Structural basis of sodium-dependent bile salt uptake into the liver


Thermostable NTCP constructs

Consensus amino acids have been calculated utilizing JALVIEW50 and reported standards29 from sequences of consultant NTCP vertebrate orthologues (Prolonged Information Fig. 1), aligned utilizing Muscle51. Consensus amino acid exchanges have been concurrently launched into wild-type NTCP sequence background with N-glycosylation mutations N5T and N11T, enhancing protein stability. Deletions of N-terminal residue E2, and the unstructured C terminus (residues T329–A349) within the consensus non-glycosylated assemble additional improved homogeneity of the pattern, yielding the so-called NTCPCO.

Usually, the consensus strategy generates protein samples with total improved stability, however it’s anticipated that by concurrently introducing all consensus mutations, some destabilizing exchanges are included. To reduce the latter, we probed thermal stability of single-point NTCPCO mutants, during which we reverted consensus amino acids to the wild-type residues, utilizing fluorescence-detection size-exclusion chromatography52 (SEC). Removing of destabilizing consensus exchanges in NTCPCO, yielded a consensus design, NTCPEM, which is almost an identical to wild-type NTCP (roughly 98% id) (Prolonged Information Figs. 1, 6), whereas preserving Na+-dependent bile salt transport in addition to myr-preS1 recognition mechanisms.

Protein expression and purification

cDNAs encoding NTCP constructs have been synthesized (GenScript) and subcloned right into a pcDNA3.1(+) vector encompassing a C-terminal PreScission web site, adopted by GFP, and two Strep-tags in tandem for affinity purification. Protein expression was finished in HEK 293F cells (Thermo Fisher; cells weren’t authenticated or examined for mycoplasma contamination) by transient transfection, as described53 with small variations. Briefly, cells grown in FreeStyle 293 medium (Thermo Scientific) have been transfected with linear 25K polyethyleneimine (PEI) (Polysciences) at a cell density of two.5 × 106 cells per ml utilizing 3 µg ml−1 DNA. Valproic acid (VPA) was added to the tradition at a last focus of two.2 mM 6–12 h after transfection and cells have been grown for added 48 h earlier than assortment.

Cell pellets have been 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 particles was eliminated by ultracentrifugation. Detergent-solubilized transporters have been purified by affinity chromatography utilizing 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 beneath 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 a pair of.5 mM desthiobiotin. The eluted protein was digested with PreScission protease in a single day, concentrated to a number of mg per ml utilizing 100 kDa MWCO concentrator (Corning Spin-X UF concentrators) and injected in a Superose 6 column (GE Healthcare Life Sciences) utilizing 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 have been used instantly or flash frozen and saved at −80 °C. All purification steps have been finished at 4 °C.

NTCPEM complexes with nanobodies and megabodies, respectively, have been shaped by mixing purified protein samples at 1:1.2 (transporter:nanobody, or megabody) molar ratio, and incubated for 2h at 4 °C. Extra nanobody or megabody was eliminated by SEC utilizing SEC buffer. MSP1D1 nanodisc-scaffold protein was expressed and purified utilizing printed protocols54. Reconstitution was finished by mixing purified NTCPEM–Nb and NTCPEM–Mb complexes, respectively, with MSP1D1 and liver whole lipid extract (Avanti Polar Lipids) at 0.1:1:15 molar ratio, and incubated with methanol-activated biobeads for two h. Biobeads have been exchanged as soon as, and the combination was additional incubated in a single day. Nanodisc-reconstituted pattern was purified in a Superdex 200 enhance column (GE Healthcare Life Sciences) in buffer containing 20 mM HEPES pH 7.4, 100 mM NaCl, and 0.2 mM sodium taurocholate. Samples have been concentrated as described above, and instantly used for cryo-EM grid preparation.

Nanobody technology, expression and purification

Nanobodies in opposition to NTCPCO have been generated utilizing printed protocols55. Briefly, one llama (Lama glama) was six occasions immunized with a complete 0.9 mg of NTCPCO reconstituted in proteoliposomes. 4 days after the ultimate increase, blood was taken from the llama to isolate peripheral blood lymphocytes. RNA was purified from these lymphocytes and reverse transcribed by PCR to acquire the cDNA of the open studying frames coding for the nanobodies. The ensuing library was cloned into the phage show vector pMESy4 bearing a C-terminal His6 tag and a CaptureSelect sequence tag (Glu-Professional-Glu-Ala). Totally different nanobody households, as outlined by the distinction within the CDR3, have been chosen by biopanning. For this, NTCPCO reconstituted in proteoliposomes was strong section coated immediately on plates. NTCPCO particular phage have been recovered by restricted trypsinization, and after two rounds of choice, periplasmic extracts have been made and analysed utilizing ELISA screens. Nb87 and Nb91 have been expressed in Escherichia coli for subsequent purification from the bacterial periplasm. After Ni-NTA (Sigma) affinity purification, nanobodies have been additional purified by SEC in buffer: 10 mM HEPES pH 7.4, and 110 mM NaCl.

Nb91 was enlarged by fusion to the round permutated glucosidase of E. coli K12 (YgjK, 86 kDa) to construct the megabody known as Mb91. Mb91 was generated and purified utilizing beforehand described protocols30.

