Plant photoreceptors and their signaling components compete for COP 1 binding via VP peptide motifs
2019; Springer Nature; Volume: 38; Issue: 18 Linguagem: Inglês
10.15252/embj.2019102140
ISSN1460-2075
AutoresKelvin Lau, Roman Podolec, Richard Chappuis, Roman Ulm, Michael Hothorn,
Tópico(s)Plant Molecular Biology Research
ResumoArticle15 July 2019Open Access Source DataTransparent process Plant photoreceptors and their signaling components compete for COP1 binding via VP peptide motifs Kelvin Lau Kelvin Lau orcid.org/0000-0002-9040-7597 Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland Search for more papers by this author Roman Podolec Roman Podolec orcid.org/0000-0003-2998-7892 Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, Geneva, Switzerland Search for more papers by this author Richard Chappuis Richard Chappuis Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland Search for more papers by this author Roman Ulm Corresponding Author Roman Ulm [email protected] orcid.org/0000-0001-8014-7392 Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, Geneva, Switzerland Search for more papers by this author Michael Hothorn Corresponding Author Michael Hothorn [email protected] orcid.org/0000-0002-3597-5698 Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland Search for more papers by this author Kelvin Lau Kelvin Lau orcid.org/0000-0002-9040-7597 Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland Search for more papers by this author Roman Podolec Roman Podolec orcid.org/0000-0003-2998-7892 Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, Geneva, Switzerland Search for more papers by this author Richard Chappuis Richard Chappuis Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland Search for more papers by this author Roman Ulm Corresponding Author Roman Ulm [email protected] orcid.org/0000-0001-8014-7392 Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, Geneva, Switzerland Search for more papers by this author Michael Hothorn Corresponding Author Michael Hothorn [email protected] orcid.org/0000-0002-3597-5698 Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland Search for more papers by this author Author Information Kelvin Lau1,3,‡, Roman Podolec1,2,‡, Richard Chappuis1, Roman Ulm *,1,2 and Michael Hothorn *,1 1Department of Botany and Plant Biology, Section of Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland 2Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, Geneva, Switzerland 3Present address: Protein Production and Structure Core Facility, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +41-22-3793650; E-mail: [email protected] *Corresponding author. Tel: +41-22-3793013; E-mail: [email protected] The EMBO Journal (2019)38:e102140https://doi.org/10.15252/embj.2019102140 See also: Q Wang & C Lin (September 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Plants sense different parts of the sun's light spectrum using distinct photoreceptors, which signal through the E3 ubiquitin ligase COP1. Here, we analyze why many COP1-interacting transcription factors and photoreceptors harbor sequence-divergent Val-Pro (VP) motifs that bind COP1 with different binding affinities. Crystal structures of the VP motifs of the UV-B photoreceptor UVR8 and the transcription factor HY5 in complex with COP1, quantitative binding assays, and reverse genetic experiments together suggest that UVR8 and HY5 compete for COP1. Photoactivation of UVR8 leads to high-affinity cooperative binding of its VP motif and its photosensing core to COP1, preventing COP1 binding to its substrate HY5. UVR8–VP motif chimeras suggest that UV-B signaling specificity resides in the UVR8 photoreceptor core. Different COP1–VP peptide motif complexes highlight sequence fingerprints required for COP1 targeting. The blue-light photoreceptors CRY1 and CRY2 also compete with transcription factors for COP1 binding using similar VP motifs. Thus, our work reveals that different photoreceptors and their signaling components compete for COP1 via a conserved mechanism to control different light signaling cascades. Synopsis Light-activated plant photoreceptors bind and inhibit the E3 ubiquitin ligase COP1, thus protecting downstream transcription factors from degradation. X-ray structures, quantitative binding assays and reverse