Structure of human NTCP reveals the basis of recognition and sodium-driven transport of bile salts into the liver

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equilibrated with HBS buffer (25 mM Hepes (pH 7.5), 150 mM NaCl) and were added to a concentration of 0.8 g/mL to the nanodisc mixture and incubated overnight at 4 °C with gentle stirring. Bio-Beads were removed by passing through a gravity column (Bio-Rad), and the mixture was briefly spun at 4000×g at 4 °C to remove excess lipids. The sample was concentrated using a 50 kDa molecular weight cut-off centrifugal filter (Amicon), followed by size exclusion chromatography ran with HBS buffer. Peak fractions containing nanodiscreconstituted NTCP were collected and concentrated accordingly.

Cellular uptake assay
Cells stably expressing the NTCP fusion construct with the C-terminal Avi-tag were induced with 3 µg/mL doxycycline and grown for 24 h in supplemented DMEM with 2% fetal bovine serum (FBS, Thermo Fisher Scientific), 100 units/mL penicillin (Gibco) and 100 µg/mL streptomycin (Gibco) under standard conditions. Cells were gently detached and seeded at a density of 150,000 cells per well in a Poly-L-Lysine 24-well plate (Greiner Bio-One). After 2 hours (to aid adherence), the growth media was exchanged with a pre-warmed uptake buffer, consisting of DMEM supplemented with 1 µM sodium taurocholate (TC). The added TC contained a mix of radiolabeled TC ( 3 H-TC) and non-labeled TC in a 1:20 mol/mol ratio. The uptake buffer was further supplemented with 750 nM NTCP_Fab12, 100 µM sodium glycochenodeoxycholic (GCDC, Sigma) or 100 µM CHS.
To verify sodium-dependent uptake of TC by NTCP, choline uptake buffer containing 142.9 mM choline chloride, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.8 mM CaCl2 and 20 mM HEPES, pH 7.4 was supplemented with 1 µM TC as described above, and used for the uptake assay.
The cells in the uptake buffer were incubated at 37 °C, and the reaction was stopped after 10 minutes by washing the cells twice with ice-cold phosphate buffered saline (PBS, Gibco). Cells were lysed upon addition of 100 µL 1% Triton X-100 in H2O for 5 minutes. Lysed cells were added to 2 mL scintillation fluid and radioactivity was measured using a scintillation counter (Perkin Elmer 2450 Microbeta2). For assays using choline uptake buffer, cells were washed twice with choline buffer, instead of PBS.
Data were analyzed using GraphPad Prism 8. Background subtracted from all values was calculated by taking the y-intercept of the linear regression through values of TC uptake for induced cells at timepoints between 2 and 20 minutes. Data were normalized to the value of TC uptake under the induced condition. The statistical significance between conditions was determined using Dunnett's test. p values are depicted in GraphPad style (GP) as 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***) and <0.0001 (****), and are shown in Fig. 1b.

Enzymatic Biotinylation of NTCP
3C-cleaved NTCP was biotinylated via BirA-mediated biotinylation of the NTCP Avi-tag construct, as described previously 1 . The yield of labeling was verified by a streptavidin pulldown assay.

Phage display
Fab Library E 2 , a phage library expressing Fab-fragments on the surface, was used for biopanning (DNA was kindly provided by S. Koide). All five rounds of biopanning were performed in a selection buffer (HBS supplemented with 0.5% bovine serum albumin (BSA) and detergent mixture of 0.02%/0.004% DDM/CHS). In the first round, NTCP was immobilized onto magnetic beads, followed by manual biopanning. Beads were washed three times with the selection buffer, whereafter only phage expressing Fab-fragments specific to immobilized NTCP remained attached. The beads were resuspended and used to infect log-phase Escherichia coli XL-1 Blue cells. Phage were amplified overnight in media containing M13-KO7 helper phage (10 9 pfu/mL) and ampicillin (100 μg/mL) and used as input for subsequent rounds. Four additional rounds of biopanning were performed with decreasing target concentrations. The reduction of nonspecific binders was achieved by preclearing phage pools from each of 2 to 5 rounds with 100 μL streptavidin. The phage pools from the fourth and fifth round were screened for individual clones and validated by a single-point phage ELISA in a 96-well plate (Nunc). Plates were coated with 2 μg/mL neutravidin and blocked with the selection buffer. Individual phage were amplified by inoculating 400 μL media (supplemented with M13-KO7 and 100 μg/mL ampicillin) with E. coli XL-1 colonies harboring individual phagemids and incubated overnight in 96-well deep blocks at 37 °C. The supernatant containing the phage particles was collected and diluted tenfold in the selection buffer prior to a phage ELISA assay. Detergent-solubilized NTCP was immobilized on a coated plate at 50 nM concentration for 30 min, followed by 30 min incubation with diluted phage. After 30 min incubation with HRP-conjugated anti-M13 monoclonal antibody (GE Healthcare), TMB substrate (Thermo Fisher Scientific) was added in order to detect signal corresponding to bound phage particles. The selection and ELISA steps were performed at 4 °C.
The clones that bound specifically were sequenced at the University of Chicago Comprehensive Cancer Center DNA Sequencing Facility.

