PHY Domain Research Articles

Two-photon Absorption and Photoionization of a Bacterial Phytochrome

Phytochromes constitute a family of photosensory proteins that are utilized by various organisms to regulate several physiological processes. Phytochromes bind a bilin pigment that switches its isomeric state upon absorption of red or far-red photons, resulting in protein conformational changes that are sensed by the organism. Previously, the ultrafast dynamics in bacterial phytochrome was resolved to atomic resolution by time-resolved serial femtosecond X-ray diffraction (TR-SFX), showing extensive changes in its molecular conformation at 1 picosecond delay time. However, the large excitation fluence of mJ/mm2 used in TR-SFX questions the validity of the observed dynamics. In this work, we present an excitation-dependent ultrafast transient absorption study to test the response of a related bacterial phytochrome to excitation fluence. We observe excitation power-dependent sub-picosecond dynamics, assigned to the population of high-lying excited state Sn through resonantly enhanced two-photon absorption, followed by rapid internal conversion to the low-lying S1 state. Inspection of the long-lived spectrum under high fluence shows that in addition to the primary intermediate Lumi-R, spectroscopic signatures of solvated electrons and ionized chromophore radicals are observed. Supported by numerical modelling, we propose that under excitation fluences of tens of μJ/mm2 and higher, bacterial phytochrome partly undergoes photoionization from the Sn state in competition with internal conversion to the S1 state in 300 fs. We suggest that the extensive structural changes of related, shorter bacterial phytochrome, lacking the PHY domain, resolved from TR-SFX may have been affected by the ionized species. We propose approaches to minimize the two-photon absorption process by tuning the excitation spectrum away from the S1 absorption or using phytochromes exhibiting minimized or shifted S1 absorption.

Time-resolved fluorescence anisotropy with Atto 488-labeled phytochrome Agp1 from Agrobacterium fabrum.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time-resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto-488 was attached to each mutant, and time-resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full-length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr-to-Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr.

Long-Distance Protonation-Conformation Coupling in Phytochrome Species

Phytochromes are biological red/far-red light sensors found in many organisms. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY, PGP) and the C-terminal transmitter (output) module. We recently showed a direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains of the prototypical phytochrome Cph1 PGP. These results suggested that the transient phycocyanobilin (PCB) chromophore deprotonation is closely associated with a higher protein mobility both in proximal and distal protein sites, implying a causal relationship that might be important for the global large-scale protein rearrangements. Here, we investigate the prototypical biliverdin (BV)-binding phytochrome Agp1. The structural changes at various positions in Agp1 PGP were investigated as a function of pH using picosecond time-resolved fluorescence anisotropy and site-directed fluorescence labeling of cysteine variants of Agp1 PGP. We show that the direct correlation of chromophore deprotonation with pH-dependent conformational changes does not occur in Agp1. Together with the absence of long-range effects between the PHY domain and chromophore pKa, in contrast to the findings in Cph1, our results imply phytochrome species-specific correlations between transient chromophore deprotonation and intramolecular signal transduction.

Influence of the PHY domain on the ms-photoconversion dynamics of a knotless phytochrome

The ability of some knotless phytochromes to photoconvert without the PHY domain allows evaluation of the distinct effect of the PHY domain on their photodynamics. Here, we compare the ms dynamics of the single GAF domain (g1) and the GAF-PHY (g1g2) construct of the knotless phytochrome All2699 from cyanobacterium Nostoc punctiforme. While the spectral signatures and occurrence of the intermediates are mostly unchanged by the domain composition, the presence of the PHY domain slows down the early forward and reverse dynamics involving chromophore and protein binding pocket relaxation. We assign this effect to a more restricted binding pocket imprinted by the PHY domain. The photoproduct formation is also slowed down by the presence of the PHY domain but to a lesser extent than the early dynamics. This indicates a rate limiting step within the GAF and not the PHY domain. We further identify a pH dependence of the biphasic photoproduct formation hinting towards a pKa dependent tuning mechanism. Our findings add to the understanding of the role of the individual domains in the photocycle dynamics and provide a basis for engineering of phytochromes towards biotechnological applications.Graphical abstract

