Structural basis of the activation of c-MET receptor.

Constructs design

All primers are listed in Supplementary Table 1 . C-MET927 Human cDNA encoding the c-MET extracellular region (amino acids 1–927) followed by the Human Rhinovirus 3 C (HR3C) recognition site, Tsi3 tag 36 , and the 6-histidine-tag (His6) tag, was cloned into the pEZT-BM, which is a BacMan vector that can infect both Sf9 insect and HEK293F cells (Supplementary Table 1 ). C-MET927-LZ c-MET (amino acids 1–927) followed by a GGGGS linker, a GCN4 zipper sequence (RMKQL EDKVE ELLSK NYHLE NEVAR LKKLV GER), HR3C, Tsi3 tag, and His6 tag, was cloned into the pEZT-BM vector (Supplementary Table 1 ). HGF Full-length human HGF followed by HR3C site, and a His6 tag was cloned into the pEZT-BM vector (Supplementary Table 1 ). All HGF mutants were generated using site-directed mutagenesis (NEB Q5 Site-Directed Mutagenesis). For the phosphorylation assay, a full-length c-MET receptor followed by five or two repeats of the Myc tag sequence was cloned into pLVX-IRES-Puro. To introduce mutations to c-MET, short gene fragments (100–150 nt) containing mutations were synthesized (IDT Inc.), and the corresponding gene fragment in the wild-type c-MET was replaced by the synthesized gene fragment using HiFi DNA assembly enzyme (NEB). Expression of c-MET, HGF, and NK1 C-MET927, c-MET927-LZ, and HGF The plasmid was transformed into the DH10Bac bacteria (Geneva Biotech) for the production of bacmid DNA. Recombinant baculovirus was produced by transfecting Sf9 cells with the bacmid DNA using Cellfectin (Cellfectin Reagent Invitrogen). Protein was expressed with suspension-adapted HEK293F cells by infecting the virus at a ratio of 1:10 (virus:cell, v/v). The infected cells were supplemented with 5 mM sodium butyrate to increase protein expression. For HGF expression, 5% of FBS (Sigma) was supplemented to the culture media. The cells were cultured for 96 h at 37 °C and 8% CO 2 . NK1 Human HGF (amino acids 31–209) was tagged at the N-terminus with Thiol:disulfide interchange protein (DsBC) and His6 followed by HR3C recognition site cloned into pET22b vector. The plasmid was transformed into the Rosetta-gami 2 (DE3) (Milipore sigma) and cultured at 37 °C. When the OD600 reached 0.6, the cells induced with Isopropyl β-d-1-thiogalactopyranoside (IPTG), and expressed at 22 °C for 16 h.

Protein purification C-MET927 and c-MET927-LZ purification

Culture media containing the secreted protein of interest was harvested by centrifugation and filtered using a glass fiber filter. The cleared media was incubated with the INDIGO-Ni resin (Cube Biotech Inc.) for 30 min on the bottle roller. The resin was collected using a chromatography column. The resin was washed with 10 resin volumes of the washing buffer (20 mM Hepes pH 7.5, 400 mM NaCl, 20 mM Imidazole) and eluted with the elution buffer (20 mM Hepes pH 7.5, 400 mM NaCl, 400 mM Imidazole). The eluted protein was dialyzed against the dialysis buffer (20 mM Hepes pH 7.5, 400 mM NaCl) at 4 °C for 16 h. Tsi3 and His6 tags were cleaved with an HR3C protease during the dialysis. The protein was further purified by using SEC (Superose 6 increase 100/30 Cytiva) with the SEC buffer (20 mM Hepes at pH 7.5 and 400 mM NaCl).

HGF purification

Culture media containing the secreted HGF was harvested by centrifugation and filtered using a glass fiber filter. The cleared media was adjusted to a final concentration of 50 mM sodium/potassium phosphate at pH 6.5 and applied to the cation exchange column (Hitrap SP HP, Cytiva). The protein was eluted with a linear gradient over 50% buffer B (20 mM Sodium/potassium phosphate buffer pH 6.5 and 2 M NaCl). The eluate was further purified using SEC (Superose 6 increase 100/30 Cytiva) with the SEC buffer (20 mM Hepes pH7.5 and 400 mM NaCl).

