A Photo-Labile Thioether Linkage to Phycoviolobilin Provides the Foundation for the Blue/Green Photocycles in DXCF-Cyanobacteriochromes

190

307

Many aspects of plant photomorphogenesis are controlled by the phytochrome (Phy) family of bilin-containing photoreceptors that detect red and far-red light by photointerconversion between a dark-adapted Pr state and a photoactivated Pfr state. Whereas 3D models of prokaryotic Phys are available, models of their plant counterparts have remained elusive. Here, we present the crystal structure of the photosensing module (PSM) from a seed plant Phy in the Pr state using the PhyB isoform from Arabidopsis thaliana. The PhyB PSM crystallized as a head-to-head dimer with strong structural homology to its bacterial relatives, including a 5(Z)syn, 10(Z)syn, 15(Z)anti configuration of the phytochromobilin chromophore buried within the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain, and a well-ordered hairpin protruding from the Phy-specific domain toward the bilin pocket. However, its Per/Arnt/Sim (PAS) domain, knot region, and helical spine show distinct structural differences potentially important to signaling. Included is an elongated helical spine, an extended β-sheet connecting the GAF domain and hairpin stem, and unique interactions between the region upstream of the PAS domain knot and the bilin A and B pyrrole rings. Comparisons of this structure with those from bacterial Phys combined with mutagenic studies support a toggle model for photoconversion that engages multiple features within the PSM to stabilize the Pr and Pfr end states after rotation of the D pyrrole ring. Taken together, this Arabidopsis PhyB structure should enable molecular insights into plant Phy signaling and provide an essential scaffold to redesign their activities for agricultural benefit and as optogenetic reagents.G iven the importance of sunlight to their survival and growth, plants have adopted a collection of photoreceptors and interconnected signaling cascades to optimize their photosynthetic potential and synchronize their lifecycles with circadian and seasonal rhythms. Chief among these are the phytochromes (Phys), a family of bilin (or open-chain tetrapyrrole)-containing red/far-red light-absorbing photoreceptors that provides spatial and time-dependent information by sensing the fluence rate, direction, duration, and color of a plant's light environment (1, 2). This information then regulates nearly all aspects of plant growth and development from seed germination to senescence. Notably, seed plants typically express three Phy isoforms (PhyA, PhyB, and PhyC) that control distinct and overlapping photoresponses, with PhyB having a dominant role in green tissues (2, 3).Phys are homodimers with each sister polypeptide divided into an N-terminal photosensory module (PSM) that absorbs light followed by an output module (OPM) that promotes dimerization and presumably, relays the light signals (1, 4). The PSM sequentially contains a Per/Arnt/Sim (PAS) domain of unknown function, a cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain that cradles the bilin, and a Phy-specific (PHY) domain that stabilizes the photoactivated ...

Section: Resultssupporting

confidence: 74%

“…2C). A 180°rotation would then expose the D-ring functionalities to a nonideal environment, which recovers by sliding the bilin within the GAF pocket as illustrated in the paired end-state GAF domain models from the Phy relative PixJ from Thermosynechococcus elongatus and the PSM of Pa-BphP (8,13,15,19).…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of the photosensing module from a red/far-red light-absorbing plant phytochrome

Burgie

Bussell

Walker

et al. 2014

190

307

“…The mechanistic basis for sensing blue light with bilins is well documented through studies of CBCRs: a second Cys residue forms a covalent linkage to the C10 methine bridge of the bilin chromophore, shortening the conjugated system and blue-shifting the absorption (42,43,67,68). One such naturally occurring two-Cys photocycle has been reported in phytochromes to date, in the cyanobacterial protein NpF1183 (42).…”

