Tips and turns of bacteriophytochrome photoactivation

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC 4.0 https://creativecommons.org/licenses/by-nc/4.0/ Tips and turns of bacteriophytochrome photoactivation © The Royal Society of Chemistry and Owner Societies 2020 Published version Takala, Heikki; Edlund, Petra; Ihalainen, Janne A.; Westenhoff, Sebastian Takala, H., Edlund, P., Ihalainen, J. A., & Westenhoff, S. (2020). Tips and turns of bacteriophytochrome photoactivation. Photochemical and Photobiological Sciences, 19(11), 1488-1510. https://doi.org/10.1039/D0PP00117A 2020 Photochemical & Photobiological Sciences PERSPECTIVE Cite this: DOI: 10.1039/d0pp00117a Received 27th March 2020, Accepted 4th September 2020 DOI: 10.1039/d0pp00117a rsc.li/pps Tips and turns of bacteriophytochrome photoactivation Heikki Takala, *† a,b Petra Edlund,† c Janne A. Ihalainen a and Sebastian Westenhoff* c Phytochromes are ubiquitous photosensor proteins, which control the growth, reproduction and movement in plants, fungi and bacteria. Phytochromes switch between two photophysical states depending on the light conditions. In analogy to molecular machines, light absorption induces a series of structural changes that are transduced from the bilin chromophore, through the protein, and to the output domains. Recent progress towards understanding this structural mechanism of signal transduction has been manifold. We describe this progress with a focus on bacteriophytochromes. We describe the mechanism along three structural tiers, which are the chromophore-binding pocket, the photosensory module, and the output domains. We discuss possible interconnections between the tiers and conclude by presenting future directions and open questions. We hope that this review may serve as a compendium to guide future structural and spectroscopic studies designed to understand structural signaling in phytochromes. Discovery and function of phytochromes Brief history of the discovery of plant and fungal phytochromes Multiple responses of plants depend on environmental light conditions. First observed in the 1930s, 1 and further rationalized in the 1950s, 2 the photoperiodic control of flowering time and seed germination in plants were found to be sensitive to illumination by red and far-red light. The responsible photoconvertible protein was identified in 1959 by absorption spectroscopy and termed phytochrome. 3 In 1983, an oat phytochrome was purified 4 and two years later, the primary amino acid sequence was revealed. 5 Phytochromes have been shown to be important for many properties of plants, such as shade avoidance, plant cycle, stem elongation, and flowering time. 6 A characteristic feature of phytochromes is that they can photoswitch between two photochemical states. Depending on the light conditions, they adopt a state that absorbs red light, termed “Pr”and one that absorbs far-red light, termed “Pfr” (Fig. 1c). The two states have different biochemical activity, which leads to different cellular responses. The hypothesis that plant chloroplasts have developed from a photosynthetic bacterium led to the speculations that prokaryotic genes represent the evolutionary origin of plant phytochromes. 7–9 Indeed, the first prokaryotic phytochrome to be discovered was Cph1 in the cyanobacterium Synechocystis sp. PCC6803 in 1997, when two groups showed that it could be expressed in Escherichia coli and autoassembled with a bilin cofactor to show phytochrome-typical photoswitching. 10,11 In 1999, the rather more divergent group of bacteriophytochromes (BphP) was discovered in the non-photosynthetic bacterium Deinoccocus radiodurans. 12 Although the gene rcaE in the cyanobacterium Fremyella displosisphon was proposed to encode a phytochrome in 1996 on the basis of genetic evidence, 13 it was shown only recently on the basis of a revised sequence that it indeed encodes a bona fide photoreceptor. 14 In 2005, a phytochrome was described in the fungus Aspergillus nidulans. 15 Phytochrome-related proteins exist in eukaryotic algae, where their absorption maxima can span the entire visible region. 16 Plant phytochromes are important because they control the growth, reproduction, and development of virtually all vegetation on Earth. Uncovering in detail how phytochromes achieve this, is fundamentally important for understanding the basis of life on Earth. Moreover, the knowledge could enable modification of plants to control their growth and development. 