Structural insights into the HBV receptor and bile acid transporter NTCP

Abstract

Around 250 million people are infected with hepatitis B virus (HBV) worldwide1, and 15 million may also carry the satellite virus hepatitis D virus (HDV), which confers even greater risk of severe liver disease2. The HBV receptor has been identified as sodium taurocholate co-transporting polypeptide (NTCP), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large protein3. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria4,5, and these models are believed to resemble closely both NTCP and ASBT. Here we have used cryo-electron microscopy to solve the structure of NTCP bound to an antibody, clearly showing that the transporter has no equivalent of the first transmembrane helix found in other SLC10 proteins, and that the N terminus is exposed on the extracellular face. Comparison of our structure with those of related proteins indicates a common mechanism of bile acid transport, but the NTCP structure displays an additional pocket formed by residues that are known to interact with preS1, presenting new opportunities for structure-based drug design.

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

“…Multiple sequence alignment analysis revealed that all three tyrosine residues of this motif, namely Y139, Y141, and Y146, are evolutionarily conserved among human NTCP, as well as chimpanzee, rhesus monkey, rat, and mouse Ntcps (Figure 1A). Based on the recent cryo-EM structures of human NTCP [13][14][15], the 139YIYSRGIY146 motif is localized at the transition of transmembrane domain 4 to extracellular loop 2, and thereby is exposed to the extracellular surface of NTCP that is relevant for preS1-peptide binding (Figure 1B).…”

“…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.…”