genetics show that photoreceptors and transcription factors compete for COP1 binding via Val-Pro (VP) motifs. COP1 binds to VP motifs of photoreceptors (e.g. UVR8, CRY1 and CRY2) and downstream transcription factors, such as HY5, HFR1 and CO. COP1 binds more strongly to its targets HY5 and CO than to the corresponding photoreceptors UVR8 and CRY2 in the absence of light stimulation. UV-B-activated UVR8 exhibits high-affinity, cooperative binding to COP1, allowing it to displace HY5 from COP1. Light activation also increases CRY2 affinity towards COP1, suggesting a common regulatory mechanism. Introduction Flowering plants etiolate in darkness, manifested by the rapid elongation of the embryonic stem, the hypocotyl, and closed and underdeveloped embryonic leaves, the cotyledons. Under light and upon photoreceptor activation, seedlings de-etiolate and display a photomorphogenic phenotype, characterized by a short hypocotyl and open green cotyledons, enabling a photosynthetic lifestyle (Gommers & Monte, 2018). The constitutively photomorphogenic 1 (cop1) mutant displays a light-grown phenotype in the dark, including a short hypocotyl, and open and expanded cotyledons. COP1 is thus a crucial repressor of photomorphogenesis (Deng et al, 1991). COP1 contains an N-terminal zinc finger, a central coiled-coil, and a C-terminal WD40 domain, which is essential for proper COP1 function (Deng et al, 1992; McNellis et al, 1994). Light-activated phytochrome, cryptochrome, and UVR8 photoreceptors inhibit COP1's activity (von Arnim & Deng, 1994; Hoecker, 2017; Podolec & Ulm, 2018). Although COP1 can act as a stand-alone E3 ubiquitin ligase in vitro (Saijo et al, 2003; Seo et al, 2003), it forms higher-order complexes in vivo, for example, with SUPPRESSOR OF PHYA-105 (SPA) proteins (Hoecker & Quail, 2001; Zhu et al, 2008; Ordoñez-Herrera et al, 2015). COP1 can also act as a substrate adaptor in CULLIN4–DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1)-based heteromeric E3 ubiquitin ligase complexes (Chen et al, 2010). These different complexes may finetune COP1's activity toward different substrates (Ren et al, 2019). COP1 regulates gene expression and plays a central role as a repressor of photomorphogenesis by directly modulating the stability of transcription factors that control the expression of light-regulated genes (Lau & Deng, 2012; Podolec & Ulm, 2018). For example, the bZIP transcription factor ELONGATED HYPOCOTYL 5 (HY5) acts antagonistically with COP1 (Ang et al, 1998). COP1 binding to HY5 leads to its subsequent degradation via the 26S proteasome in darkness, a process that is inhibited by light (Osterlund et al, 2000). In addition to HY5, other COP1 targets have been identified including transcriptional regulators, such as the HY5 homolog HYH (Holm et al, 2002), CONSTANS (CO) and other members of the BBX protein family (Jang et al, 2008; Liu et al, 2008; Khanna et al, 2009; Xu et al, 2016; Lin et al, 2018; Ordoñez-Herrera et al, 2018), and others such as LONG HYPOCOTYL IN FAR-RED 1 (HFR1) (Jang et al, 2005; Yang et al, 2005) and SHI-RELATED SEQUENCE 5 (SRS5) (Yuan et al, 2018). It has been suggested that specific Val-Pro (VP)-peptide motifs with a core sequence V-P-E/D-Φ-G, where Φ designated a hydrophobic residue, are able to bind the COP1 WD40 domain (Holm et al, 2001; Uljon et al, 2016). Deletion of regions containing the VP peptide motifs results in the loss of interaction of COP1 substrates with the COP1 WD40 domain (Holm et al, 2001; Jang et al, 2005; Datta et al, 2006). Recently, it has been reported that the human COP1 WD40 domain directly binds VP motifs, such as the one from the TRIB1 pseudokinase that acts as a scaffold facilitating ubiquitination of human COP1 substrates (Uljon et al, 2016; Durzynska et al, 2017; Newton et al, 2018; Kung & Jura, 2019). Arabidopsis photoreceptors for UV-B radiation (UV RESISTANCE LOCUS 8, UVR8), for blue light (cryptochrome 1 and 2, CRY1/CRY2) and for red/far-red light (phytochromes A–E), are known to repress COP1 activity in a light-dependent fashion (Yang et al, 2000, 2018a; Wang et al, 2001; Yu et al, 2007; Favory et al, 2009; Jang et al, 2010; Lian et al, 2011; Liu et al, 2011; Zuo et al, 2011; Viczián et al, 2012; Huang et al, 2013; Lu et al, 2015; Sheerin et al, 2015). UVR8 itself