17 Bacteriophytochromes are particularly interesting, because they have a relatively simple modular architecture †Equal contribution. a Department of Biological and Environmental Science, Nanoscience Center, University of Jyvaskyla, Box 35, 40014 Jyvaskyla, Finland. E-mail: [email protected] b Department of Anatomy, Faculty of Medicine, University of Helsinki, Box 63, 00014 Helsinki, Finland c Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, 40530 Gothenburg, Sweden. E-mail: [email protected] This journal is © The Royal Society of Chemistry and Owner Societies 2020 Photochem. Photobiol. Sci. Open Access Article. Published on 27 October 2020. Downloaded on 10/28/2020 10:23:41 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal and photoswitch with red light. This makes them interesting targets for optogenetic applications, imaging in tissues, lightdependent gene expression, and possible medical applications. 18–23 These applications can be achieved by coupling the light-sensing part of phytochrome to the proteins of interest with a desired output signal. 21 Out of all of these motivations, it is important to understand how phytochromes function at the atomic level. Plant phytochromes are involved in a complex signaling network within the cell. They function as serine/threonine kinases, 24 probably facilitated by a family of nuclear protein kinases (photoregulatory protein kinases 1–4, also known as MUT9-like kinases) 25 and also have several additional biological outputs in vivo. 26–28 Curiously, the physiological function of cyanobacterial and bacteriophytochromes is less well understood compared to plant phytochromes. For example, the Rhodopseudomonas palustris phytochrome regulates the photosynthetic apparatus, 29 and in the cyanobacterium Leptolyngbya sp. strain JSC-1, a phytochrome controls remodeling of the photosynthetic apparatus to adapt to far-red light conditions. 30 In non-photosynthetic prokaryotes, the physiological function of BphPs remains even more unclear, with some notable exceptions. 31,32 Interestingly, prokaryotic phytochromes have a variety of different output domains, which include histidine kinases (HK and HWE), diguanylyl cyclases (GGDEF), phosphodiesterases (EAL), metal-dependent Ser/Thr protein phosphatases (PPM), as well as domains without any enzymatic activity (e.g., PAS and HOS). 33 The discovery of prokaryotic phytochromes marked a turning point in the structural biology of phytochromes, because it was now possible to express and purify the proteins at larger quantities. 34 Thereby, crystal structures could be determined, and more extensive spectroscopic and biochemical analyses could be undertaken. 35,36 Information obtained on bacteriophytochromes can often be transferred to plant phytochromes. This is especially true for the photosensing part of the phytochrome, which is homologous across the kingdoms of life in which phytochromes exist. Indeed, the first crystal structure of a phytochrome fragment from the plant Arabidopsis thaliana shows similarities to its prokaryotic counterparts in terms of the domain structure and arrangement. 37 Differences are also observed, for example in the thioether linkage of the chromophore 38 and the photoactivation mechanisms of the D-ring, which in Pfr is the likely oriented in a β-facial position in plant phytochromes, compared to α-facial in bacterial phytochromes. 39 The extensive interest in phytochromes has led to decades of research and several reviews on phytochromes. 9,32,33,40–44 Specific reviews on plant phytochromes 28,45–48 and fungal phytochromes 49 are also available. Here we summarize the current understanding of how phytochromes remodel their threedimensional structure when they photoconvert between Pr and Pfr. We concentrate on bacteriophytochromes because the most structural dynamic data has been reported for these species. We start by describing the photocycle and chromophore and we continue to discuss the structural features that change upon photoactivation. It is currently not fully understood how the biochemical activity of phytochromes is controlled, but we discuss a few potential explanations. We end by providing an outlook on the future of the structural biology of phytochromes. Chromophore and photocycle Chromophore Phytochromes carry a bilin chromophore that absorbs light at the red/far-red wavelength region. It is an open tetrapyrrole, which originates from heme catabolism in all organisms (Fig. 1a). The phytochromes from bacteria, cyanobacteria and plants bind biliverdin (BV), phycocyanobilin (PCB) or phytochromobilin (PΦB), respectively. 