Plant Phytochrome B Assembles an Asymmetric Dimer with Unique Signaling Potential

Phytochromes (Phy) sense red and far‐red light and reversibly interconvert between inactive Pr and active Pfr conformers. Phys typically consist of a photo sensory module (PSM) formed by sequential N‐terminal PAS, GAF, and PHY domains, followed by a C‐terminal histidine kinase‐related domain (HKRD). There are four plant isoforms Phy A through D, and they all contain two additional PAS domains (PAS1 and PAS2) inserted between PSM and HKRD. Despite extensive biochemical and structural studies, there has been a lack of a full‐length structure, hindering our understanding of plant Phy signaling. Here we report a 3.3‐Å cryo‐EM structure of Arabidopsis PhyB dimer. Instead of the widely anticipated parallel association of the two monomers resembling the prokaryotic relatives, Plant PhyB adopts a topologically complex dimeric architecture with the two PSMs in a head‐to‐tail antiparallel form while the two HKRDs in a head‐to‐head parallel form. The PAS2 mediates the antiparallel dimerization of PSM that leads to a parallelogram‐shaped platform. And a preceding ‘modulator’ loop assembles tightly with the PSM of its own protomer. We show that these inter‐ and intra‐protomer connections contribute to stabilizing of dimer and accelerate thermal reversion of Pfr back to Pr. Lean of HKRDs towards the platform results in profound asymmetry of PhyB that might enable distinct signaling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate‐sensitive surface for interaction with their downstream signaling partners.

Identification and characterisation of blue light photoreceptor gene family and their expression in tomato (Solanum lycopersicum) under cold stress.

The Arabidopsis thaliana L. photoreceptor genes homologues in tomato (Solanum lycopersicum L.) genome were analysed using bioinformatic tools. The expression pattern of these genes under cold stress was also evaluated. Transcriptome analysis of the tomato sequence revealed that the photoreceptor gene family is involved in abiotic stress tolerance. They participate in various pathways and controlling multiple metabolic processes. They are structurally related to PAS, LIGHT-OXYGEN-VOLTAGE-SENSING (LOV), DNA photolyase, 5,10-methenyl tetrahydrofolate (MTHF), flavin-binding kelch F-box, GAF, PHY, Seven-bladed β-propeller and C27 domains. They also interact with flavin adenine dinucleotide (FAD), (5S)-5-methyl-2-(methylsulfanyl)-5-phenyl-3-(phenylamino)-3,5-dihydro-4H-imidazol-4-one (FNM) and Phytochromobilin (PϕB) ligands. These interactions help to create a cascade of protein phosphorylation involving in cell defence transcription or stress-regulated genes. They localisation of these gene families on tomato chromosomes appeared to be uneven. Phylogenetic tree of tomato and Arabidopsis photoreceptor gene family were classified into eight subgroups, indicating gene expression diversity. Morphological and physiological assessment revealed no dead plant after 4h of cold treatment. All the plants were found to be alive, but there were some variations in the data across different parameters. Cold stress significantly reduced the rate of photosynthesis from 10.06 to 3.16μmolm-2 s-1 , transpiration from 4.6 to 1.3mmolm-2 s-1 , and stomatal conductance from 94.6 to 25.6mmolm-2 s-1 . The cold stressed plants also had reduced height, root/shoot length, and fresh/dry biomass weight than the control plants. Relative expression analysis under cold stress revealed that after 4h, light stimulates the transcript level of Cry2 from 1.9 to 5.7 and PhyB from 0.98 to 6.9 compared to other photoreceptor genes.

