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Thermostable NTCP constructs

Consensus amino acids were calculated using JALVIEW50 and reported criteria29 from sequences of representative NTCP vertebrate orthologues (Extended Data Fig. 1), aligned using Muscle51. Consensus amino acid exchanges were simultaneously introduced into wild-type NTCP sequence background with N-glycosylation mutations N5T and N11T, improving protein stability. Deletions of N-terminal residue E2, and the unstructured C terminus (residues T329–A349) in the consensus non-glycosylated construct further improved homogeneity of the sample, yielding the so-called NTCPCO.

In general, the consensus approach generates protein samples with overall improved stability, but it is expected that by simultaneously introducing all consensus mutations, some destabilizing exchanges are included. To minimize the latter, we probed thermal stability of single-point NTCPCO mutants, in which we reverted consensus amino acids to the wild-type residues, using fluorescence-detection size-exclusion chromatography52 (SEC). Removal of destabilizing consensus exchanges in NTCPCO, yielded a consensus design, NTCPEM, which is nearly identical to wild-type NTCP (approximately 98% identity) (Extended Data Figs. 1, 6), while preserving Na+-dependent bile salt transport as well as myr-preS1 recognition mechanisms.

Protein expression and purification

cDNAs encoding NTCP constructs were synthesized (GenScript) and subcloned into a pcDNA3.1(+) vector encompassing a C-terminal PreScission site, followed by GFP, and two Strep-tags in tandem for affinity purification. Protein expression was done in HEK 293F cells (Thermo Fisher; cells were not authenticated or tested for mycoplasma contamination) by transient transfection, as described53 with small variations. In brief, cells grown in FreeStyle 293 medium (Thermo Scientific) were transfected with linear 25K polyethyleneimine (PEI) (Polysciences) at a cell density of 2.5 × 106 cells per ml using 3 µg ml−1 DNA. Valproic acid (VPA) was added to the culture at a final concentration of 2.2 mM 6–12 h after transfection and cells were grown for additional 48 h before collection.

Cell pellets were resuspended and lysed in buffer containing 50 mM HEPES pH 7.4, 200 mM NaCl, 5% v/v glycerol, 1 mM EDTA, 1 mM TCEP, 0.5 mM sodium taurocholate and supplemented with protease inhibitors (1 mM PMSF and protease inhibitor cocktail from Sigma), 1% dodecyl-β-d-maltopyranoside (DDM) (Anatrace) and 0.2% cholesteryl hemi-succinate tris salt (CHS) (Anatrace), and incubated for 1 h. Cell debris was removed by ultracentrifugation. Detergent-solubilized transporters were purified by affinity chromatography using streptactin sepharose resin (Cytiva Life Sciences). Resin was pre-equilibrated in buffer A containing 50 mM HEPES pH 7.4, 200 mM NaCl, 5% v/v glycerol, 0.017% DDM, 0.0034% CHS, and 0.2 mM sodium taurocholate, and incubated with transporters for 1 h under rotation. Resin was extensively washed with buffer A, and protein was eluted in buffer B containing 50 mM HEPES pH 7.4, 200 mM NaCl, 5% v/v glycerol, 0.017% DDM, 0.0034% CHS, 0.2 mM sodium taurocholate, and 2.5 mM desthiobiotin. The eluted protein was digested with PreScission protease overnight, concentrated to several mg per ml using 100 kDa MWCO concentrator (Corning Spin-X UF concentrators) and injected in a Superose 6 column (GE Healthcare Life Sciences) using SEC buffer containing 20 mM HEPES pH 7.4, 100 mM NaCl, 0.017% DDM, 0.0034% CHS, and 0.2 mM sodium taurocholate. Purified transporters were used immediately or flash frozen and stored at −80 °C. All purification steps were done at 4 °C.

NTCPEM complexes with nanobodies and megabodies, respectively, were formed by mixing purified protein samples at 1:1.2 (transporter:nanobody, or megabody) molar ratio, and incubated for 2h at 4 °C. Excess nanobody or megabody was removed by SEC using SEC buffer. MSP1D1 nanodisc-scaffold protein was expressed and purified using published protocols54. Reconstitution was done by mixing purified NTCPEM–Nb and NTCPEM–Mb complexes, respectively, with MSP1D1 and liver total lipid extract (Avanti Polar Lipids) at 0.1:1:15 molar ratio, and incubated with methanol-activated biobeads for 2 h. Biobeads were exchanged once, and the mixture was further incubated overnight. Nanodisc-reconstituted sample was purified in a Superdex 200 increase column (GE Healthcare Life Sciences) in buffer containing 20 mM HEPES pH 7.4, 100 mM NaCl, and 0.2 mM sodium taurocholate. Samples were concentrated as described above, and immediately used for cryo-EM grid preparation.

