Crystal structure of the ZNRF3-RSPO1 complex.

<div><p>A.Cα trace with transparent surface representation of ZNRF3 (green) and RSPO1 (orange). ZNRF3 makes contacts to the β-hairpins 1-2 of the Fu1 domain of ZNRF3.</p> <p>B.Two views of the binding sites with interface residues indicated.</p> <p>C.Identical (red) and conserved (orange) residues between ZNRF3 and RNF43 are shown in surface representation; two views of ZNRF3 are shown. Identical residues are labeled.</p></div

ZNRF3-RSPO1 binding site coincides with LGR5-RSPO1 ‘trans’ site.

<p>Shown in cartoon with transparent surface representation are the crystal structure of the 2:2 LGR5-RSPO1 complex (PDB code 4BSR) and the structure of ZNRF3-RSPO1 superimposed on RSPO1 on the right-hand side (orange), only the Cα trace of ZNRF3 (red) is shown for clarity. The overlapping LGR5 chain is shown in blue. The remaining part of the 2:2 LGR5-RSPO1, i.e. left-hand side RSPO1 and right-hand side LGR5, is shown in grey.</p

Flexible hinge in RSPO1 and binding of ZNRF3 to RSPO1 and LGR5-RSPO1.

<div><p>A.Overlay of four representative RSPO1 structures in two orientations with free RSPO1 (grey; PDB code 4BSO), RSPO1 in LGR5-RSPO1-RNF43 complex (blue, PDB code 4KNG) and RSPO1 in complex with ZNRF3 (orange and red).</p> <p>B.Representative SPR dose-response curve used to determine equilibrium binding affinity of LGR5-RSPO1 or RSPO1 to ZNRF3, as described in Material and Methods. Standard deviations are calculated from four experiments.</p></div