The PHY Domain Dimer Interface of Bacteriophytochromes Mediates Cross-talk between Photosensory Modules and Output Domains

Protein dynamics play a major role for the catalytic function of enzymes, the interaction of protein complexes or signal integration in regulatory proteins. In the context of multi-domain proteins involved in light-regulation of enzymatic effectors, the central role of conformational dynamics is well established. Light activation of sensory modules is followed by long-range signal transduction to different effectors; rather than domino-style structural rearrangements, a complex interplay of functional elements is required to maintain functionality. One family of such sensor-effector systems are red-light-regulated phytochromes that control diguanylate cyclases involved in cyclic-dimeric-GMP formation. Based on structural and functional studies of one prototypic family member, the central role of the coiled-coil sensor-effector linker was established. Interestingly, subfamilies with different linker lengths feature strongly varying biochemical characteristics. The dynamic interplay of the domains involved, however, is presently not understood. Here we show that the PHY domain dimer interface plays an essential role in signal integration, and that a functional coupling with the coiled-coil linker element is crucial. Chimaeras of two biochemically different family members highlight the phytochrome-spanning helical spine as an essential structural element involved in light-dependent upregulation of enzymatic turnover. However, isolated structural elements can frequently not be assigned to individual characteristics, which further emphasises the importance of global conformational dynamics. Our results provide insights into the intricate processes at play during light signal integration and transduction in these photosensory systems and thus provide additional guidelines for a more directed design of novel sensor-effector combinations with potential applications as optogenetic tools.

Impact of amino acid substitutions on the behavior of a photoactivatable near infrared fluorescent protein PAiRFP1

A photoactivatable near-infrared fluorescent protein (NIR-FP) PAiRFP1 has been developed by 15 amino acid substitutions in its nonfluorescent template Agp2. In our previous communication, we investigated the role of three amino acids in PHY domain distal from BV molecule. The impact of the twelve amino acids in GAF domain, especially five residues near BV-binding pocket is unclear. In this paper, PCR based reverse mutagenesis, spectroscopic methods, molecular modelling and simulations have been employed to explore the roles of these substitutions during the molecular evolution of PAiRFP1. It was found that the residue L163 is important for protein folding in PAiRFP1. The residues F244 and C280 exerted remarkable effects on molar extinction coefficient, NIR fluorescence quantum yield, molecular brightness, fluorescence fold, and dark recovery rate. The residues F244 and V276 modulate the maximum absorption and emission peak position. The reverse mutant L168M exhibited a higher fluorescence fold than PAiRFP1. Additionally, the reverse mutants V203A, V294E, S218G and D127G possessed better spectral properties than PAiRFP1. This study is important for the rational design of a better BphP-based photoactivatable NIR-FPs.

The structural changes in the signaling mechanism of bacteriophytochromes in solution revealed by a multiscale computational investigation.

Phytochromes are red-light sensing proteins, with important light-regulatory roles in different organisms, which are capturing an increasing interest in bioimaging and optogenetics. Upon absorption of light by the embedded bilin chromophore, they undergo structural changes that extend from the chromophore to the protein and finally drive the biological function. Up to now, the underlying mechanism still has to be characterized fully. Here we investigate the Pfr activated form of a bacterial phytochrome, by combining extensive molecular dynamics simulations with a polarizable QM/MM description of the spectroscopic properties, revealing a large structure relaxation in solution, compared to the crystal structure, both in the chromophore-binding pocket and in the overall structure of the phytochrome. Our results indicate that the final opening of the dimeric structure is preceded by an important internal reorganization of the phytochrome specific (PHY) domain involving a bend of the helical spine connecting the PHY domain with the chromophore-binding domain, opening the way to a new understanding of the activation pathway.

Effect of the PHY Domain on the Photoisomerization Step of the Forward Pr →Pfr Conversion of a Knotless Phytochrome.

Phytochrome photoreceptors operate via photoisomerization of a bound bilin chromophore. Their typical architecture consists of GAF, PAS and PHY domains. Knotless phytochromes lack the PAS domain, while retaining photoconversion abilities, with some being able to photoconvert with just the GAF domain. Therefore, we investigated the ultrafast photoisomerization of the Pr state of a knotless phytochrome to reveal the effect of the PHY domain and its “tongue” region on the transduction of the light signal. We show that the PHY domain does not affect the initial conformational dynamics of the chromophore. However, it significantly accelerates the consecutively induced reorganizational dynamics of the protein, necessary for the progression of the photoisomerization. Consequently, the PHY domain keeps the bilin and its binding pocket in a more reactive conformation, which decreases the extent of protein reorganization required for the chromophore isomerization. Thereby, less energy is lost along nonproductive reaction pathways, resulting in increased efficiency.

