrate release. Collectively, this work provides a comprehensive atomistic description of the entire transport cycle of ABCB11 and highlights the impact of substrate and cofactor binding on conformational transition that facilitates bile acid translocation.