contains a conserved C-terminal VP peptide motif that is critical for UV-B signaling (Cloix et al, 2012; Yin et al, 2015). Moreover, overexpression of the UVR8 C-terminal 44 amino acids results in a cop-like phenotype (Yin et al, 2015). A similar phenotype has been observed when overexpressing the COP1-interacting CRY1 and CRY2 C-terminal domains (CCT; Yang et al, 2000, 2001). Indeed, CRY1 and CRY2 also contain potential VP peptide motifs within their CCT domains, but their function in blue-light signaling has not been established (Lin & Shalitin, 2003; Müller & Bouly, 2015). The presence of VP peptide motifs in different light signaling components suggests that COP1 may use a common targeting mechanism to interact with downstream transcription factors and upstream photoreceptors. Here, we present structural, quantitative biochemical, and genetic evidence for a VP peptide based competition mechanism, enabling COP1 to play a crucial role in different photoreceptor pathways in plants. Results The COP1 WD40 domain binds VP motifs from UVR8 and HY5 The WD40 domains of human and Arabidopsis COP1 directly interact with VP-containing peptides (Uljon et al, 2016). Such a VP peptide motif can be found in the UVR8 C-terminus that is not part of the UV-B-sensing β-propeller domain (Fig 1A; Kliebenstein et al, 2002; Rizzini et al, 2011; Christie et al, 2012; Wu et al, 2012), but is essential for UV-B signaling (Cloix et al, 2012; Yin et al, 2015). HY5 (Oyama et al, 1997), which is a COP1 target acting downstream of UVR8 in the UV-B signaling pathway (Ulm et al, 2004; Brown et al, 2005; Oravecz et al, 2006; Binkert et al, 2014), also contains a VP peptide motif (Fig 1A; Holm et al, 2001). UV-B absorption leads to UVR8 monomerization, COP1 binding, and subsequent stabilization of HY5 (Favory et al, 2009; Rizzini et al, 2011; Huang et al, 2013). Mutation of the HY5 VP pair to alanine (AA) stabilizes the HY5 protein (Holm et al, 2001). Figure 1. The Arabidopsis COP1 WD40 domain binds peptides representing the core VP motifs of UVR8 and HY5 with different affinities A. 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 (2,500 μM in 175 μM); HY5–COP1 (1,500 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 labeled). The UVR8 peptide from the UVR8–COP1 complex is superimposed on top in purple (with Tyr407 labeled), 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 (colors as in panel D). The following concentrations were typically used (titrant into cell): UVR8–COP1Lys422Ala (1,000 μM in 100 μM); UVR8–COP1Tyr441Ala (2,500 μM in 90 μM); UVR8–COP1Trp467Ala (2,500 μM in 90 μM); HY5–COP1Lys422Ala (1,500 μM in 175 μM); HY5–COP1Tyr441Ala (1,600 μM in 138 μM); and HY5–COP1Trp467Ala (1,600 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; and HY5–COP1Tyr441Ala = 0.92 ± 0.3 (all measurements ± standard deviation; n.d.: no detectable binding). F, G. Images of representative individuals (F) and quantification of hypocotyl lengths (G) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bars 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. H, I. Quantitative real-time PCR analysis of (H) HY5 and (I) RUP2 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 h (+ UV-B) or not (− UV-B). Error bars represent SEM of three biological replicates. Data Information: In (F–I), 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. Download figure Download PowerPoint In order to compare how the VP peptide motifs from different plant light signaling components bind COP1, we quantified the interaction of the UVR8 and HY5 VP peptides with the recombinant Arabidopsis COP1349–675 WD40 domain (termed COP1 thereafter) using isothermal titration calorimetry (ITC). We find that both peptides bind COP1 with micromolar affinity and with HY539–48 binding ~ 8 times stronger than UVR8406–413 (Fig 1B). Next, we solved crystal structures of the COP1 WD40 domain–VP peptide complexes representing UVR8406–413–COP1 and HY539–48–COP1 interactions, to 1.3 Å resolution (Fig 1C). Structural superposition of the two complexes (r.m.s.d. is ~ 0.2 Å comparing 149 corresponding Cα atoms) reveals an overall conserved mode of VP peptide binding (r.m.s.d is ~ 1.2 Å comparing six corresponding Cα atoms), with the central VP residues making hydrophobic interactions with COP1Trp467 and COP1Phe595 (buried surface area is ~ 500 Å2 in COP1; Fig 1D and Appendix Fig S1). COP1Lys422 and COP1Tyr441 form hydrogen bonds and salt bridges with either UVR8Tyr407 or HY5Arg41, both being anchored to the COP1 WD40 core (Fig 1C and D, and Appendix Fig S1), as previously seen for the corresponding TRIB1 Gln356 residue in the COP1–TRIB1 peptide complex (Uljon et al, 2016). In our HY539–48–COP1 structure, an additional salt bridge is formed between HY5Glu45 and COP1His528 (Fig 1D). In the peptides, the residues surrounding the VP core adopt different conformations in UVR8 and HY5, which may explain their different binding affinities (Fig 1B and C). We tested this by mutating residues Lys422, Tyr441, and Trp467 in the VP peptide binding pocket of COP1. Mutation of COP1Trp467 to alanine disrupts binding of COP1 to either UVR8- or HY5-derived peptides (Fig 1B and E). Mutation of COP1Tyr441 to alanine abolishes binding of COP1 to the UVR8 peptide and greatly reduces binding to the HY5 peptide (Fig 1B and E), in good agreement with our structures (Fig 1D). The COP1Lys422Ala mutant binds HY539–48 as wild type, but increases the binding affinity of UVR8406–413 ~ 10-fold (Fig 1B and E). Interestingly, COP1Lys422Ala interacts with full-length UVR8 also in the absence of UV-B in yeast two-hybrid assays, which is not detectable for wild type COP1 (Appendix Fig S2A; Rizzini et al, 2011). Moreover, COP1Lys422Ala also interacts more strongly with the constitutively interacting UVR8C44 fragment (corresponding to the C-terminal UVR8 tail containing the VP motif) when compared to wild type COP1 in yeast two-hybrid assays (Appendix Fig S2B). In contrast, COP1Tyr441Ala and COP1Trp467Ala show reduced interaction to both UVR8 and HY5 (Appendix Fig S2). A UVR8406–413–COP1Lys422Ala complex structure reveals the UVR8 VP peptide in a different conformation, with UVR8Tyr407 binding at the surface of the VP-binding pocket (Appendix Fig S3A–E). In contrast, a structure of HY539–48–COP1Lys422Ala closely resembles the wild type complex (Appendix Fig S3F). We next assessed the impact of COP1 VP peptide binding pocket mutants in UV-B signaling assays in planta. The seedling-lethal cop1-5 null mutant can be complemented by expression of YFP-COP1 driven by the CaMV 35S promoter. We introduced COP1 mutations into this construct and isolated transgenic lines in the cop1-5 background. All lines expressed the YFP-fusion protein and complemented the seedling lethality of cop1-5 (Figs 1F and EV1A). We found that cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5/Pro35S:YFP-COP1Lys422Ala transgenic lines have constitutively shorter hypocotyls when compared to wild type or cop1-5/Pro35S:YFP-COP1 control plants (Fig 1F and G), in agreement with previous work (Holm et al, 2001), suggesting partially impaired COP1 activity. This is similar to the phenotype of cop1-4 (Figs 1F and G, and EV1A–E), a weak cop1 allele that is viable but fully impaired in UVR8-mediated UV-B signaling (McNellis et al, 1994; Oravecz et al, 2006; Favory et al, 2009). In contrast, cop1-5/Pro35S:YFP-COP1Tyr441Ala showed an elongated hypocotyl phenotype when compared to wild type (Fig 1F and G), suggesting enhanced COP1 activity. Importantly, in contrast to YFP-COP1, transcriptional responses for UV-B-induced marker genes like HY5, RUP2, ELIP2, and CHS are clearly abolished in YFP-COP1Lys422Ala, YFP-COP1Tyr441Ala, or YFP-COP1Trp467Ala after 2 h of UV-B treatment (Figs 1H and I, and EV1B and C). These responses represent an early read-out of UV-B signaling, which has been previously linked to UVR8-COP1-HY5 (Brown et al, 2005; Oravecz et al, 2006; Favory et al, 2009; Binkert et al, 2014). Surprisingly, however, the YFP-COP1Lys422Ala line showed strongly reduced UVR8 levels (Fig EV1A), despite showing normal UVR8 transcript levels (Fig EV1F and G), precluding any conclusion of the mutation's effect on UV-B signaling per se. In contrast, YFP-COP1Tyr441Ala and YFP-COP1Trp467Ala were impaired in UV-B signaling, despite showing wild type UVR8 protein levels (Fig EV1A). This indicates strongly reduced UVR8 signaling, in agreement with the reduced affinity of the COP1 mutant proteins versus UVR8406–413 in vitro (Fig 1E). Together, our crystallographic, quantitative biochemical, and functional assays suggest that UVR8 and