Fab Expression and Purification.
Clones were subcloned into a pRH2.2 plasmid using an In-Fusion Cloning kit (Takara). E. coli BL21-Gold cells (Agilent) were transformed using the plasmids expressing the Fab-fragments. Cells were grown in 1 L media supplemented with 100 μg/mL ampicillin to OD600 of 0.8, induced with 1 mM IPTG and maintained for a further 4 h at 37 °C. The cells were harvested and stored at -80 °C. The antigen-binding fragments were purified by Protein A chromatography, followed by ion-exchange chromatography as described previously 3 .
To estimate the NTCP:Fab binding affinity, a protein ELISA was conducted. Briefly, the same neutravidin-coated 96-well plates were used to immobilize 50 nM detergentsolubilized NTCP. Fab-fragments were subsequently diluted and assayed against the immobilized protein. Binder detection was done using HRP-conjugated mouse anti-human IgG F(ab′)2 (Jackson) and TMB substrate (Thermo Fisher Scientific). The results were plotted and, for the NTCP_Fab12, which was used for structure determination, presented in Fig. S2.

Fab-binding Nanobody Expression and Purification
Fab-binding nanobody 4 , fusing one N-terminal His-tag and TEV protease cleavage site in pET26b (+) vector, was transformed into E. coli BL21 (DE3) cells for expression with Lysogeny broth medium. The cells were grown to OD600 of 0.8 at 37 °C, and induced by addition of 1 mM IPTG and grown for 20 hrs at 20 °C. The cells were harvested and periplasmic protein was obtained via osmotic shock by sucrose gradient. The lysate was purified by Ni-NTA chromatography, and the His-tag was removed with TEV protease followed by a Ni-NTA chromatography.

EM Sample Preparation
Nanodisc-reconstituted NTCP was prepared as described above, and Fab fragments (NTCP_Fab12) and Fab-binding nanobody 4 were added to the sample with 1:1:1.2 molar ratio, before the final SEC. SEC-purified human NTCP in a complex with NTCP_Fab12 and a Fab-binding nanobody, at a concentration of ~0.7 mg/mL, was equilibrated with 100 μM sodium glyco-chenodeoxycholic (GCDC, Sigma) for 5 min. Samples were applied onto glow discharged Quantifoil R1.2/1.3 carbon/copper 300 mesh grids, followed by plunge freezing in liquid ethane/propane mixture using Vitrobot Mark IV (FEI) at 4 °C and 100% humidity.

EM Data Acquisition and Processing
Data were collected semi-automatically with EPU2 (Thermo Fisher Scientific) on a Titan Krios 300 kV microscope (Thermo Fisher Scientific) equipped with a Gatan K3 camera and a Gatan Biocontinuum energy filter. Image stacks (40 frames) were collected at nominal magnification of 130,000k and corresponding pixel size of 0.33 Å/pix (super-resolution mode). The defocus was in the range of -0.6 to -2 µm, flux was set to 15 e -/pix/s and total dose was 64 e -/Å 2 We collected a total of 13,208 multiframe micrographs.
Data processing details are presented in Fig. S3. In short, the multiframe micrographs were imported into RELION 4.0 5 , motion corrected with MotionCor 2 6 and binned by a factor of 2 to a pixel size of 0.66 Å/pix. Contrast Transfer Function (CTF) parameters were estimated with Gctf 7 . Particles were picked using the reference-free Laplacian-of-Gaussian routine. Binned particles (factor of 4) were extracted and subjected to several rounds of 2D classification. The best classes were then used to generate an initial model ab initio and were further subjected to several rounds of 3D classification. The best particles were used for an initial 3D refinement. The particles were then unbinned (to 0.66 Å/pix) and subjected to several additional rounds of 3D refinement and classification. The final set of particles (161,093) were CTF refined and polished prior to a last round of 3D refinement (with a mask that excluded the density for the nanodisc and the constant domain of NTCP_Fab12) and post processed (with B-factor set to -52 Å 2 and ad-hoc low-pass filter to 2.7 Å). The final EM density map had an overall resolution of 2.88 Å, as estimated by the 0.143 Fourier Shell Correlation criterion. Local resolution estimation was performed in RELION and the results are shown in Fig. S3.

Model Building and Refinement
Model building was performed in Coot 8 . The final density map featured well-resolved TM helices (Fig. S4) that allowed for de novo model building based on the protein sequence. The N-terminal (residues 1-18) and C-terminal (312-349) are highly flexible and were not resolved. Refinement of the models was performed in Phenix 9 and validation in MolProbity 10 and presented in Table S1. The atomic coordinates and geometrical restraint of the ligands (cholesterol and GCDC) were used from the ligand library (ligand codes: CLR, CHO).

Figure Preparation
Graph preparation and data analysis were performed in GraphPad Prism version 8.0.0 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com. The images of models and the EM density map were prepared in UCSF Chimera 11 and UCSF ChimeraX 12 .