Lyophilization Reveals a Multitude of Structural Conformations in the Chromophore of a Cph2-like Phytochrome.

Cyanobacteria sense and respond to various colors of light employing a large number of bilin-based phytochrome-like photoreceptors. All2699 from Nostoc 7120 has three consecutive GAF domains with GAF1 and GAF3 binding a phycocyanobilin chromophore. GAF1, even when expressed independently, can be photoconverted between red-absorbing Pr and far-red-absorbing Pfr states, while the nonphotosensory GAF2 domain is structurally and functionally homologous to the PHY domains in canonical and Cph2-like phytochromes. Here, we characterize possible bilin chromophore conformers using solid-state NMR spectroscopy on the two lyophilized All2699 samples (GAF1-only and GAF1-PHY constructs). On the basis of complete 1H, 13C, and 15N assignments for the chromophore obtained on the two Pr lyophilizates, multiple static conformations of the chromophore in both cases are identified. Moreover, most atoms of the chromophore in the bidomain sample show only subtle changes in the mean chemical shifts relative to those in frozen solution (FS), indicating an optimized interaction of the GAF2 domain with the GAF1-bound chromophore. Our results confirm the conservation of key chromophore-protein interactions and the photoreversibility in both All2699 lyophilizates, offering the possibility to investigate conformational distributions of the heterogeneous chromophore and its functional consequences in phytochromes and other bilin-dependent photoreceptors intractable by the solid-state NMR technique as FSs.

Dynamic Properties of the Photosensory Domain of Deinococcus radiodurans Bacteriophytochrome.

Phytochromes are biological photoreceptors found in all kingdoms of life. Numerous physicochemical and spectroscopic studies of phytochromes have been carried out for many decades, both experimentally and computationally, with the main focus on the photoconversion mechanism involving a tetrapyrrole chromophore. In this computational work, we concentrate on the long-scale dynamic motion of the photosensory domain of Deinococcus radiodurans by means of classical all-atom molecular dynamics (MD) simulations. Conventional and accelerated MD methods in combination with two different force fields, CHARMM27 and AMBER ff14SB, are tested in long atomistic simulations to confront the dynamics of monomer and dimer forms. These calculations highlight dissimilar equilibrium conformations in aqueous solutions and, in turn, different large-scale dynamic behaviors of the monomer form vs the dimer form. While the phytochrome in a monomer form tends to close the cavity entailed between the GAF and PHY domains, the opposite trend is predicted for the phytochrome dimer, which opens up as a consequence of the formation of strong salt bridges between the PHY domains of two molecules in water.

Transient Deprotonation of the Chromophore Affects Protein Dynamics Proximal and Distal to the Linear Tetrapyrrole Chromophore in Phytochrome Cph1.

Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes, including Cph1 from the cyanobacterium Synechocystis 6803, act as photochemical switches that interconvert between stable red (Pr)- and metastable far-red (Pfr)-absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr → Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr state, involving pH titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain), and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and in distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.

Modulation of Structural Heterogeneity Controls Phytochrome Photoswitching

Phytochromes sense red/far-red light and control many biological processes in plants, fungi, and bacteria. Although the crystal structures of dark- and light-adapted states have been determined, the molecular mechanisms underlying photoactivation remain elusive. Here, we demonstrate that the conserved tongue region of the PHY domain of a 57-kDa photosensory module of Deinococcus radiodurans phytochrome changes from a structurally heterogeneous dark state to an ordered, light-activated state. The results were obtained in solution by utilizing a laser-triggered activation approach detected on the atomic level with high-resolution protein NMR spectroscopy. The data suggest that photosignaling of phytochromes relies on careful modulation of structural heterogeneity of the PHY tongue.

Effects of reverse genetic mutations on the spectral and photochemical behavior of a photoactivatable fluorescent protein PAiRFP1.

