Structural basis for hepatitis B virus restriction by a viral receptor homologue

Abstract

Macaque restricts hepatitis B virus (HBV) infection because its receptor homologue, NTCP (mNTCP), cannot bind preS1 on viral surface. To reveal how mNTCP loses the viral receptor function, we here solve the cryo-electron microscopy structure of mNTCP. Superposing on the human NTCP (hNTCP)-preS1 complex structure shows that Arg158 of mNTCP causes steric clash to prevent preS1 from embedding onto the bile acid tunnel of NTCP. Cell-based mutation analysis confirms that only Gly158 permitted preS1 binding, in contrast to robust bile acid transport among mutations. As the second determinant, Asn86 on the extracellular surface of mNTCP shows less capacity to restrain preS1 from dynamic fluctuation than Lys86 of hNTCP, resulting in unstable preS1 binding. Additionally, presence of long-chain conjugated-bile acids in the tunnel induces steric hindrance with preS1 through their tailed-chain. This study presents structural basis in which multiple sites in mNTCP constitute a molecular barrier to strictly restrict HBV.

Fig. 1. Overall structure and substrate binding of mNTCP.

A The overall structure and cryo-EM density map of the macaque NTCP (mNTCP)/YN69083Fab complex. The 14 nonconserved residues with human NTCP (hNTCP) are highlighted in green. Cyan, mNTCP; orange, Fab heavy chain; blue, Fab light chain. B Structural comparison of taurocholic acid (TCA)-bound mNTCP (cyan) with apo-state hNTCP (green, PDB:7FCI). Root mean square deviation (RMSD) for all Cα atoms. C Vertical slice-through of a hydrophobicity surface representation of mNTCP, showing two TCAs and the tunnel. Black dashed boxes indicate the binding interfaces of the two TCAs. Hydrophobic and hydrophilic areas are shown in orange and cyan, respectively. D Residues interacting with the two TCAs in the tail and head interfaces. Residues are shown as sticks. Carbon, nitrogen, and oxygen atoms are in yellow, blue, and red, respectively. E Schematic diagrams showing front and side views of TCAs bound in mNTCP. The planar angle and distances between TCA-1 and TCA-2 are measured. The atoms of TCA-1 and TCA-2 used to measure distances are labeled in yellow and orange, respectively. Carbon, nitrogen, oxygen, and chlorine atoms are in black, blue, red, and green, respectively. F Schematic diagrams of the interactions between TCA and mNTCP, drawn using Ligplot+. Carbon, nitrogen, oxygen, and chlorine atoms are in black, blue, red, and green, respectively. Ball and stick model showing residues in mNTCP that form hydrogen bonds with TCA. G Cartoon and surface representation of TCA bound wild-type mNTCP and S267F mutant homology model. S267 and R158 are represented using a stick model. The distance between sulfur of TCA-1 and oxygen of hydroxyl group in S267F are measured. The region of steric hindrance with upper TCA (TCA-1) is highlighted by a black dashed box. Please note that hydrogens are not modelled in S267F homology model.

Fig. 2. Structural comparison of mNTCP and hNTCP.

A Structural comparison of TCA-bound mNTCP (cyan) with preS1 (pink)-bound hNTCP (green, PDB:8HRY). The RMSD values for all Cα atoms are indicated. The movements of TM1 and TM5 by preS1 binding are indicated by red arrows. B Close-up view of preS1 binding cavity surrounded by TM1 and TM5. The region of steric clash with preS1 is highlighted by a black dashed box. TCA-bound mNTCP (cyan), preS1 (pink)-bound hNTCP (green), and preS1 are shown as cartoons. C Structural comparison of extracellular surface binding interfaces of preS1 in mNTCP with preS1-bound hNTCP. Hydrophobic and hydrophilic areas are shown in orange and cyan, respectively. Residues comprising loop TM2–TM3 are represented by a stick model. D Close-up view of extracellular surface binding interfaces of preS1. mNTCP and preS1-bound hNTCP are superimposed and represented as cyan and green cartoon models, respectively.

Fig. 3. R158G substitution in mNTCP is responsible but not sufficient for supporting preS1 binding and HBV infection.