Fluorescent substrate analogue transport assay

Sodium-dependent substrate uptake was measured in HEK 293F cells transfected with 2 µg μl−1 cDNA utilizing the above-mentioned protocol with small modifications. Forty-eight h after transfection, round 1 million cells have been 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 management buffer during which NaCl was substituted with choline chloride (ChCl). To probe the impact of nanobodies on bile salt transport, cells have been incubated with nanobodies for 1.5 h, adopted by addition of the fluorescent substrate analogue tauro-nor-THCA-24-DBD56,57 (tebu-bio) to a last focus of 10 μM for 30 min at 37 °C. Extra fluorescent analogue was eliminated by centrifugation (13,000g for 30 s), and 1 wash with the above-mentioned management buffer. Then, cells have been resuspended and lysed utilizing Pierce IP lysis buffer (Thermo Fisher). Lastly, lysates have been 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) utilizing excitation at 454 nm and emission of 570 nm. Three biologically unbiased experiments have been quantified in triplicate samples. Nb titrations information have been fitted in Prism 8.0.1 (GraphPad) to the next dose-respond curve:

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

The place Ymin corresponds to fraction of transport at saturating Nb concentrations, IC50 is the half-maximal inhibitory focus, 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 area (myr-GTNLSVPNPLGFFPDHQLDPAFRANSNNPDWDFNPNKDHWPEANKVG) was synthesized (GenScript) and subcloned right into a pcDNA3.1(+) vector encompassing a C-terminal GFP, and poly Histidine-tag (specifically, 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 have been lysed by 3–5 passes by way of a homogenizer (EmulsiFlex-C5, Avestin) and membrane fraction was collected by ultracentrifugation. Membranes have been 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 after which purified by affinity chromatography utilizing 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 beneath 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 a number of mg ml−1 utilizing a 30 kDa MWCO concentrator (Corning Spin-X UF concentrators) and injected right into a Superose 6 column (GE Healthcare Life Sciences) utilizing a SEC buffer containing 20 mM HEPES pH 7.4, 200 mM NaCl, 0.013% DDM, and 0.0027% CHS. Myristoylation of the pattern was confirmed by mass spectrometry. Purified myr-preS148–GFP was flash frozen and saved at −80 °C. All purification steps have been finished at 4 °C.

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

Electron microscopy pattern preparation and information acquisition

Purified NTCPEM–Nb or –Mb complexes have been utilized to glow-discharged Au 300 mesh Quantifoil R1.2/ 1.3. Usually, 4 µl of pattern at 3-4 mg ml−1 was utilized to the grids, and the Vitrobot chamber was maintained at 100% humidity and 4 °C. Grids have been screened in 200 kV Talos Arctica microscope (ThermoFisher) on the IECB cryo-EM imaging facility. Last information assortment was carried out in 300 kV Titan Krios microscope (ThermoFisher) at EMBL-Heidelberg Cryo-Electron Microscopy Service Platform, geared up with K3 direct electron detector (Gatan). Last pictures have been recorded with SerialEM58 at a pixel dimension of 0.504 Å. Dose price was 15–20 e pixel s−1.

For NTCPEM–Nb87 complexes, 21,792 pictures have been recorded with −0.5 to −1.5 µm defocus vary. Pictures have been collected with 0.7-s subframes (whole 40 subframes), akin to a complete dose of 57.8 e Å−2. For NTCPEM–Mb91 complexes, 21,390 pictures have been recorded with −0.6 to −1.75-µm defocus vary. Pictures have been collected with 0.7 s subframes (whole 40 subframes), akin to a complete dose of 56.5 e Å−2.

Cryo-EM information processing, mannequin constructing and construction evaluation

All datasets have been processed with cryoSPARC v2 and v359. Motion pictures have been achieve corrected, and aligned utilizing in-built patch-motion correction routine. Distinction switch perform (CTF) parameters have been estimated utilizing the in-built patch-CTF routine in cryoSPARC. Low-quality pictures have been discarded manually upon visible inspection.

For the NTCPEM–Mb91 advanced, 5,796,802 particles have been template-picked from 21,390 micrographs, and chosen by way of a number of rounds of 2D, in addition to 3D ab initio classifications. Particles from 3D ab initio courses displaying interpretable density for transmembrane helices have been pooled, and used for homogenous refinement (Prolonged Information Fig. 2). Cryo-EM density akin to each detergent micelle and megabody scaffold have been masked out, and particles have been additional subjected to native refinement utilizing a fulcrum that localized to middle of NTCPEM transmembrane area. Centered refinement yielded a last map at an total decision of three.3 Å, based mostly on the gold-standard 0.143 Fourier shell correlation (FSC) cut-off.

For the NTCPEM–Nb87 advanced, 6,535,687 particles have been template-picked from 21,792 micrographs, and categorized by way of a number of rounds of 2D and 3D ab initio classifications (Prolonged Information Fig. 3). Round 220,000 chosen particles have been additional categorized by heterogenous refinement, yielding a last set of 61,053 particles that have been processed by non-uniform refinement60. Additional targeted refinement excluding nanodisc scaffold yielded a last map at an total decision of three.7 Å, based mostly on the gold-standard 0.143 FSC cut-off. Maps have been visualized utilizing UCSF Chimera61 and ChimeraX62.

The cryo-EM map of the NTCPEM–Mb91 advanced confirmed clear density for many sidechains within the transmembrane helices, though TM1 and TM6 within the panel area displayed fewer molecular options, and was used to construct an atomic mannequin of NTCPEM utilizing Coot63,64. Secondary construction predictions utilizing Psipred65 and bacterial homologue construction (Protein Information Financial institution ID 3ZUY) have been used to assist preliminary sequence task. Preliminary Nb fashions have been created with I-TASSER66, after which match as inflexible our bodies into the density, adopted by handbook constructing and modification in Coot63,64. The inward-facing conformation within the NTCPEM–Nb87 advanced was constructed by becoming core and panel domains from NTCPEM–Mb91 construction as separate inflexible our bodies into the density, adopted by handbook modification in Coot. All atomic fashions have been refined utilizing PHENIX67.

Structural analyses have been 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 floor mapping with ConSurf71.

Reporting abstract

Additional info on analysis design is accessible within the Nature Analysis Reporting Abstract linked to this paper.

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