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.

中文翻译：

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人 NTCP 中 Na+ 驱动的胆汁酸转运的分子机制

人牛磺胆酸钠共转运蛋白（hNTCP）是维持肠肝循环的关键胆汁盐转运蛋白，负责识别乙型肝炎和丁型肝炎病毒。尽管具有里程碑意义的冷冻电子显微镜研究揭示了抗体稳定的 hNTCP 的开孔和向内状态，但运输机制仍然很大程度上未知。为了解决这一知识差距，我们使用分子动力学和增强的采样元动力学模拟来阐明 Na 结合驱动的 hNTCP 介导的牛磺胆酸 (TCA) 运输的内在机制。我们发现了三种 TCA 结合模式，其中一种与开孔 hNTCP 中观察到的有限冷冻电子显微镜密度非常匹配。我们还捕获了底物运输循环中的几个关键 hNTCP 构象，特别是包括面向外的底物结合状态。此外，我们提供了热力学证据，支持 Na 结合状态的变化通过利用底物的两亲性并调节蛋白质环境来驱动 TCA 运输，从而使 TCA 分子翻转。了解 Na 驱动的胆汁酸转运的这些机制细节可能有助于开发针对肝病的 hNTCP 靶向疗法。