44 The cofactor is covalently attached to a cysteine residue that resides in the PAS domain in bacteria and fungi, and in the GAF domain in cyanobacteria Fig. 1 Biliverdin structure and the photocycle of BphPs. (a) The structure of biliverdin and some of its amino acid interactions in the binding pocket of Deinococcus radiodurans bacteriophytochrome. Selected atom names, the four bilin rings, and interacting residues are indicated. (b) The photocycle of a canonical BphP with two parent states, which are called Pr and Pfr after their absorption properties. The protein can be switched between the states with red/far-red light or by thermal dark reversion. The photocycle also shows the kinetics for the intermediates identified for the photoconversions pathways and the direction of the thermal dark reversion for canonical phytochromes. (c) Absorption spectra of a bacteriophytochrome from D. radiodurans in its Pr state (black) and mixed Pr/Pfr state after red light illumination (red). Perspective Photochemical & Photobiological Sciences Photochem. Photobiol. Sci. This journal is © The Royal Society of Chemistry and Owner Societies 2020 Open Access Article. Published on 27 October 2020. Downloaded on 10/28/2020 10:23:41 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online and plants (Fig. 2), but is embedded inside the GAF domain in all occasions. In bacteria, the gene for expression of the enzyme hemeoxygenase, which converts the heme precursor into a biliverdin, is located in the same operon as BphPs. 34,50,51 In recombinant expression, hemeoxygenase can be co-expressed with phytochrome to yield the holoprotein. 34 An extensive review on the synthesis of bilin is available. 52 The photocycle The photocycle of phytochromes has been studied extensively with spectroscopy. A hallmark feature of the superfamily is that the proteins can adopt two spectroscopically different metastable states (Fig. 1b). For canonical phytochromes, these are called the red light-absorbing Pr state and the far-red lightabsorbing Pfr state. The absorption maximum of bacteriophytochrome is usually at around 700 nm for the Pr state and 750 nm for the Pfr state (Fig. 1c). This however may vary between the phytochrome species, reaching the blue-most absorption for a Pr state at 610 nm and Pfr state at 670 nm, 53 albeit in this extreme case the bilin chromophore is phycocyanobilin instead of biliverdin. Phytochromes can be actively switched between the Pr and Pfr states with red/far-red light, or by thermal reversion in the dark. This dark reversion occurs in minutes, hours, or even days depending on the species and construct. The dark reversion time depends on cellular conditions, such as pH, ionic strength, reducing agents concentrations of metal ions, 54 and on temperature. 55,56 Generally, the resting state of phytochromes is the Pr state. However, a number of bacteriophytochromes show reversed thermal dark reversion into the Pfr state. These are called bathy phytochromes, discovered first in phytochromes from Bradyrhizobium and Agrobacterium fabrum. 29,32,50 Bathy phytochromes are primarly found in nitrogen-fixing plants, which is logical since far-red light penetrates the soil more effectively and can be transduced in the roots of plants. 57 Some bacterial species express both canonical and bathy phytochromes. One widely studied example is from the soil bacterium A. fabrum, with a canonical phytochrome Agp1 and a bathy phytochrome Agp2. These proteins have been proposed to complement each other. 58 The structural and spectroscopic properties of the Pr and Pfr states are retained in bathy phytochromes and it is merely the relative free energy of the two states which is interchanged. 59 Intermediate states When switching between the two metastable states, phytochromes pass through a number of intermediate states. A different number of intermediates have been reported for plant, cyanobacterial, and bacteriophytochromes, with plant phytochromes having the most complex photocycle. 