Fig. 2: Binding of Axin and GSK3 to the PPPSPAT element of LRP6.
a Top, cartoon of Axin1 (as in Fig. 1a), with extents of A3Axin (A3) and A5Axin (A5) fragments indicated; below, Lip-A3Axin comprising helical elements (LIRα, GIR) as predicted by AF2 and AF3, with sequence underneath; conserved residues mutated in DED4 > A and RKR > A in bold; red, Axin LIR. b BEST-TROSY spectra of 80 μM 15N-labeled Lip-L1LRP6 recorded by itself (black) or incubated with 80 μM ubiquitin, wt Lip-A5Axin or its DED4 > A mutant (red), as indicated in color key above panels. c Peak heights in BEST-TROSY spectra of 80 μM 15N-labeled Lip-L1LRP6 by itself (brown) or incubated with 80 μM Lip-A3Axin (blue) and corresponding bleach map underneath, depicting percentage attenuation of peak height by incubation with ligand ranging from strong (red) to weak (yellow); mean attenuations (x̅) and means + 2 standard deviations (+2σ) are given on the right in this and subsequent bleach maps. Gray bars indicate residues for which NMR peaks are not assigned or obscured by overlap. d Bleach map generated from BEST-TROSY spectrum of 80 μM 15N-labeled Lip-L1LRP6 + /−80 μM of GSK3β-K85R. e Bleach map generated from 2D-CON spectrum of 150 μM 13C/15N-labeled Lip-L1LRP6 + /−150 μM Lip-A3Axin (see also Supplementary Fig. 2d, e). Bleach maps generated from BEST-TROSY spectra of 80 μM 15N-labeled Lip-L1LRP6 + /−80 μM (f) Axin Lip-LIRα, (g) Lip-DED4 > A, (h) Lip-DE > A, (i) Lip-D4 > A or (j) Lip-RKR > A (all mutants in A5Axin). Bottom, sequence of Lip-L1LRP6 with PPPSPxS motifs A and B (bold), PPPSPAT element (gray underlay) and S1490 (red); blue, YYYF > A substitution, to render Lip-L1LRP6 soluble in aqueous buffer (see Methods).
