Unusual Spectral Properties of Bacteriophytochrome Agp2 Result from a Deprotonation of the Chromophore in the Red-absorbing Form Pr

Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pK_a is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pK_a shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion.     

Arabidopsis PHYTOCHROME INTERACTING FACTOR Proteins Promote Phytochrome B Polyubiquitination by COP1 E3 Ligase in the Nucleus

Many plant photoresponses from germination to shade avoidance are mediated by phytochrome B (phyB). In darkness, phyB exists as the inactive Pr in the cytosol but upon red (R) light treatment, the active Pfr translocates into nuclei to initiate signaling. Degradation of phyB Pfr likely regulates signal termination, but the mechanism is not understood. Here, we show that phyB is stable in darkness, but in R, a fraction of phyB translocates into nuclei and becomes degraded by 26S proteasomes. Nuclear phyB degradation is mediated by COP1 E3 ligase, which preferentially interacts with the PhyB N-terminal region (PhyB-N). PhyB-N polyubiquitination by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in vitro can be enhanced by different PHYTOCHROME INTERACTING FACTOR (PIF) proteins that promote COP1/PhyB interaction. Consistent with these results, nuclear phyB accumulates to higher levels in pif single and double mutants and in cop1-4. Our results identify COP1 as an E3 ligase for phyB and other stable phytochromes and uncover the mechanism by which PIFs negatively regulate phyB levels.     

Structure of the Biliverdin Cofactor in the Pfr State of Bathy and Prototypical Phytochromes

Phytochromes act as photoswitches between the red- and far-red absorbing parent states of phytochromes (Pr and Pfr). Plant phytochromes display an additional thermal conversion route from the physiologically active Pfr to Pr. The same reaction pattern is found in prototypical biliverdin-binding bacteriophytochromes in contrast to the reverse thermal transformation in bathy bacteriophytochromes. However, the molecular origin of the different thermal stabilities of the Pfr states in prototypical and bathy bacteriophytochromes is not known. We analyzed the structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa, the very good agreement between calculated and experimental Raman spectra of the biliverdin cofactor is in line with important conclusions of previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its mode of covalent attachment to the protein. The highly homogeneous chromophore conformation seems to be a unique property of the Pfr states of bathy phytochromes. This is in sharp contrast to the Pfr states of prototypical phytochromes that display conformational equilibria between two sub-states exhibiting small structural differences at the terminal methine bridges A-B and C-D. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp-194 that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore pocket opening a reaction channel for the thermal (ZZE → ZZZ) Pfr to Pr back-conversion.     

Light-induced global structural changes in phytochrome A regulating photomorphogenesis in plants

Phytochromes are photoreceptor proteins that monitor the light environment and regulate a variety of photomorphogenic responses to optimize the growth and development of plants. Phytochromes comprise N-terminal photosensory and C-terminal regulatory domains. They are mutually photoconvertible between a red-light-absorbing (Pr) and a far-red-light-absorbing (Pfr) form. Their interconversion by light stimuli initiates downstream signaling cascades. Here we report the molecular structures of pea phytochrome A lacking the N-terminal 52 amino-acid residues in the Pr and Pfr forms studied by small-angle X-ray scattering. A new purification protocol yielded monodispersive sample solutions. The molecular mass and the maximum dimension of Pr determined from scattering data indicated its dimeric association. The molecular structure of Pr predicted by applying the ab initio simulation method to the scattering profile was approximated as a stack of two flat bodies, comprising two lobes assignable to the functional regions. Scattering profiles recorded under red-light irradiation showed small but definite changes from those of Pr. The molecular dimensions and predicted molecular structure of Pfr suggest global structural changes such as movement of the C-terminal domains in the Pr-to-Pfr phototransformation. Red-light-induced structural changes in Pfr were reversible, mostly due to thermal relaxation processes.     