NK1 purification

Cells were harvested and lysed by sonication in sonication buffer (20 mM Hepes pH 7.5, 400 mM NaCl, 5% glycerol). After centrifugation, the supernatant was applied to the Ni Sepharose resin (Cytiva). The DsBC and His6 tag were cleaved by adding HR3C protease to the resin and incubating for 16 h at 4 °C. Eluted protein was further purified with SEC (Superdex 75, Cytiva) in the SEC buffer (20 mM Hepes pH 7.5 and 400 mM NaCl). C-MET927-LZ/HGF/heparin and c-MET927-LZ/NK1/heparin complex reconstitution c-MET927-LZ was mixed with HGF or NK1 in the presence of heparin (Tinzaparin sodium; MilliporeSigma) in a 1:1:4 molecular ratio. The complex sample was concentrated and loaded onto SEC (SRT SEC-500 Sepax) in the buffer containing 20 mM Hepes pH 7.5 and 100 mM NaCl. The fractions containing the complex were pooled and concentrated using an Amicon Ultra concentrator with a 100 kDa cut-off (Millipore) to ~5 mg/ml.

EM data acquisition

Immediately before preparing the cryo-EM grids, 2 mM fluorinated Fos-Choline-8 (Anatrace) was added to the purified sample to prevent the protein from being damaged by the hydrophobic air–water interface on a cryo-EM grid. The cryo-EM grid was prepared by applying 3 μl of the protein samples to glow-discharged Quantifoil R1.2/1.3 300-mesh gold holey carbon grids (Quantifoil, Micro Tools GmbH, Germany). Grids were blotted for 4.0 s under 100% humidity at 4 °C before being plunged into the liquid ethane using a Mark IV Vitrobot (FEI). Micrographs were acquired on a Titan Krios microscope (FEI) operated at 300 kV with a K3 direct electron detector (Gatan), using a slit width of 20 eV on a GIF-Quantum energy filter. SerialEM was used for the data collection 37 . A calibrated magnification of 46,296 was used for imaging, yielding a pixel size of 1.08 Å on images. The defocus range was set from −1.6 to −2.6 μm. Each micrograph was dose-fractionated to 30 frames under a dose rate of 18 e − /pixel/s, with a total exposure time of 4 s, resulting in a total dose of about 60 e − /Å 2 .

