The Arabidopsis COP1 WD40 domain binds peptides representing the core VP motifs of UVR8 and HY5 with different affinities.Figure 1A. Alignment of the UVR8 and HY5 VP-peptide motifs. The conserved VP pairs are highlighted in red and the anchor residues in orange. B. ITC binding assays of the UVR8 and HY5 VP-peptides versus the COP1 WD40 domain. The top panel represents the heats detected during each injection. The bottom panel represents the integrated heats of each injection, fitted to a one-site binding model (solid line). The following concentrations were typically used (titrant into cell): UVR8 - COP1 (2500 μM in 175 μM); HY5 - COP1 (1500 μM in 175 μM). The insets show the dissociation constants (Kd) and stoichiometries of binding (N) (± standard deviation). C. Superposition of the X-ray crystal structures of the HY5 and UVR8 peptides in the VP-peptide binding site of the COP1 WD40 domain. COP1 is depicted in surface representation and belongs to the HY5 - COP1 complex. The HY5 peptide is depicted in green in ball-and-stick representation (with Arg41 labelled). The UVR8 peptide from the UVR8 - COP1 complex is superimposed on top in purple (with Tyr407 labelled), depicted in ball-and-stick representation. The surface of COP1 has been clipped to better visualize the anchor residue in the COP1 WD40 domain. D. Ribbon diagram depicting the VP-binding site of COP1 (blue) bound to the HY5 peptide (green). Residues Lys422, Tyr441 and Trp467 are highlighted with a colored box in cyan, magenta and red, respectively. E. ITC assays of the HY5 and UVR8 VP-peptides versus the different COP1 WD40 domain mutants The following concentrations were typically used (titrant into cell): UVR8 - COP1Lys422Ala (1000 μM in 100 μM); UVR8 - COP1Tyr441Ala (2500 μM in 90 μM); UVR8 - COP1Trp467Ala (2500 μM in 90 μM); HY5 - COP1Lys422Ala (1500 μM in 175 μM); HY5 - COP1Tyr441Ala (1600 μM in 138 μM); HY5 - COP1Trp467Ala (1600 μM in 112 μM). The insets show the dissociation constants (Kd). The stoichiometries of binding (N) for UVR8 - COP1Lys422Ala = 0.87 ± 0.2; HY5 - COP1Lys422Ala = 0.83 ± 0.2; HY5 - COP1Tyr441Ala = 0.92 ± 0.3 (All measurements ± standard deviation; n.d.: no detectable binding). Images of representative individuals (F) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Data Information , lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines. quantification of hypocotyl lengths (G) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines. Quantitative real-time PCR analysis of (H) HY5 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines. Quantitative real-time PCR analysis of (I) RUP2 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.