Inter-domain crosstalk in the phytochrome molecules

Phytochromes are bifunctional photoreceptors with a two-domain structure, consisting of the N-terminal photosensory domain and the C-terminal regulatory domain. The photo-induced Pr <--> Pfr phototransformation accompanies subtle conformational changes, primarily triggered by the apoprotein-chromophore interactions in the N-terminal domain. The conformational signals are subsequently transmitted to the C-terminal domain through various inter-domain crosstalks, resulting in the interaction of the activated C-terminal domain with phytochrome interacting factors. Thus the inter-domain crosstalks play critical roles in the photoactivation of the phytochromes. Protein phosphorylation, such as that of Ser-598, is implicated in this process by inducing conformational changes and by modulating inter-domain signaling.     

Functional analysis of a 450-amino acid N-terminal fragment of phytochrome B in Arabidopsis

Phytochrome, a major photoreceptor in plants, consists of two domains: the N-terminal photosensory domain and the C-terminal domain. Recently, the 651-amino acid photosensory domain of phytochrome B (phyB) has been shown to act as a functional photoreceptor in the nucleus. The phytochrome (PHY) domain, which is located at the C-terminal end of the photosensory domain, is required for the spectral integrity of phytochrome; however, little is known about the signal transduction activity of this domain. Here, we have established transgenic Arabidopsis thaliana lines expressing an N-terminal 450-amino acid fragment of phyB (N450) lacking the PHY domain on a phyB-deficient background. Analysis of these plants revealed that N450 can act as an active photoreceptor when attached to a short nuclear localization signal and beta-glucuronidase. In vitro spectral analysis of reconstituted chromopeptides further indicated that the stability of the N450 Pfr form, an active form of phytochrome, is markedly reduced in comparison with the Pfr form of full-length phyB. Consistent with this, plants expressing N450 failed to respond to intermittent light applied at long intervals, indicating that N450 Pfr is short-lived in vivo. Taken together, our findings show that the PHY domain is dispensable for phyB signal transduction but is required for stabilizing the Pfr form of phyB.     

Protein conformational changes of Agrobacterium phytochrome Agp1 during chromophore assembly and photoconversion

Phytochromes are widely distributed photochromic biliprotein photoreceptors. Typical bacterial phytochromes such as Agrobacterium Agp1 have a C-terminal histidine kinase module; the N-terminal chromophore module induces conformational changes in the protein that lead to modulation of kinase activity. We show by protein cross-linking that the C-terminal histidine kinase module of Agp1 mediates stable dimerization. The fragment Agp1-M15, which comprises the chromophore module but lacks the histidine kinase module, can also form dimers. In this fragment, dimer formation was stronger for the far-red-absorbing form Pfr than for the red-absorbing form Pr. The same or similar behavior was found for Agp1-M15Delta9N and Agp1-M15Delta18N, which lack 9 and 18 amino acids of the N-terminus, respectively. The fragment Agp1-M20, which is derived from Agp1-M15 by truncation of the C-terminal "PHY domain" (191 amino acids), can also form dimers, but dimerization is independent of irradiation conditions. The cross-linking data also showed that the PHY domain is in tight contact with Lys 16 of the protein and that the nine N-terminal amino acids mediate oligomer formation. Limited proteolysis shows that the hinge region between the chromophore module and the histidine kinase and a part of the PHY domain become exposed upon Pr to Pfr photoconversion.     

Assembly of synthetic locked chromophores with agrobacterium phytochromes Agp1 and Agp2

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15=C16 double bond is fixed in either the E or Z configuration and the C14-C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4=C5 double bond is fixed in the Z configuration, and the C5-C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4=C5 Z C5-C6 syn C15=C16 Z C14-C15 anti stereochemistry and that in the Pfr chromophore the C15=C16 double bond has isomerized to the E configuration, whereas the C14-C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5-C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs.     

