Constructs design

All primers are listed in Supplementary Table ¹.

Human cDNA encoding the c-MET extracellular region (amino acids 1–927) followed by the Human Rhinovirus 3 C (HR3C) recognition site, Tsi3 tag³⁶, 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-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).

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).