Image processing

Movie frames of c-MET927-LZ/HGF micrographs were motion-corrected and binned twofold, resulting in a pixel size of 1.08 Å, and dose-weighted using MotionCor2 38 . CTF correction was performed using GCTF 39 . The rest of the image processing steps were carried out using RELION 3.1 40 . Particles were first roughly picked by using the Laplacian-of-Gaussian blob method and then subjected to 2D classification. Class averages representing projections of the c-MET/HGF in different orientations were used as templates for reference-based particle picking. A total of 3,287,093 particles were picked from 8100 micrographs. Particles were extracted and binned by three times (leading to 3.24 Å/pixel) and subjected to another round of 2D classification. Particles in good 2D classes were chosen (1,367,618 in total) for 3D classification using an initial model generated from a subset of the particles in RELION. After the initial 3D classification c-MET927-LZ/HGF particles set, two major classes were identified showing good secondary structural features (namely holo-complex and sub-complex, respectively). The holo-complex comprises two c-MET and two HGF molecules; while the sub-complex only comprises one c-MET and two HGF. Particles for holo-complex or sub-complex were selected separately and re-extracted into the original pixel size of 1.08 Å. Subsequently, we performed finer 3D classification for each particle subset by using local search in combination with small angular sampling, resulting in improved density for both assembly states. The final reconstructions of holo-complex and sub-complex were resolved at 4.8 and 4 Å resolution, respectively. To improve the resolution for c-MET I and HGF I in holo-complex, we performed focused 3D refinement, by using a soft mask around c-MET I and HGF I, leading to a 3D reconstruction at an overall resolution of 4.5 Å. Finally, we performed another round of finer 3D classification on the particle set of sub-complex. After the further classification, we could identify a new class where HGF II adopts a rigid conformation. The subsequent 3D refinement of this class yielded a cryo-EM at 4.9 Å resolution, resolving a complete version of HGF II. A total of 1,342,144 particles were picked from 3644 micrographs of c-MET/NK1. A total of 656,258 particles were selected by 2D classification. Subsequent 3D classification revealed one good class showing good density. Next, we performed two rounds of finer 3D classification by using local search in combination with small angular sampling, resulting in a new class showing improved density for the entire protein. 3D refinement of this class, along with CTF refinement and particle polishing, yielded a structure at 5 Å resolution. The resolution was estimated by applying a soft mask around the protein density. The Fourier Shell Correlation (FSC) 0.143 criterion was used. Local resolution was calculated in RELION. Model building, refinement, and validation Model buildings of both c-MET/HGF and c-MET/NK1 complexes were initiated by rigid-body docking of individual domains from the crystal structures of SEMA, PSI, IPT1, and IPT2 domains of c-MET and N, K1, K2, and SPH domains of HGF 14 , 24 , 25 , 30 . The models of K3 and K4 of HGF were first built by homology modeling using the structures of K1 of HGF as a template (50% sequence identity between K1 and K3; 36.1% sequence identity between K1 and K3), and then rigid-body fitted into the cryo-EM density in Chimera 41 . The manual building was carried out using the program Coot. The model was refined by using the real-space refinement module in the Phenix package (V1.17) 42 . Restraints on secondary structure, backbone Ramachandran angels, and residue sidechain rotamers were used during the refinement to improve the geometry of the model. MolProbity 4.5 as a part of the Phenix validation tools was used for model validation (Table 1 ). Figures were generated in Chimera 1.14. c-MET activation assay Naive H1299 cells were gifted from John Minna (UT Southwestern Medical Center, Dallas) and cultured in RPMI 1640 medium (Thermofisher) supplemented with 10% (vol/vol) FBS at 37 °C and 5% CO 2 . Lentiviruses encoding myc-tagged c-MET (myc-c-MET, WT, or mutant cloned into the pLVx-IRES-puro vector) were generated in HEK 293 T packaging cells following standard transfection protocol 43 . Titrations of lentiviruses were used to infect H1299 cells, and infected cells were selected using 2 μg/ml puromycin for 2–3 days. Cells stably express near-endogenous myc-c-MET (WT or mutant) were selected for the following assays. To examine the agonist activities of HGF mutants, naive H1299 cells were plated 200,000 cells/well on six-well plates (Corning) and starved in an FBS-free medium for 16 h. The starved cells were stimulated with 2 ml, 100 ng/ml HGF (WT or mutant) for 10 min at 37 °C. After stimulation, cells were washed once with 1x PBS and then collected in 150 µl 2x Laemmli Sample Buffer supplemented with 5% 2-Mercaptoethanol. SDS-PAGE and Western Blotting were used to probe c-MET (8198 S, Cell Signaling, 1:1000 dilution in 5% milk), phospho-c-MET (Tyr1234/1235, 3077 S, Cell Signaling, 1:1000 dilution in 5% milk), and loading control β-actin (A1978, Sigma, 1:5000 dilution in 5%BSA). Blotting results were analyzed using ImageJ. To examine the phosphorylation capacities of c-MET mutants, 3′UTR siRNA (hs.Ri.MET.13.1 from IDT) was used for selective knockdown of endogenous c-MET in H1299 cell lines that stably express near-endogenous myc-c-MET (WT or mutant). Transfections of siRNA were carried out using Lipofectamine RNAi-Max (Life Technologies) following the manufacturer’s protocol. After depletion of endogenous c-MET, 100 ng/ml HGF WT was used to stimulate the phosphorylation of myc-c-MET following the protocol described above.

Pull-down assay

Five micrograms of the bait protein c-MET927-TSI3-His was immobilized to TSE resin by incubating for 30 min on ice and washed with binding buffer PBS (Gibco) supplemented with10 µM Tinzaparin and 2 mM CaCl2. The resin was incubated with 5 µg of the prey proteins HGF wild type or mutants in 50 µl of binding buffer for 1 h at 4°. Each tube was tap gently but thoroughly every 10 min. The resin was washed with binding buffer to wash away prays. The resin was boiled in SDS loading buffer (Bio-rad) and loaded on the SDS-PAGE. The intensity of the bands was calculated using ImageJ. Reporting Summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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