Chromophore: apoprotein interactions in phytochrome A*

Phytochromes are molecular light switches by virtue of their photochromic red/far-red reversibility. The His-324 residue next to the chromophore-linked Cys-323 plays a critical role in conferring photochromism to the tetrapyrrole chromophore in native phytochrome A. The chromophore appears to be enclosed between the amphiphilic α-helical chains in a hydrophobic pocket. The absorbance maxima of both the Pr and the Pfr forms of pea phytochrome A are blue-shifted by 10 and 20 nm, respectively, upon C-terminal truncation. We speculate that the quaternary structure of the phytochrome A molecule involves some interactions of the C-terminal half with the chromophore domain. The Pfr conformation of phytochrome includes an amphiphilic α-helix of the amino terminal chain, which occurs in 113 ms after picosecond photoisomerization of the Pr form. Compared to α-helical folding, unfolding of the α-helix occurs faster in about 310 μs upon phototransformation of the Pfr form of phytochrome A. The photochromic transformation of phytochrome A modulates protein kinase-catalysed phosphorylation sites in vivo and in vitro, but only a subtle local change in conformation is detectable in the phosphorylated phytochromes. This suggests that the post-translational modification serves as a surface label, rather than a transducer-activating trigger, for the recognition of a putative phytochrome receptor.     

Light-dependent, dark-promoted interaction between Arabidopsis cryptochrome 1 and phytochrome B proteins

Plant photoreceptors transduce environmental light cues to downstream signaling pathways, regulating a wide array of processes during growth and development. Two major plant photoreceptors with critical roles in photomorphogenesis are phytochrome B (phyB), a red/far-red absorbing photoreceptor, and cryptochrome 1 (CRY1), a UV-A/blue photoreceptor. Despite substantial genetic evidence for cross-talk between phyB and CRY1 pathways, a direct interaction between these proteins has not been observed. Here, we report that Arabidopsis phyB interacts directly with CRY1 in a light-dependent interaction. Surprisingly, the interaction is light-dissociated; CRY1 interacts specifically with the dark/far-red (Pr) state of phyB, but not with the red light-activated (Pfr) or the chromophore unconjugated form of the enzyme. The interaction is also regulated by light activation of CRY1; phyB Pr interacts only with the unstimulated form of CRY1 but not with the photostimulated protein. Further studies reveal that a small domain extending from the photolyase homology region (PHR) of CRY1 regulates the specificity of the interaction with different conformational states of phyB. We hypothesize that in plants, the phyB/CRY1 interaction may mediate cross-talk between the red/far-red- and blue/UV-sensing pathways, enabling fine-tuning of light responses to different spectral inputs.     

Nuclear localization activity of phytochrome B

Phytochromes are soluble red/far-red-light photoreceptor proteins which mediate various photomorphogenic responses of plants. Despite much effort, the signal transduction mechanism of phytochrome has remained obscure. Phytochromes are encoded by a small multigene family in Arabidopsis . Among the members of the family, phytochrome A (phyA) and B (phyB) are the best characterized. PhyB contains putative nuclear localization signals within its C-terminal region. Transgenic Arabidopsis plants were produced which expressed a fusion protein consisting of GUS and C-terminal fragments of phyB. GUS staining from the fusion protein in these transgenic plants was observed in the nucleus, which suggests that the nuclear localization signal of the fragment is functional. Next, it was examined whether the endogenous phyB was detected in the nucleus. Nuclei were isolated from the light-grown wild-type Arabidopsis leaves and subjected to the immunoblot analysis. The result indicated that a substantial fraction of total phyB was recovered in the isolated nuclei. This result was further confirmed by the immunocytochemical analysis of the protoplasts. Finally, the effects of light treatments on the levels of phyB in the isolated nuclei were examined. Dark adaptation of the plants before the nuclear isolation reduced the levels of phyB. The reduction was accelerated by irradiation of plants with far-red light before the transfer to darkness. Thus, nuclear localization of phyB was suggested to be light-dependent.     

Differential interactions of phytochrome A (Pr vs. Pfr) with monoclonal antibodies probed by a surface plasmon resonance technique.

