Abstract
Human Na(+) taurocholate co-transporting protein (hNTCP) is a key bile salt transporter to maintain enterohepatic circulation and is responsible for the recognition of hepatitis B and D viruses. Despite landmark cryoelectron microscopy studies revealing open-pore and inward-facing states of hNTCP stabilized by antibodies, the transport mechanism remains largely unknown. To address this knowledge gap, we used molecular dynamics and enhanced sampling metadynamics simulations to elucidate the intrinsic mechanism of hNTCP-mediated taurocholate acid (TCA) transport driven by Na(+) binding. We uncovered three TCA-binding modes, including one that closely matched the limited cryoelectron microscopy density observed in the open-pore hNTCP. We also captured several key hNTCP conformations in the substrate transport cycle, particularly including an outward-facing, substrate-bound state. Furthermore, we provided thermodynamic evidence supporting that changes in the Na(+)-binding state drive the TCA transport by exploiting the amphiphilic nature of the substrate and modulating the protein environment, thereby enabling the TCA molecule to flip through. Understanding these mechanistic details of Na(+)-driven bile acid transport may aid in the development of hNTCP-targeted therapies for liver diseases.