WNKs bind to OSR1 CCT domain, promoting the interaction between OSR1 and MO25.
A, structural comparison of OSR1 kinase-CCT domain and OSR1-MO25 complex. The disordered loop (294–433) between OSR1 kinase domain and CCT domain is shown as dotted lines. B, crystal structure of OSR1 CCT domain (cyan) in complex with a WNK4-derived RFXV-containing peptide (pink) (PDB ID: 3DAK). C, purified recombinant proteins of His-tagged OSR1, including full-length (1–527), kinase domain plus (1–338), CCT domain (434–527), and no-tagged WNK1-(1–663) proteins were subjected to His pull-down analysis. D–E, His pull-down assays were performed using purified His-tagged MO25 and untagged OSR1 proteins in the presence or absence of WNK1-(1–663) or WNK4 peptide (GRFQVT). Pull-down samples were subjected to SDS-PAGE. The bottom panel indicates the amount of full-length OSR1 proteins used in each reaction.