Phytochromes are red- and far-red light-reversible photoreceptors for photomorphogenesis in plants. Phytochrome A is a dimeric chromopeptide that mediates very low fluence and high irradiance responses. To analyze the surface properties of phytochrome A (phyA), the epitopes of 21 anti-phyA monoclonal antibodies were determined by variously engineered recombinant phyA proteins and the dissociation constants of seven anti-phyA monoclonal antibodies with phyA were measured using a surface plasmon resonance (SPR)-based resonant mirror biosensor (IAsys). Purified oat phyA was immobilized on the sensor surface using a carboxymethyl dextran cuvette in advance, and the interactions of each chosen monoclonal antibody against phyA in either red light absorbing form (Pr) or far-red light absorbing form (Pfr) at different concentrations were monitored. The binding profiles were analyzed using the FAST Fit program of IAsys. The resultant values of dissociation constants clearly demonstrated the differential affinities between the phyA epitopes and the monoclonal antibodies dependent upon Pr vs. Pfr conformations. Monoclonal antibody mAP20 preferentially recognized the epitope at amino acids 653-731 in the Pr form, whereas mAA02, mAP21 and mAR07/mAR08 displayed preferential affinities for the Pfr's surfaces at epitopes 494-601 (the hinge region between the N- and C-terminal domains), 601-653 (hinge in PASI domain), and 772-1128 (C-terminal domain), respectively. The N-terminal extension (1-74) was not recognized by mAP09 and mAP15, suggesting that the N-terminal extreme is not exposed in the native conformation of phyA. On the other hand, the C-terminal domain becomes apparently exposed on Pr-to-Pfr phototransformation, suggesting an inter-domain cross-talk. The use of surface plasmon resonance spectroscopy offers a new approach to study the surface properties of phytochromes associated with the photoreversible structural changes, as well as for the study of protein-protein interactions of phytochromes with their interacting proteins involved in light signaling events in plants.     

Nucleo-cytoplasmic partitioning of the plant photoreceptors phytochromes

Phytochromes in harmony with blue light photoreceptors play a major role in controlling plant growth and development from germination to seed maturation. Light absorption by phytochromes triggers a signaling cascade, phototransduction, which culminates in regulated gene expression. A major regulatory step at the cellular level, which affects specificities of light-induced physiological responses, seems to be the light-quality and light-quantity dependent nuclear import of the phytochromes themselves. The correlations found between the nuclear import of phytochromes (phyA and phyB) and various physiological responses regulated by these photoreceptors provides strong support for this hypothesis.     

Directed dimerization: an in vivo expression system for functional studies of type II phytochromes

Type II phytochromes (phy) in Arabidopsis form homodimers and heterodimers, resulting in a diverse collection of light‐stable red/far‐red (R/FR) sensing photoreceptors. We describe an in vivo protein engineering system and its use in characterizing the activities of these molecules. Using a phyB null mutant background, singly and doubly transgenic plants were generated that express fusion proteins containing the phyB–phyE N–terminal photosensory regions (NB–NE PSRs), a nuclear localization sequence, and small yeast protein domains that mediate either homodimerization or heterodimerization. Activity of NB/NB homodimers but not monomeric NB subunits in control of seedling and adult plant responses to R light is demonstrated. Heterodimers of the NB sequence with the chromophoreless NB^C357S sequence, which mimic phyB Pfr/Pr photo‐heterodimers, mediate R sensitivity in leaves and petioles but not hypocotyls. Homodimerization of the NC, ND and NE sequences and directed heterodimerization of these photosensory regions with the NB region reveal form‐specific R‐induced activities for different type II phy dimers. The experimental approach developed here of directed assembly of defined protein dimer combinations in vivo may be applicable to other systems.     

Two-photon conversion of a bacterial phytochrome

In nature, sensory photoreceptors underlie diverse spatiotemporally precise and generally reversible biological responses to light. Photoreceptors also serve as genetically encoded agents in optogenetics to control by light organismal state and behavior. Phytochromes represent a superfamily of photoreceptors that transition between states absorbing red light (Pr) and far-red light (Pfr), thus expanding the spectral range of optogenetics to the near-infrared range. Although light of these colors exhibits superior penetration of soft tissue, the transmission through bone and skull is poor. To overcome this fundamental challenge, we explore the activation of a bacterial phytochrome by a femtosecond laser emitting in the 1 μm wavelength range. Quantum chemical calculations predict that bacterial phytochromes possess substantial two-photon absorption cross sections. In line with this notion, we demonstrate that the photoreversible Pr ↔ Pfr conversion is driven by two-photon absorption at wavelengths between 1170 and 1450 nm. The Pfr yield was highest for wavelengths between 1170 and 1280 nm and rapidly plummeted beyond 1300 nm. By combining two-photon activation with bacterial phytochromes, we lay the foundation for enhanced spatial resolution in optogenetics and unprecedented penetration through bone, skull, and soft tissue.     