HY5 can specifically interact with the COP1 WD40 domain using sequence-divergent VP motifs and that mutations in the COP1 VP-binding site can modulate these interactions and impair UVR8 signaling. Click here to expand this figure. Figure EV1. Characterization of COP1 mutant lines A. Immunoblot analysis of YFP-COP1, UVR8, and actin (loading control) protein levels in lines shown in Fig 1F. Seedlings were grown for 4 days under white light. B, C. Quantitative real-time PCR analysis of (B) ELIP2 and (C) CHS expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 h (+ UV-B) or not (− UV-B). Error bars represent SEM of three biological replicates. D, E. Images of representative individuals (D) and quantification of hypocotyl lengths (E) of 4-day-old seedlings grown in darkness. The scale bar represents 5 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. F, G. Quantitative real-time PCR analysis of (F) UVR8 and (G) COP1 expression in wild type (Ws), uvr8-7, cop1-4 and cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, and cop1-5/Pro35S:YFP-COP1Trp467Ala seedlings grown for 4 days under white light. Error bars represent SEM of 3 biological replicates. Data Information: In (A–G), 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, and cop1-5/Pro35S:YFP-COP1Trp467Ala seedlings. #1 and #2: independent transgenic lines. Source data are available online for this figure. Download figure Download PowerPoint High-affinity, cooperative binding of photoactivated UVR8 HY5 levels are stabilized in a UVR8-dependent manner under UV-B light (Favory et al, 2009; Huang et al, 2013). We hypothesized that COP1 is inactivated under UV-B light, by activated UVR8 preventing HY5 from interacting with COP1. Our analysis of the isolated VP peptide motifs of UVR8 and HY5 suggests that UVR8 cannot efficiently compete with HY5 for COP1 binding. However, it has been previously found that the UVR8 β-propeller core can interact independently of its VP motif with the COP1 WD40 domain (Yin et al, 2015). We thus quantified the interaction of UV-B-activated full-length UVR8 with the COP1 WD40 domain. Recombinant UVR8 expressed in insect cells was purified to homogeneity, monomerized under UV-B, and analyzed in ITC and grating-coupled interferometry (GCI) binding assays. We found that UV-B-activated full-length UVR8 binds COP1 with a dissociation constant (Kd) of ~ 150 nM in both quantitative assays (Fig 2A and B) and ~ 10 times stronger than non-photoactivated UVR8 (Appendix Fig S4A). This ~ 1,000-fold increase in binding affinity compared to the UVR8406–413 peptide indicates cooperative binding of the UVR8 β-propeller core and the VP peptide motif. In line with this, UV-B-activated UVR8 monomers interact with the COP1 WD40 domain in analytical size-exclusion chromatography experiments, while the non-activated UVR8 dimer shows no interaction in this assay (Fig EV2A). Figure 2. High-affinity cooperative binding of activated full-length UVR8 to the COP1 WD40 domain is mediated by its UV-B-activated β-propeller core and its C-terminal VP peptide motif ITC assay between the COP1 WD40 domain and full-length UVR8 pre-monomerized by UV-B light. Integrated heats are shown in solid, purple squares. For comparison, an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain (from Fig 1B) is also shown in open, purple squares. The following concentrations were typically used (titrant into cell): COP1–UVR8 + UV-B (130 μM in 20 μM). The inset shows the dissociation constant (Kd), stoichiometry of binding (N) (± standard deviation). Binding kinetics of UVR8 pre-monomerized by UV-B versus the COP1 WD40 domain obtained by grating-coupled interferometry (GCI). Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand—COP1 (2,000 pg/mm2); analyte—UVR8 + UV-B (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant. The domain organization of Arabidopsis UVR8. It consists of a UV-B-sensing β-propeller core (residues 12–381) and a long C-terminus containing the VP motif. Constructs and peptides used and their residue endings are indicated. Binding kinetics of UVR8ValPro/AlaAla pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand—COP1 (2,000 pg/mm2); analyte—UVR8ValPro/AlaAla +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant. Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of HY5 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand—COP1 (2,000 pg/mm2); analyte—HY5 (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant. Yeast 3-hybrid analysis of the COP1–HY5 interaction in the presence of UVR8. (Top) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (− UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (− Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for three biological repetitions are shown. (Bottom) representative filter lift assays. AD, activation domain; BD, DNA binding domain; Met, methionine. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Only UV-B-activated UVR8 and mutants are able to bind COP1 in size-exclusion chromatography binding assays Coomassie-stained SDS–PAGE gels from a size-exclusion chromatography binding assay between the COP1 WD40 domain and UVR8 in the presence and absence of UV-B. Purified monomeric UVR8 ˜ 50 kDa. Four μM of each protein or a mix of proteins was loaded on to a Superdex 200 Increase 10/300 GL column. Indicated fractions were taken each of the size-exclusion chromatography runs and separated on a 10% SDS–PAGE gel. Coomassie-stained SDS–PAGE gels from a size-exclusion chromatography binding assay between the COP1 WD40 domain and UVR812–381 in the presence and absence of UV-B. Purified monomeric UVR812–381 ˜ 40 kDa. Four μM of each protein or a mix of proteins was loaded on to a Superdex 200 Increase 10/300 GL column. Indicated fractions were taken each of the size-exclusion chromatography runs and separated on a 10% SDS–PAGE gel. Coomassie-stained SDS–PAGE gels from a size-exclusion chromatography binding assay between COP1 and UVR8ValPro/AlaAla pre-monomerized by UV-B. Purified monomeric UVR8ValPro/AlaAla ˜ 50 kDa, COP1 WD40 a smear ˜ 25–40 kDa. Four μM of each protein was loaded independently or mixed together. Indicated fractions were taken each of the size-exclusion chromatography runs and separated on a 10% SDS–PAGE gel. Source data are available online for this figure. Download figure Download PowerPoint As the interaction of full-length UVR8 is markedly stronger than the isolated UVR8 VP peptide, we next dissected the contributions of the individual UVR8 domains to COP1 binding (Fig 2C). We find that the UV-B-activated UVR8 β-propeller core (UVR812–381) binds COP1 with a Kd of ~ 0.5 μM and interacts with the COP1 WD40 domain in size-exclusion chromatography experiments (Fig EV2B and Appendix Fig S4B). The interaction is strengthened when the C-terminus is extended to include the VP peptide motif (UVR812–415; Appendix Fig S4B and C). Mutation of the UVR8 VP peptide motif to alanines results in ~ 20-fold reduced binding affinity when compared to the wild type protein (Fig 2D). However, the mutant photoreceptor is still able to form complexes with the COP1 WD40 domain in size-exclusion chromatography assays (Fig EV2C). We could not detect sufficient binding enthalpies to monitor the binding of UVR8ValPro/AlaAla to COP1 in ITC assays nor detectable signals in GCI experiments in the absence of UV-B (Appendix Fig S5). The COP1Lys422Ala mutant binds UV-B-activated full-length UVR8 with wild type affinity, while COP1Trp467Ala binds ~ 5 times more weakly (Appendix Fig S6A and B). Mutations targeting both COP1 and the UVR8 C-terminal VP peptide motif decrease their binding affinity even further (Appendix Fig S6C). Thus, full-length UVR8 uses both its β-propeller photoreceptor core and its C-terminal VP peptide to cooperatively bind the COP1 WD40 domain when activated by UV-B light. We next asked whether UV-B-activated full-length UVR8 could compete with HY5 for binding to COP1. We produced the full-length HY5 protein in insect cells and found that it binds the COP1 WD40 domain with a Kd of ~ 1 μM in GCI assays (Fig 2E). For comparison, the isolated HY5 VP peptide binds COP1 with a Kd of ~ 20 μM (Fig 1B). This would indicate that only the UV-B-activated UVR8 and not ground-state UVR8 (Kd ~ 150 nM versus ~ 1 μM, see above) can efficiently compete with HY5 for COP1 binding. We tested this hypothesis in yeast 3-hybrid experiments. We confirmed that HY5 interacts with COP1 in the absence of UVR8 and that this interaction is specifically abolished in the presence of UVR8 and UV-B light (Fig 2F). We
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