A, B PreS1 binding capacity of wild-type hNTCP (hNTCP WT), mNTCP (mNTCP WT), and G158-substituted mNTCP (mNTCP R158G). HepG2 cells overexpressing the indicated NTCPs were incubated with TAMRA-conjugated myristoylated preS1 (2–48)-peptide (preS1-TAMRA) to detect cell surface-bound preS1 (red) and NTCP (green). Pictures of immunofluorescence analysis with 40 nM preS1-TAMRA incubation are shown in A. Scale bar: 100 μm. Quantification of preS1-TAMRA fluorescence intensities is indicated in B. Dashed line indicated the background level. C HBV infection. HepG2 cells overexpressing the indicated NTCPs were inoculated with HBV, washed, and cultured for 12 d. HBV infection was evaluated by monitoring HBsAg in the culture supernatant. D Quantification of fluorescence intensities for preS1-TAMRA bound to NTCP-expressing cells in the preS1 binding assay at different concentrations of exposed preS1-TAMRA (10, 40, 160, and 640 nM). E Bile acid uptake activity. HepG2 cells overexpressing indicated NTCPs were incubated with [³H]-taurocholic acid (TCA) in the presence (dark gray) or absence (light gray) of sodium to measure intracellular radioactivity. F Cell surface NTCP protein production. HepG2 cells overexpressing indicated NTCPs were incubated in biotinylation buffer, washed, pulled down with streptavidin beads. Cell surface NTCP in the pull down fraction (upper) and actin in total cell lysate as internal control (lower) were detected by immunoblotting using anti-myc and anti-actin antibodies, respectively. The gel data are shown from one representative experiment. Uncropped gels are shown in Source data. G, H PreS1 binding capacity of mNTCP WT and its 19 variants. HepG2 cells overexpressing NTCP WT or mutants at position 158 were incubated with preS1-TAMRA. Fluorescence pictures with 40 nM preS1-TAMRA incubation are shown in G. Scale bar: 100 μm. Quantification of preS1-TAMRA fluorescence intensities when treated with 40 (light gray) and 640 nM (dark gray) of preS1-TAMRA are indicated in H. I Bile acid uptake activity of NTCPs. HepG2 cells overexpressing indicated NTCPs were incubated with [³H]-TCA in the presence (dark gray) or absence (light gray) of sodium to measure intracellular radioactivity. Bars showed the means of three independent experiments (n = 3) and error bars represent standard deviation (SD). The dashed line indicates the background levels of the assay based on the control groups. Statistical significance of p values is indicated as follows: ****p < 0.0001. Source data are provided in a Source Data file. hNTCP, human NTCP; mNTCP, macaque NTCP; WT, wild type; MyrB, Myrcludex B; SD, standard deviation.

Fig. 4. Lys at position 86 enhances NTCP ability for supporting preS1 binding and HBV infection.

A Mutated sites of mNTCP 4 mut (I29V/Q84R/N86K/L161I) and 5 mut (I29V/Q84R/N86K/R158G/L161I) are shown in green. B–D PreS1 binding (B, C) and HBV infection (D) were examined using HepG2 cells overexpressing the indicated mNTCPs, as well as hNTCP WT. Green and red signals indicate NTCP and preS1-TAMRA, respectively. Scale bar: 100 μm. ns indicates not significant. E–G The preS1 binding capacity of mNTCP WT or its indicated variants (R158G, I29V, Q84R, N86K, and L161I, as well as double mutants I29/R158G, Q84/R158G, N86/R158G, and L161/R158G) and hNTCP WT (E, F) and HBV infection (G) was examined with HepG2 cells overexpressing the indicated NTCPs. Scale bar: 100 μm. Bars showed the means of three independent experiments (n = 3) and error bars represent SD. Statistical significance of p values is indicated as follows: ****p < 0.0001 in (F), **p = 0.0023 in (G), and ns indicates not significant. Source data are provided in a Source Data file. hNTCP, human NTCP; mNTCP, macaque NTCP; WT, wild type; MyrB, Myrcludex B; SD, standard deviation.

Fig. 5. MD simulations showing the stabilization of the preS1 binding pose by the N86K substitution in NTCP.

A Impairment of preS1 fluctuation by the N86K substitution in mNTCP R158G. Root-mean-square-fluctuation (RMSF) of preS1 relative to NTCP is shown for each amino acid residue in preS1 (2–48 aa) during MD simulations. Average RMSF values for mNTCP R158G, mNTCP N86K/R158G, and hNTCP in three independent simulations are shown in cyan, purple, and green, respectively. B Distribution of the preS1 binding pose on the principal component (PC)1–PC2 surface. The binding poses sampled by three MD simulations for hNTCP, mNTCP R158G, and mNTCP N86K/R158G are shown in green, cyan, and green, respectively. The preS1 binding pose of the hNTCP-preS1 complex solved by cryo-EM are shown as a black square. C PC1 and PC2 vectors for the analysis in (B) are shown colored in orange and blue, respectively. D Histograms of MM/PBSA binding energy during MD simulations for hNTCP (green), mNTCP R158G (cyan), and mNTCP N86K/R158G (purple). E Augmented contact frequency of the preS1 44–48 aa residues to NTCP, by N86K substitution in mNTCP R158G. Frequencies of preS1-NTCP contact over time during the three independent MD simulations are shown for each preS1 amino acid position.

Fig. 6. Higher anti-HBV activity of bile acids with long conjugated-chains at position 17.

A, B Tripartite superposed image of mNTCP (cyan), TCA (yellow), and preS1 (pink). B indicates a steric clash of the side chain at position 17 of 2 TCAs with the counterpart preS1 (magenta). C Chemical structures of bile acids or their derivatives. D Activity of the compounds to inhibit preS1 binding was examined at 10, 20, 40, and 80 μM in the preS1 binding assay using HepG2-hNTCP-C4 cells. The values for 50% inhibitory concentration (IC₅₀) are indicated (μM). Bars indicate the means of three independent experiments (n = 3) and error bars represent SD. The dashed line indicates the 50% of the levels for the DMSO-treated control group. Source data are provided in a Source Data file. MyrB Myrcludex B, SD standard deviation, CA cholic acid, CDCA chenodeoxycholic acid, UDCA ursodeoxycholic acid, DCA deoxycholic acid, LCA lithocholic acid, GDCA glycodeoxycholic acid, GUDCA glycoursodeoxycholic acid, GCDCA glycochenodeoxycholic acid, TDCA taurodeoxycholic acid, TUDCA tauroursodeoxycholic acid.