Section: Discussionmentioning

confidence: 99%

Eukaryotic algal phytochromes span the visible spectrum

Rockwell

Duanmu

Martin

et al. 2014

158

177

Plant phytochromes are photoswitchable red/far-red photoreceptors that allow competition with neighboring plants for photosynthetically active red light. In aquatic environments, red and far-red light are rapidly attenuated with depth; therefore, photosynthetic species must use shorter wavelengths of light. Nevertheless, phytochrome-related proteins are found in recently sequenced genomes of many eukaryotic algae from aquatic environments. We examined the photosensory properties of seven phytochromes from diverse algae: four prasinophyte (green algal) species, the heterokont (brown algal) Ectocarpus siliculosus, and two glaucophyte species. We demonstrate that algal phytochromes are not limited to red and far-red responses. Instead, different algal phytochromes can sense orange, green, and even blue light. Characterization of these previously undescribed photosensors using CD spectroscopy supports a structurally heterogeneous chromophore in the far-red–absorbing photostate. Our study thus demonstrates that extensive spectral tuning of phytochromes has evolved in phylogenetically distinct lineages of aquatic photosynthetic eukaryotes.

“…CBCR subfamilies that sense light in the near-UV to blue region (330-470 nm) have been studied extensively. Such CBCRs combine bilin photoisomerization with subsequent formation or elimination of a second thioether linkage to the bilin C10 atom, shortening the conjugated π system to induce a remarkable spectral shift (19,21,23,(25)(26)(27)(28)(29). In such "two-Cys photocycles," the reactive thiol group is supplied by a second conserved Cys residue within the GAF domain.…”

mentioning

confidence: 99%

Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle

Hirose

Rockwell

Nishiyama

et al. 2013

142

264

Cyanobacteriochromes (CBCRs) are cyanobacterial members of the phytochrome superfamily of photosensors. Like phytochromes, CBCRs convert between two photostates by photoisomerization of a covalently bound linear tetrapyrrole (bilin) chromophore. Although phytochromes are red/far-red sensors, CBCRs exhibit diverse photocycles spanning the visible spectrum and the near-UV (330-680 nm). Two CBCR subfamilies detect near-UV to blue light (330-450 nm) via a "two-Cys photocycle" that couples bilin 15Z/15E photoisomerization with formation or elimination of a second bilincysteine adduct. On the other hand, mechanisms for tuning the absorption between the green and red regions of the spectrum have not been elucidated as of yet. CcaS and RcaE are members of a CBCR subfamily that regulates complementary chromatic acclimation, in which cyanobacteria optimize light-harvesting antennae in response to green or red ambient light. CcaS has been shown to undergo a green/red photocycle: reversible photoconversion between a green-absorbing 15Z state ( 15Z P g ) and a red-absorbing 15E state ( 15E P r ). We demonstrate that RcaE from Fremyella diplosiphon undergoes the same photocycle and exhibits light-regulated kinase activity. In both RcaE and CcaS, the bilin chromophore is deprotonated as 15Z P g but protonated as 15E P r . This change of bilin protonation state is modulated by three key residues that are conserved in green/red CBCRs. We therefore designate the photocycle of green/red CBCRs a "protochromic photocycle," in which the dramatic change from green to red absorption is not induced by initial bilin photoisomerization but by a subsequent change in bilin protonation state.light sensing | phycobiliprotein | signal transduction | spectral tuning | two-component signaling P hytochrome photosensors initially were discovered in plants and later found in cyanobacteria, nonoxygenic photosynthetic bacteria, nonphotosynthetic bacteria, fungi, and algae (1, 2). These photoreceptors bind linear tetrapyrrole (bilin) chromophores within a conserved GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domain via a covalent thioether linkage to a conserved Cys residue (Fig. S1A)(3-6). Upon illumination, phytochromes reversibly convert between a red-absorbing dark state and a far-red-absorbing photoproduct. This red/far-red photocycle is triggered by photoisomerization of the bilin 15,16-double bond between the 15Z and 15E configurations (7,8), with 15Z giving red absorption and 15E far-red absorption (4, 6, 9). In phytochromes, the conjugated π system of the bilin is protonated in both photostates, and this protonation is necessary to maintain the red and far-red absorption (10-12). Conserved GAF residues supply a hydrogen bond network to tune the chemical and spectral properties of the bilin (Fig. S1B).* Cyanobacteriochromes (CBCRs) are widespread cyanobacterial photosensors with phytochrome-related GAF domains (1,2,13,14). Although CBCRs also convert between two photostates via bilin photoisomerization at C15, they exhibit much more spe...