Nanobody generation, expression and purification

Nanobodies against NTCPCO were generated using published protocols55. In brief, one llama (Lama glama) was six times immunized with a total 0.9 mg of NTCPCO reconstituted in proteoliposomes. Four days after the final boost, blood was taken from the llama to isolate peripheral blood lymphocytes. RNA was purified from these lymphocytes and reverse transcribed by PCR to obtain the cDNA of the open reading frames coding for the nanobodies. The resulting library was cloned into the phage display vector pMESy4 bearing a C-terminal His6 tag and a CaptureSelect sequence tag (Glu-Pro-Glu-Ala). Different nanobody families, as defined by the difference in the CDR3, were selected by biopanning. For this, NTCPCO reconstituted in proteoliposomes was solid phase coated directly on plates. NTCPCO specific phage were recovered by limited trypsinization, and after two rounds of selection, periplasmic extracts were made and analysed using ELISA screens. Nb87 and Nb91 were expressed in Escherichia coli for subsequent purification from the bacterial periplasm. After Ni-NTA (Sigma) affinity purification, nanobodies were further purified by SEC in buffer: 10 mM HEPES pH 7.4, and 110 mM NaCl.

Nb91 was enlarged by fusion to the circular permutated glucosidase of E. coli K12 (YgjK, 86 kDa) to build the megabody referred to as Mb91. Mb91 was generated and purified using previously described protocols30.

Fluorescent substrate analogue transport assay

Sodium-dependent substrate uptake was measured in HEK 293F cells transfected with 2 µg μl−1 cDNA using the above-mentioned protocol with small modifications. Forty-eight h after transfection, around 1 million cells were pelleted, washed, and resuspended in 500 µl of transport buffer (110 mM NaCl, 4 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 45 mM mannitol, 5 mM glucose and 10 mM HEPES pH 7.4), or control buffer in which NaCl was substituted with choline chloride (ChCl). To probe the effect of nanobodies on bile salt transport, cells were incubated with nanobodies for 1.5 h, followed by addition of the fluorescent substrate analogue tauro-nor-THCA-24-DBD56,57 (tebu-bio) to a final concentration of 10 μM for 30 min at 37 °C. Excess fluorescent analogue was removed by centrifugation (13,000g for 30 s), and 1 wash with the above-mentioned control buffer. Then, cells were resuspended and lysed using Pierce IP lysis buffer (Thermo Fisher). Finally, lysates were centrifuged (13,000g for 10 min), and transferred to black 96-well flat-bottom plates (Grenier), and quantified by fluorescence in a micro-plate reader (CLARIOstar-Plus) using excitation at 454 nm and emission of 570 nm. Three biologically independent experiments were quantified in triplicate samples. Nb titrations data were fitted in Prism 8.0.1 (GraphPad) to the following dose-respond curve:

$${rm{Fractional; transport}}=1+frac{{Y}_{min }-1}{1+{10}^{log {{rm{IC}}}_{50}-x}}$$

Where Ymin corresponds to fraction of transport at saturating Nb concentrations, IC50 is the half-maximal inhibitory concentration, and x is log[Nb].

Myr-preS1 purification and binding assay

cDNA encoding the N-terminal myristoylated consensus residues 2–48 of human HBV myr-preS1 domain (myr-GTNLSVPNPLGFFPDHQLDPAFRANSNNPDWDFNPNKDHWPEANKVG) was synthesized (GenScript) and subcloned into a pcDNA3.1(+) vector encompassing a C-terminal GFP, and poly Histidine-tag (namely, myr-PreS148–GFP). Myr-preS148–GFP was expressed in HEK 293F cells (Thermo Fisher) by transient transfection, as described for expression of NTCPEM purification. Cells were lysed by 3–5 passes through a homogenizer (EmulsiFlex-C5, Avestin) and membrane fraction was collected by ultracentrifugation. Membranes were resuspended in a buffer containing 50 mM HEPES pH 7.4, 200 mM NaCl, 5% v/v glycerol, 1 mM EDTA, protease inhibitors (1 mM PMSF and protease-inhibitor cocktail from Sigma), 1% dodecyl-β-d-maltopyranoside (Anatrace), and 0.2% cholesteryl hemi-succinate tris salt (Anatrace), and incubated for 1 h. Solubilized myr-preS148–GFP was subjected to ultracentrifugation and then purified by affinity chromatography using anti-His affinity resin (Sigma). Resin was pre-equilibrated in a buffer containing 50 mM HEPES pH 7.4, 200 mM NaCl, 5% v/v glycerol, 0.013% DDM, 0.0027% CHS, and incubated with detergent-solubilized myr-preS148–GFP for 1 h under rotation. Resin was extensively washed with buffer containing 50 mM HEPES pH 7.4, 200 mM NaCl, 5% v/v glycerol, 0.013% DDM, 0.0027% CHS and 50 mM imidazole. Myr-preS148–GFP was eluted in buffer containing 50 mM HEPES pH 7.4, 200 mM NaCl, 5% v/v glycerol, 0.013% DDM, 0.0027% CHS, and 250 mM imidazole. The eluted protein was concentrated to several mg ml−1 using a 30 kDa MWCO concentrator (Corning Spin-X UF concentrators) and injected into a Superose 6 column (GE Healthcare Life Sciences) using a SEC buffer containing 20 mM HEPES pH 7.4, 200 mM NaCl, 0.013% DDM, and 0.0027% CHS. Myristoylation of the sample was confirmed by mass spectrometry. Purified myr-preS148–GFP was flash frozen and stored at −80 °C. All purification steps were done at 4 °C.

