Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP

Abstract

Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na⁺-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor's extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.

Fig. 1. Schematic of human NTCP-mediated HBV infection.

Bile salt uptake into hepatocytes is mediated by NTCP, located in the basolateral membrane of hepatocytes. HBV interacts with heparan sulfate proteoglycans (HSPGs) by low-affinity attachment, followed by interaction with the NTCP receptor protein, initiating the entry of viral particles into the hepatocytes. HBV-NTCP interaction occurs via high-affinity binding of the myristyolated preS1 domain of LHBs to NTCP. This process can be inhibited by bulevirtide, a preS1-derived peptide.

Fig. 2. Binding of preS1 and patient-derived SVPs as model systems to study HBV-NTCP interaction.

a Fluorescence microscopy and (b) fluorescence detection of HEK-NTCP cells and non-NTCP expressing HEK293 cells, incubated with or without 200 nM fluorescent preS1-AX568 peptide (representing genotype D, GtD) in the presence and absence of sodium. Expression levels of NTCP in HEK-NTCP cells were confirmed with qPCR. NBD-TC was used to demonstrate NTCP-mediated bile salt transport, whereas 250 μM TC was used as an inhibitor of preS1-peptide binding. Data in (b) present the means ± SD of quadruplicate measurements. *Significantly higher preS1-AX568 fluorescence compared to all other columns, ^§significantly different preS1 binding and ^#significant preS1 binding inhibition, all p < 0.0001. c Schematic of HBV subviral particles. The hepatitis B virus surface proteins LHBs, MHBs, and SHBs differ in the N-terminal additions (preS1, preS2) and N-glycosylation pattern within the S-domain. The antigenic loop (AGL) within the S-domain and the N-terminal preS1, together with the myristic acid covalently attached to Gly2, are determinants for HBV infectivity. d Silver-stained polyacrylamide gels of highly purified SVP preparations from two different patients: K826 HBV-GtA and ID1 HBV-GtD. Both show clear bands of non- and N-glycosylated SHBs (p24/gp27 kDa), single and double N-glycosylated MHBs (gp33/ggp36 kDa), and non- and N-glycosylated preS1-containing LHBs (p39/gp42 kDa). Non-reduced SHBs-dimers (48 kDa or 54 kDa, depending on glycosylation status) and human serum albumin (67 kDa) are also indicated. e, f Binding of nanodisc-reconstituted wild-type or G158R mutant of NTCP-eYFP to SVPs from (e) patient K826 (HBV GtA) and (f) patient ID1 (HBV GtD). NTCP-eYFP fluorescence was detected using a fluorescence microtiter plate reader at 485 nm excitation and 535 nm emission. Data present the means ± SD of triplicate measurements of background-subtracted relative fluorescence units (RFU). *Significantly higher fluorescence intensity between SVP-coated wells incubated with and without nanodisc-reconstituted NTCP-eYFP (p = 0.0013, and p = 0.0492 for panels (e) and (f), respectively, according to one-way ANOVA with Dunnett’s multiple comparison test).

Fig. 3. Cryo-EM structure and functional analysis of BLV-bound NTCP.

a Normalized inhibition of TC transport (1 μM) into Flp-In T-Rex cells stably expressing NTCP at varying concentrations of BLV. Uptake of ³H-TC was measured after 8 min, each data point indicates the mean of three independent replicates, and error bars represent the SD. b Size exclusion chromatography and SDS-PAGE analysis of purified human NTCP in a complex with BLV, Fab3, and Nb. c Cryo-EM density map (left) and model in ribbon representation (right) of NTCP-BLV-Fab3-Nb complex. The BLV peptide is colored in three sections: myristoylated glycine (magenta), plug (orange), and string (blue). The densities and models are colored as follows: NTCP in green, Nb in gray and Fab3 in purple (heavy chain) and coral (light chain).

Fig. 4. Molecular interaction between BLV and NTCP.

a Surface representation of NTCP (green), with the bound BLV peptide: myristoylated glycine (magenta), residues T3-G20 (orange), and residues P21-G48 (blue). b 2D schematic of the full BLV peptide, coloring is the same as in (a). c Sequence alignment of BLV and the preS1 peptides from the ten genotypes of HBV. Purple shading indicates amino acids conservation between BLV and different HBV preS1 genotypes. Black asterisks denote residues of BLV that are exposed to the solvent and therefore do not interact with NTCP. Red font indicates the HBV genotype used for the design of the BLV peptide. d–g Interactions between BLV and NTCP. Coloring as in panel (a). The EM density for BLV is displayed as blue mesh. BLV residues are labeled black.

Fig. 5. Comparison of BLV-bound NTCP and substrate-bound NTCP.

a, b Superposed BLV-bound NTCP (green) and substrate-bound NTCP (PDB ID: 7ZYI, gray) structures shown in ribbon representation. Two molecules of the substrate glycochenodeoxycholic acid (GCDC) are shown as gray sticks and the BLV peptide is colored as before. Close-up views show a clash between the substrate GCDC and TM6 of the BLV-bound NTCP structure (a), and a clash between GCDC substrates and the BLV peptide (b).

Fig. 6. NTCP species specificity against HBV infection.

a Sequence alignment between human NTCP and NTCP from two different species of monkeys: Cynomolgus monkey (Old World) and Common squirrel monkey (New World). Highlighted amino acids indicate the residues described in the text: the Gly/Arg variation at position 158, and Ser267 and its genetic variant Phe267, which has been linked to HBV resistance. b A close-up view of residue Gly158 (sphere representation) and its proximity to BLV (orange and blue transparent surface). c A close-up view of residue Ser267 (sphere representation) and its proximity to BLV (orange and blue transparent surface). d Topology diagram of human NTCP. Sodium binding sites (unoccupied in the NTCP-BLV structure) and substrate binding regions are indicated as empty circles and yellow diamonds, respectively. ECL extracellular loop, ICL intracellular loop. The crossing motif (X-motif) is formed by TM helices 3 and 8. Glycosylation sites are shown and numbered. Colored arches indicate the regions of NTCP interacting with distinct regions of the BLV peptide.