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. 2017 Mar 15;6:e20882. doi: 10.7554/eLife.20882

Figure 6. HD3 binds directly to ChiLS.

(A) Top, sequence alignments of HD3; yellow, conserved residues; grey, semi-conserved residues. Bottom, position-specific alignment (HMMER; Finn et al., 2011) for B9L HD3; yellow, conserved tryptophan and phenylalanine/tyrosine doublet. (B) Predicted structure of HD3 (by I-TASSER), with heat-map indicating relative line broadenings upon incubation of 15N-HD3 with ChiLS (see D), ranging from 80% (red) to 40% (yellow); grey, proline (not detectable). (C) TALOS+ predictions of α-helicity of HD3, based on backbone secondary chemical shifts in Figure 6—figure supplement 1; for each position, the rigidity index (RCI-S2) and helical probability (p) are indicated (pink, N-terminal linker residues). (D–F) Overlays of HSQC spectra of 100 μM 15N-labeled (D, E) wt HD3 or (F) W520R mutant alone (red), and probed with 300 μM (D, F) MBP-Chip205-436-Lip-SSDP1-92 or (E) Lip-SSDP1-92 (blue); line broadening owing to ChiLS binding to wt HD3 (D) results in the disappearance of selected resonances in the blue (HD3 + ChiLS) spectrum, revealing the corresponding resonances from the red (HD3-only) spectrum.

DOI: http://dx.doi.org/10.7554/eLife.20882.016

Figure 6.

Figure 6—figure supplement 1. Assignment of the [1H-15N]-HSQC spectrum of HD3.

Figure 6—figure supplement 1.

Assignments of 1H-15N correlations for the backbone amide resonances of [15N-13C]-Lip-HD3 residues, after overlay with [15N-13C]-Lip (to identify Lip residues).
DOI: http://dx.doi.org/10.7554/eLife.20882.017
Figure 6—figure supplement 2. Modification of the chip phenotype by loss of HD3.

Figure 6—figure supplement 2.

(A) Genetic interactions between chipe55/+ heterozygosity (causing wing margin defects, marked by arrowheads) and heterozygosity of Wnt pathway components; representative wings are shown. Note that this phenotype is exacerbated by lowering the dose of SSDP protein (as expected, given that Chip and SSDP form an obligatory complex; see Fiedler et al., 2015), but ameliorated by lowering the dose of associated proteins such as dTCF, Armadillo, Pygo, Legless or Groucho, possibly because normal levels of the latter sequester some of the limiting amounts of Chip (which is reduced in these heterozygotes). Note the strong suppression by the HD3 allele, possibly indicative of the direct binding of Chip to this domain. (B) Restoration of normal wing margins by heterozygosity of genes, as indicated (see also A); black, partial restoration (only anterior margin); white, restoration of both margins.
DOI: http://dx.doi.org/10.7554/eLife.20882.018