Temperature effects on Agrobacterium phytochrome Agp1

Phytochromes are widely distributed biliprotein photoreceptors with a conserved N-terminal chromophore-binding domain. Most phytochromes bear a light-regulated C-terminal His kinase or His kinase-like region. We investigated the effects of light and temperature on the His kinase activity of the phytochrome Agp1 from Agrobacterium tumefaciens. As in earlier studies, the phosphorylation activity of the holoprotein after far-red irradiation (where the red-light absorbing Pr form dominates) was stronger than that of the holoprotein after red irradiation (where the far red-absorbing Pfr form dominates). Phosphorylation activities of the apoprotein, far red-irradiated holoprotein, and red-irradiated holoprotein decreased when the temperature increased from 25 °C to 35 °C; at 40 °C, almost no kinase activity was detected. The activity of a holoprotein sample incubated at 40 °C was nearly completely restored when the temperature returned to 25 °C. UV/visible spectroscopy indicated that the protein was not denatured up to 45 °C. At 50 °C, however, Pfr denatured faster than the dark-adapted sample containing the Pr form of Agp1. The Pr visible spectrum was unaffected by temperatures of 20-45 °C, whereas irradiated samples exhibited a clear temperature effect in the 30-40 °C range in which prolonged irradiation resulted in the photoconversion of Pfr into a new spectral species termed Prx. Pfr to Prx photoconversion was dependent on the His-kinase module of Agp1; normal photoconversion occurred at 40 °C in the mutant Agp1-M15, which lacks the C-terminal His-kinase module, and in a domain-swap mutant in which the His-kinase module of Agp1 is replaced by the His-kinase/response regulator module of the other A. tumefaciens phytochrome, Agp2. The temperature-dependent kinase activity and spectral properties in the physiological temperature range suggest that Agp1 serves as an integrated light and temperature sensor in A. tumefaciens.     

Spectroscopy and a high-resolution crystal structure of Tyr263 mutants of cyanobacterial phytochrome Cph1

Phytochromes are biliprotein photoreceptors that can be photoswitched between red-light-absorbing state (Pr) and far-red-light-absorbing state (Pfr). Although three-dimensional structures of both states have been reported, the photoconversion and intramolecular signaling mechanisms are still unclear. Here, we report UV-Vis absorbance, fluorescence and CD spectroscopy along with various photochemical parameters of the wild type and Y263F, Y263H and Y263S mutants of the Cph1 photosensory module, as well as a 2.0-Å-resolution crystal structure of the Y263F mutant in its Pr ground state. Although Y263 is conserved, we show that the aromatic character but not the hydroxyl group of Y263 is important for Pfr formation. The crystal structure of the Y263F mutant (Protein Data Bank ID: 3ZQ5) reaffirms the ZZZssa chromophore configuration and provides a detailed picture of its binding pocket, particularly conformational heterogeneity around the chromophore. Comparison with other phytochrome structures reveals differences in the relative position of the PHY (phytochrome specific) domain and the interaction of the tongue with the extreme N-terminus. Our data support the notion that native phytochromes in their Pr state are structurally heterogeneous.     

Light-dependent dimerisation in the N-terminal sensory module of cyanobacterial phytochrome 1

Phytochromes, photoreceptors controlling important physiological processes in plants and many prokaryotes, are photochromic biliproteins. The red-absorbing Pr ground state is converted by light into the farred-absorbing Pfr which can be photoconverted back to Pr. In plants at least Pfr is the physiologically active signalling state. Here, we show that the N-terminal photochromic module of Cph1 homodimerises reversibly and independently in Pr and Pfr, Pfr-dimers being significantly more stable. Implications for the mechanism of signal transduction are discussed.