Figure 4: Biophysical characterization of the ZNRF3ecto dimer and interface mutants.

(a) Sedimentation velocity experiments. A plot of c(s) (in arbitrary units) against s (in svedbergs). Shown in each case are individual data points and the fit of an appropriate number of Gaussian distributions. All samples were adjusted to a concentration of 350 μM. Also shown arrowed are the expected sedimentation coefficients for the different complexes observed in the crystal structures as predicted using HYDROPRO (see Methods). (b–h) SPR experiments using mZNRF3_ecto (b–e) or mRspo2_Fu1–Fu2 (f–h) as analyte and interface mutants/variants as immobilized ligands. (b) mZNRF3_ecto binds to mRspo2_Fu1–Fu2 (I39-G144) and retains high affinity to Fu1 (I39-R95) but not to Fu2 (A94-G144). Fu1–Fu2 polypeptides of human or mouse homologues (hRspo1: I32-S143, hRspo3: R32-H147, mRspo4: T29-Q136) bind with different affinity to mZNRF3_ecto. (c) Mutations of the Met-finger impact affinity. (d) Anonychia mutations of RSPO4 introduced to mRspo2_Fu1–Fu2 drastically impair binding. (e) Three additional interface mutants of which two (L63F and S53R) have been found in tumour tissues. (f) As the immobilized ligand mZNRF3 binds with lower affinity to the mRspo_2Fu1–Fu2 analyte. Of the three interface mutants, two (E109K and M98T) have been identified in tumour tissues. (g) Three interface mutants of hRNF3_ecto have been identified in tumours, one of which completely disrupts binding. (h) Binding of mRspo2_Fu1–Fu2 to ZNRF3_ecto dimer interface mutants. (i) Binding of mRspo2_Fu1–Fu2, mRspo4Fu1–Fu2 and chimeras to hLGR5_ecto. Single dilution series. (j) Binding of Rspo_Fu1–Fu2 chimeras to ZNRF3_ecto. (k) Binding of the preformed hLGR5_ecto,lr–Rspo2_Fu1–Fu2 complex to ZNRF3_ecto dimer interface mutants.