Native Mass Spectrometry Reveals the Conformational Diversity of the UVR8 Photoreceptor

Abstract

UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light resulting in dissociation into monomers, which are considered to be the active state and comprise a β-propeller core domain and intrinsically disordered N-and C-terminal tails. The C-terminus is required for functional binding to signalling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photo-activation. We show that, whilst truncated UVR8 photoconverts from a single conformation of dimers to a single monomer conformation, the full-length protein exist in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C-terminus is primed for activation. In the monomer the extended C-terminus destabilises the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature. TOC Graphic

Plant photoreceptors and their signaling components compete for binding to the ubiquitin ligase COP1 using their VP-peptide motifs

“…For both types of photoreceptors, interaction through a linear VP-peptide motif and a folded, light-regulated interaction domain leads to cooperative, high-affinity binding of the activated photoreceptor to COP1. We propose that in response to UV-B light, UVR8 dimers monomerize, exposing a new interaction surface that binds to the COP1 WD40 domain and releases the UVR8 C-terminal VP motif from structural restraints that prevent its interaction with COP1 in the absence of UV-B (Yin et al, 2015;Heilmann et al, 2016;Wu et al, 2019;Camacho et al, 2019). Similarly, the VP motif in the CCT domain of cryptochromes may become exposed and available for interaction upon blue-light activation of the photoreceptor (Müller and Bouly, 2015;Wang et al, 2018).…”

Plant photoreceptors and their signaling components compete for COP 1 binding via VP peptide motifs

“…For both types of photoreceptors, interaction through a linear VP peptide motif and a folded, light-regulated interaction domain leads to cooperative, high-affinity binding of the activated photoreceptor to COP1. We propose that in response to UV-B light, UVR8 dimers monomerize, exposing a new interaction surface that binds to the COP1 WD40 domain and releases the UVR8 C-terminal VP motif from structural restraints that prevent its interaction with COP1 in the absence of UV-B (Yin et al, 2015;Heilmann et al, 2016;Camacho et al, 2019;Wu et al, 2019). Similarly, the VP motif in the CCT domain of cryptochromes may become exposed and available for interaction upon blue-light activation of the photoreceptor (Mü ller & Bouly, 2015; Wang et al, 2018).…”

A FRET method for investigating dimer/monomer status and conformation of the UVR8 photoreceptor

“…Several previous studies using a number of techniques have provided evidence for conformational change of UVR8 associated with UV-B photoreception 5,10–12. In addition, a recent report shows that the monomer can adopt multiple conformations, including partial unraveling of the β-propeller 13. Production of the most extended conformation may be required to facilitate interaction with other proteins, in particular at the C-terminal region, to initiate signaling.…”

“…There is evidence that UV-B photoreception by UVR8, with the resulting monomerization results in conformational changes to the protein 5,10–13. However, since the N- and C-termini do not appear in the crystal structures of UVR8,3,4,12 detailed information about their location and conformational changes following monomerization are lacking.…”

“…The interaction between UVR8 and COP1 is UV-B-dependent14 and involves binding to a specific region within the C-terminus 15,16. A recent study13 indicates that the UVR8 monomer can adopt multiple conformations with an extended C-terminus.…”