60 For bacteriophytochromes, a minimal and practical consensus is to consider two intermediate states, Lumi and Meta, as shown in Fig. 1b. 36 Here we primarily describe the photocycle of bacteriophytochromes. Photoexcitation of the Pr state (or Pfr state) prepares an excited state, which relaxes into a Lumi-R (or Lumi-F) intermediate. It is currently not entirely clear, how this relaxation proceeds. The predominant view is that the excited state is Fig. 2 The modular architecture of phytochromes. (a) General domain organization of homodimeric phytochromes with parallel dimeric arrangement. The N-terminal PAS, GAF and PHY domains form the photosensory module (PSM, green). The output module (OPM) is located at the C-terminus and has a variable domain organization. Despite the name of the module, the PSM and the NTE also contribute to the biological output activity of a phytochrome. (b) The classification of phytochrome-related proteins depending on the modular architecture of the PSM. The domain composition of an example protein from each group (parentheses) is shown. The chromophore is covalently bound to a cysteine in either PAS or GAF domain, as indicated. Domains are color-coded like in panel a. Domain abbreviations: diguanylate phosphodiesterase (EAL), cGMP phosphodiesterase/adenylyl cyclase/Fhl1 (GAF), diguanylate cyclase (GGDEF), histidine kinase (HK), histidine kinase-related (HKR), N-terminal extension (NTE), period/Arnt/Sim (PAS), phytochrome-specific (PHY), response regulator (RR). Photochemical & Photobiological Sciences Perspective This journal is © The Royal Society of Chemistry and Owner Societies 2020 Photochem. Photobiol. Sci. Open Access Article. Published on 27 October 2020. Downloaded on 10/28/2020 10:23:41 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online relatively long-lived (tens of picoseconds) in Pr and short-lived (around one picosecond) in Pfr. 61–65 The biliverdin D-ring undergoes Z-to-Eisomerization during the Pr-to-Pfr transition, and E-to-Zisomerization during the Pfr-to-Pr transition. 66,67 Polarization-resolved mid-infrared spectroscopy with structural refinement has confirmed that the isomerization occurs by a rotation over the C15–C16 methine-bridge between ring C and D. 68 The quantum yield of reaching the Lumi states is relatively low at less than 15%. Even though the transitions in Fig. 1b are indicated as linear forward reactions, back-reactions from intermediate states to the resting state have been recorded as well. 69,70 In bacteriophytochromes, the Lumi-R state typically lives for tens of microseconds until it converts into a Meta state, which in turn translates into the Pfr state on millisecond time scale. The Meta-R intermediate(s) has been shown to involve deprotonation and re-protonation events in several species. 71–74 This splits the Meta-R state into Meta-Ra and Meta-Rc (Fig. 1b). 75 Importantly, the chromophore is fully protonated in both Pr and Pfr states. 76 For the back reaction from Pfr to Pr, evidence by NMR suggests that the chromophore and protein stay protonated. 77 From the same study, it was also concluded that the back-isomerization occurs in two steps, which is in agreement with the crystallography of cryo-trapped intermediates of PaBphP 78 and with the fact that there are changes in the hydrogen-bonding network around the biliverdin D-ring in Meta-F. Possibly because there are no particular proton transfer reactions, the photoinduced reaction times from Pfr to Pr are somewhat faster. The Lumi-F state decays biphasic on a microsecond time-scale and the Meta-F state converts to Pr state within a millisecond time-scale. 79 Most information about the intermediate states has been obtained from spectroscopic studies, and direct structural information is unfortunately scarce. A notable exception is a recent structure of a cryo-trapped Meta-F intermediate, which reveals a twist of the biliverdin D-ring by virtually 180°, significant movements of the Cand B-ring propionates and side chain adjustments around the chromophore. 80 We note that the studied phytochrome fragment contains 24 mutations and an aberrant photocycle, raising a question whether the intermediate structure reflects the wild-type situation. In the study of cryo-trapped PaBphP intermediates, 78 the acquisition temperature resembles time, with lower temperatures trapping earlier intermediates. However, it was not possible to correlate the cryo-trapped intermediates to photocycle intermediates. These studies are a welcome start to understanding the structural mechanism of photoconversion. In the next section, we discuss what is known about this process. Three structural tiers of signal transduction Phytochromes are generally homodimeric complexes. Each subunit contains an N-terminal photosensory module (PSM), which senses light and transfers the signal, to the C-terminal output module (OPM) (Fig. 2a). Plant and fungal phytochromes have an additional N-terminal extension (NTE). The NTE has shown to stabilize their Pfr state and to participate in phosphorylation events in plants. 