Structural basis of sodium-dependent bile salt uptake into the liver

Abstract

The liver takes up bile salts from blood to generate bile, enabling absorption of lipophilic nutrients and excretion of metabolites and drugs1. Human Na+–taurocholate co-transporting polypeptide (NTCP) is the main bile salt uptake system in liver. NTCP is also the cellular entry receptor of human hepatitis B and D viruses2,3 (HBV/HDV), and has emerged as an important target for antiviral drugs4. However, the molecular mechanisms underlying NTCP transport and viral receptor functions remain incompletely understood. Here we present cryo-electron microscopy structures of human NTCP in complexes with nanobodies, revealing key conformations of its transport cycle. NTCP undergoes a conformational transition opening a wide transmembrane pore that serves as the transport pathway for bile salts, and exposes key determinant residues for HBV/HDV binding to the outside of the cell. A nanobody that stabilizes pore closure and inward-facing states impairs recognition of the HBV/HDV receptor-binding domain preS1, demonstrating binding selectivity of the viruses for open-to-outside over inward-facing conformations of the NTCP transport cycle. These results provide molecular insights into NTCP ‘gated-pore’ transport and HBV/HDV receptor recognition mechanisms, and are expected to help with development of liver disease therapies targeting NTCP.

Tyrosine 146 of the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) Is Essential for Its Hepatitis B Virus (HBV) Receptor Function and HBV Entry into Hepatocytes

“…The cryo-EM structure of human NTCP (PDB: 7PQQ) [14] was visualized with Protean 3D DNASTAR Software. Visual representation of fluctuation in protein structure and interatomic interactions at amino acid position 146 of wt and mutant NTCP proteins was performed using DynaMut2 server based on the AlphaFold model of human NTCP (AF-Q14973-F1) [26,28].…”

“…Interestingly, G158R mutation of human NTCP completely abolished susceptibility to HBV and HDV infection, while the R158G exchange was sufficient to transmute the old world monkey Ntcp to a functional HBV/HDV receptor [11]. Based on recently published cryo-electron microscopy (EM) structures [13][14][15], NTCP can undergo a conformational transition from an outward-to an inward-facing state, whereby opening of a relatively wide transmembrane pore/tunnel allows substrate translocation from the extracellular to the intracellular compartment. In addition, cryo-EM analysis of preS1-bound NTCP suggested that the myr-preS1 peptide binds preferentially to the open-pore state of the carrier and thereby competes with bile acids for binding to residues lining the same cavity [13][14][15].…”

“…Based on recently published cryo-electron microscopy (EM) structures [13][14][15], NTCP can undergo a conformational transition from an outward-to an inward-facing state, whereby opening of a relatively wide transmembrane pore/tunnel allows substrate translocation from the extracellular to the intracellular compartment. In addition, cryo-EM analysis of preS1-bound NTCP suggested that the myr-preS1 peptide binds preferentially to the open-pore state of the carrier and thereby competes with bile acids for binding to residues lining the same cavity [13][14][15]. This is in agreement with experimental data clearly demonstrating that bile acids, such as taurocholic acid (TC), efficiently block myr-preS1 peptide binding to NTCP [11,16,17].…”

“…This finally resulted in decreased susceptibility to HBV and HDV infection in humans expressing this variant with concomitant reduction of advanced stages of HBV-related liver diseases [19,20]. Thus, the S267F "loss-of-function" variant affects the same domain on the NTCP protein that interacts with bile acids and the viral preS1 peptide [13][14][15]17,21].…”

“…Taken together, based on the current knowledge of relevant NTCP domains that are important for virus binding to NTCP, a very rough map of virus/receptor interaction sites at the surface of NTCP is currently available. Although several independent cryo-EM structures of human NTCP have recently been released that confirmed these previously identified preS1 binding regions at NTCP [13][14][15], the precise molecular interaction sites between NTCP and the myr-preS1 peptide could not be completely determined. Based on this, it is very likely that there are still a number of yet undiscovered surface amino acid residues of NTCP that orchestrate together with the already identified binding regions the HBV/HDV receptor function of NTCP.…”