Bacteriophytochrome photoreceptors (BphPs) containing biliverdin (BV) have great potential for the development of genetically engineered near-infrared fluorescent proteins (NIR FPs). We investigated a photoactivatable fluorescent protein PAiRFP1, was engineered through directed molecular evolution. The coexistence of both red light absorbing (Pr) and far-red light absorbing (Pfr) states in dark is essential for the photoactivation of PAiRFP1. The PCR based site-directed reverse mutagenesis, spectroscopic measurements and molecular dynamics (MD) simulations were performed on three targeted sites V386A, V480A and Y498H in PHY domain to explore their potential effects during molecular evolution of PAiRFP1. We found that these substitutions did not affect the coexistence of Pr and Pfr states but led to slight changes in the photophysical parameters. The covalent docking of biliverdin (cis and trans form) with PAiRFP1 was followed by several 100 ns MD simulations to provide some theoretical explanations for the coexistence of Pr and pfr states. The results suggested that experimentally observed coexistence of Pr and Pfr states in both PAiRFP1 and mutants were resulted from the improved stability of Pr state. The use of experimental and computational work provided useful understanding of Pr and Pfr states and the effects of these mutations on the photophysical properties of PAiRFP1.

Hypertension specific patient-reported outcome measure. Part II: validation survey and item selection process

Aim. Improvement of the health-related quality of life (HRQoL) is one of the basic principles of value-based medicine. HRQoL could be assessed by the patient-reported outcome measures (PROMs) also in case of arterial hypertension (HTN). However for HTN patients only generic PROMs are still used. Previously the group of experts had created the primary version of HTN-specific PROM. The purpose of the second part was to conduct a validation survey and to select the items in a statistically-based manner. Material and methods. Validation survey was conducted in a large multidisciplinary center among patients with HTN stages 1-3 and healthy volunteers. Inclusion criteria were age >18 years old, ability to understand or complete the scale themselves, absence of significant illness requiring hospitalization. The items were selected according to the principles of classical test theory (CTT) and item response theory (IRT). The criteria for CTT were sensitivity (standard deviation and coefficient of variation with corresponding confidence intervals), representativeness (item-total Pearson’s correlation coefficient), internal consistency (Cronbach’s a coefficient). In IRT analysis two methods were adopted — value of four degrees of difficulty and the discrimination estimate. Each question was evaluated according to 8 criteria. An item was considered for selection when it was retained by ≥4 criteria. The expert panel considered practical significance of each item. Results. A total of 430 questionnaires were distributed and 407 (94,7%) of them were returned completed (from 359 hypertensive patients, mean age 62,3±11,7 y.o.; 48 healthy volunteers, mean age 38,8±10,5 y.o.). The average time for PROM filling was 24±4,2 minutes. Of 163 questions, 27 met all 8 criteria and 3 questions did not match any. Of the 36 HTN-specific questions, 11 matched ≥5 criteria and in the generic part there were 87 questions (33 in the PHY domain, 35 for PSY, 8 for SOC, 11 for THER). The symmetric distribution of criteria was seen in 25 questions, of which 11 were evaluated by experts and then retained. For 40 questions, <4 eligibility criteria were recorded, of which 9 were retained after expert review. The PROM draft contained 80 questions (19 questions in the physiology domain, 22 in psychology, 6 in social, 13 in therapy, 20 items are HTN-specific). Conclusion. The methods of CTT and IRT allowed to reduce the PROM volume without losing the semantic richness and the need to reorganize the conceptual structure. The next step is the validation of the scale.

Light-Driven Domain Mechanics of a Minimal Phytochrome Photosensory Module Studied by EPR

Light-exposed organisms developed photoreceptors to transduce light signals for environmental adaptation. Phytochromes, found in bacteria, fungi, and plants, can discriminate the ratio of red and far-red light using the isomerization of a bilin chromophore bound to a photosensory module to trigger downstream conformational changes in the protein. Here, we investigated by hydrogen/deuterium exchange mass spectrometry and electron paramagnetic resonancespectroscopy the light-driven domain mechanicsof a minimal monomeric photosensory module from the group II phytochrome Cph2 from Synechocystis sp. PCC 6803. We could unambiguously trace the light-driven secondary structural rearrangementof its tongue region, and we found a translational motion of the PHY domain that is related to what was found before by X-ray studies in a group I module. Our analysis demonstrates a common light response in the photosensory modules of phytochromes, orchestrated solely by the GAF-PHY bidomain independent of further quaternary interactions or the nature of downstream effector domains.