Myr-preS148–GFP binding to NTCP constructs was assayed in HEK 293F cells, grown and transfected with 1µg ml−1 DNA using the protocol described above. Forty-eight hours after transfection, cells were washed with pre-warmed PBS, and ~1 million cells were pelleted and resuspended in 1 ml of PBS. To probe the effect of nanobodies, cells expressing NTCP constructs were pre-incubated with 10 µM nanobodies for 1.5 h. They were then labelled with 10 nM (wild-type NTCP) or 50 nM (NTCPEM) purified myr-preS148–GFP for 30 min. Excess fluorescent-probe was removed by centrifugation (13,000g for 30 s), and one wash with PBS. Cells were then re-suspended in PBS and GFP fluorescence was recorded in a micro-plate reader (CLARIOstar-Plus) using excitation at 470 nm and emission at 508 nm.

Electron microscopy sample preparation and data acquisition

Purified NTCPEM–Nb or –Mb complexes were applied to glow-discharged Au 300 mesh Quantifoil R1.2/ 1.3. Typically, 4 µl of sample at 3-4 mg ml−1 was applied to the grids, and the Vitrobot chamber was maintained at 100% humidity and 4 °C. Grids were screened in 200 kV Talos Arctica microscope (ThermoFisher) at the IECB cryo-EM imaging facility. Final data collection was performed in 300 kV Titan Krios microscope (ThermoFisher) at EMBL-Heidelberg Cryo-Electron Microscopy Service Platform, equipped with K3 direct electron detector (Gatan). Final images were recorded with SerialEM58 at a pixel size of 0.504 Å. Dose rate was 15–20 e pixel s−1.

For NTCPEM–Nb87 complexes, 21,792 images were recorded with −0.5 to −1.5 µm defocus range. Images were collected with 0.7-s subframes (total 40 subframes), corresponding to a total dose of 57.8 e Å−2. For NTCPEM–Mb91 complexes, 21,390 images were recorded with −0.6 to −1.75-µm defocus range. Images were collected with 0.7 s subframes (total 40 subframes), corresponding to a total dose of 56.5 e Å−2.

Cryo-EM data processing, model building and structure analysis

All datasets were processed with cryoSPARC v2 and v359. Movies were gain corrected, and aligned using in-built patch-motion correction routine. Contrast transfer function (CTF) parameters were estimated using the in-built patch-CTF routine in cryoSPARC. Low-quality images were discarded manually upon visual inspection.

For the NTCPEM–Mb91 complex, 5,796,802 particles were template-picked from 21,390 micrographs, and selected through several rounds of 2D, as well as 3D ab initio classifications. Particles from 3D ab initio classes displaying interpretable density for transmembrane helices were pooled, and used for homogenous refinement (Extended Data Fig. 2). Cryo-EM density corresponding to both detergent micelle and megabody scaffold were masked out, and particles were further subjected to local refinement using a fulcrum that localized to center of NTCPEM transmembrane region. Focused refinement yielded a final map at an overall resolution of 3.3 Å, based on the gold-standard 0.143 Fourier shell correlation (FSC) cut-off.

For the NTCPEM–Nb87 complex, 6,535,687 particles were template-picked from 21,792 micrographs, and classified through several rounds of 2D and 3D ab initio classifications (Extended Data Fig. 3). Around 220,000 selected particles were further classified by heterogenous refinement, yielding a final set of 61,053 particles that were processed by non-uniform refinement60. Further focused refinement excluding nanodisc scaffold yielded a final map at an overall resolution of 3.7 Å, based on the gold-standard 0.143 FSC cut-off. Maps were visualized using UCSF Chimera61 and ChimeraX62.

The cryo-EM map of the NTCPEM–Mb91 complex showed clear density for most sidechains in the transmembrane helices, although TM1 and TM6 in the panel domain displayed fewer molecular features, and was used to build an atomic model of NTCPEM using Coot63,64. Secondary structure predictions using Psipred65 and bacterial homologue structure (Protein Data Bank ID 3ZUY) were used to help initial sequence assignment. Initial Nb models were created with I-TASSER66, and then fit as rigid bodies into the density, followed by manual building and modification in Coot63,64. The inward-facing conformation in the NTCPEM–Nb87 complex was built by fitting core and panel domains from NTCPEM–Mb91 structure as separate rigid bodies into the density, followed by manual modification in Coot. All atomic models were refined using PHENIX67.

Structural analyses were carried out as follows: protein cavity calculations with CASTp 3.068, pore calculations MOLEonline 2.569, protein–protein interfaces with PISA70, and amino acid conservation surface mapping with ConSurf71.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.



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