Fig. 2: Binding sites for HBV preS1 in human NTCP and mapping of key residues that maintain functionality of NTCP as HBV receptor.

a, Surface representation of the extracellular face of NTCP, coloured by electrostatic potential. Residues involved in preS1 attachment are labelled. b, Ribbon diagrams showing the core (blue) and panel (yellow) domains of NTCP. Left, NTCP is shown in the same orientation as in a. The orthogonal view (right) shows the external face at the top. Key residues mutated in this study are shown as ball-and-stick models, coloured by element. G158 and S267 are highlighted in red. c, HBV preS1 attachment (from images represented in d) and HBV infection were examined using wild-type or mutant NTCP-expressing Huh7 and HepG2 cells, respectively. Myrcludex B (500 nM) was used as a control to inhibit NTCP-mediated HBV preS1 attachment and HBV infection. Data are mean ± s.d. of three independent samples. d, Top, fluorescence images of the preS1-mediated HBV attachment assay in Huh7 cells expressing NTCP, showing the effect of NTCP mutations. Bottom, the expression level of NTCPs was monitored by Western blot. Lanes are numbered according to the corresponding images. NTCP and actin (loading control) were run on different gels. Scale bar, 100 µm. e, Electrostatic surface potential of the preS1-binding pocket of NTCP. Left, view from the cytoplasmic side. Right, view from the external side. Numbers in circles indicate the transmembrane helices lining the pocket. Leu27, Leu31 and Leu35 are shown as ball-and-stick models in pink. In a,e, Surfaces with positive and negative charges are coloured in blue and red, respectively, and electrically neutral surfaces are coloured in white.