Light-induced structural changes in a monomeric bacteriophytochrome.

Phytochromes sense red light in plants and various microorganism. Light absorption causes structural changes within the protein, which alter its biochemical activity. Bacterial phytochromes are dimeric proteins, but the functional relevance of this arrangement remains unclear. Here, we use time-resolved X-ray scattering to reveal the solution structural change of a monomeric variant of the photosensory core module of the phytochrome from Deinococcus radiodurans. The data reveal two motions, a bend and a twist of the PHY domain with respect to the chromophore-binding domains. Infrared spectroscopy shows the refolding of the PHY tongue. We conclude that a monomer of the phytochrome photosensory core is sufficient to perform the light-induced structural changes. This implies that allosteric cooperation with the other monomer is not needed for structural activation. The dimeric arrangement may instead be intrinsic to the biochemical output domains of bacterial phytochromes.

Structure of the Full-Length Bacteriophytochrome from the Plant Pathogen Xanthomonas campestris Provides Clues to its Long-Range Signaling Mechanism

Phytochromes constitute a major superfamily of light-sensing proteins that are reversibly photoconverted between a red-absorbing (Pr) and a far-red-absorbing (Pfr) state. Bacteriophytochromes (BphPs) are found among photosynthetic and non-photosynthetic bacteria, including pathogens. To date, several BphPs have been biophysically characterized. However, it is still not fully understood how structural changes are propagated from the photosensory module to the output module during the signal transduction event. Most phytochromes share a common architecture consisting of an N-terminal photosensor that includes the PAS2–GAF–PHY domain triad and a C-terminal variable output module. Here we present the crystal structure of the full-length BphP from the plant pathogen Xanthomonas campestris pv. campestris (XccBphP) bearing its photosensor and its complete output module, a PAS9 domain. In the crystals, the protein was found to be in the Pr state, whereas diffraction data together with resonance Raman spectroscopic and theoretical results indicate a ZZZssa and a ZZEssa chromophore configuration corresponding to a mixture of Pr and Meta-R state, the precursor of Pfr. The XccBphP quaternary assembly reveals a head-to-head dimer in which the output module contributes to the helical dimer interface. The photosensor, which is shown to be a bathy-like BphP, is influenced in its dark reactions by the output module. Our structural analyses suggest that the photoconversion between the Pr and Pfr states in the full-length XccBphP may involve changes in the relative positioning of the output module. This work contributes to understand the light-induced structural changes propagated from the photosensor to the output modules in phytochrome signaling.

Light Signaling Mechanism of Two Tandem Bacteriophytochromes

RpBphP2 and RpBphP3, two tandem bacteriophytochromes from the photosynthetic bacterium Rhodopseudomonas palustris, share high sequence identity but exhibit distinct photoconversion behavior. Unlike the canonical RpBphP2, RpBphP3 photoconverts to an unusual near-red-absorbing (Pnr) state; both are required for synthesis of light-harvesting complexes under low-light conditions. Here we report the crystal structures of the photosensory core modules of RpBphP2 and RpBphP3. Despite different quaternary structures, RpBphP2 and RpBphP3 adopt nearly identical tertiary structures. The RpBphP3 structure reveals tongue-and-groove interactions at the interface between the GAF and PHY domains. A single mutation in the PRxSF motif at the GAF-PHY interface abolishes light-induced formation of the Pnr state in RpBphP3, possibly due to altered structural rigidity of the chromophore-binding pocket. Structural comparisons suggest that long-range signaling involves structural rearrangement of the helical spine at the dimer interface. These structures, together with mutational studies, provide insights into photoconversion and the long-range signaling mechanism in phytochromes.