81–85 Plant phytochromes contain also two C-terminal PAS domains and a histidine kinase-like domain. Similar to other photosensor proteins, phytochromes are built up from a limited number of conserved domains. 45 Based on the composition of the PSM, the phytochrome superfamily can be divided into three classes (Fig. 2b). 46 Group I is the biggest group which describes phytochromes with three PSM domains: PAS (Per/Arnt/Sim), GAF (cGMP phosphodiesterase/adenylyl cyclase/Fhl1), and PHY (phytochrome-specific). Group I comprises of phytochromes from plants, fungi, bacteria and cyanobacteria. Group II contains phytochrome-like proteins without the PAS domain. These PAS-less phytochromes, like cyanobacterial Cph2, have a dimeric GAF-PHY structure. Group III proteins comprise of a single GAF domain and represents the cyanobacteriochromes, CBCRs. 86 PAS domains are present in many sensing proteins, in protein–protein interaction scaffolds and in transcription factors, which points towards a similar evolutionary origin. 87 Here, we focus on the group I phytochromes, which has the widest distribution across the kingdoms of life. A few years after their discovery, 10,13 several crystal and NMR structures of prokaryotic phytochromes were solved. The first crystal structure of a phytochrome was uncovered for the PAS-GAF fragment of the phytochrome from Deinococcus radiodurans. 88 The first structures of the complete phytochrome PSMs were from Cph1 from cyanobacterium Synechocystis sp. PCC6803 89 and PaBphP from Pseudomonas aeruginosa, 90 after which several other species have followed. More recently, PAS-GAF and PSM structures of plant phytochromes have been disclosed, 37,38 as well as bacteriophytochromes with an output module. 91–94 The presently solved crystal structures of PAS-GAF, PSM and full-length phytochromes from group I are summarized in Tables 1–3. Whereas many structures of the Pr and Pfr states of the PSM of bacteriophytochromes are available, the structures of the intermediate states remain to be disclosed. This means that the structural activation of phytochromes is starting to be understood, but that the mechanism of the changes is unclear. Tier one: the chromophore-binding pocket Structure of chromophore-binding domain. Several structures of the chromophore-binding PAS-GAF domains have been obtained to high resolution (Table 1). All these structures have a five stranded antiparallel β-sheet (with order 2-1-5-4-3) in the PAS domain, and a six-stranded β-sheet (with order 8-76-11-10-9) in the GAF domain, which coordinates the chromophore via hydrogen/salt-bridge bonding to the propionic side chains. The GAF domain holds three additional helices, which complete a C-shaped cavity surrounding the chromophore pocket. The PAS-GAF entities usually form dimers in solution and most, but not all, crystal structures are in dimeric arrangement (Fig. 3). Perspective Photochemical & Photobiological Sciences Photochem. Photobiol. Sci. This journal is © The Royal Society of Chemistry and Owner Societies 2020 Open Access Article. Published on 27 October 2020. Downloaded on 10/28/2020 10:23:41 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Curiously, the peptide chain in PAS-GAF forms a figure-of-eight knot (Fig. 3a), in which the N-terminal end of the chain is threaded through a loop consisting of amino acids that reside approximately several hundred residues C-terminal to them. 88 The knot has been confirmed in all PAS-GAF phytochrome structures. 31,37,89–93,96–99 So far, the knot has not been assigned a clear function for signal transduction, as structures in the Pr and Pfr states show hardly any rearrangements in this region. 89,90,100 Structural changes in the chromophore-binding pocket. The overall agreed mechanism for Pr-to-Pfr photoconversion in BphPs starts with the absorption of a photon by the biliverdin. This leads to cis-to-trans isomerization of the C15–C16 double bond between the C-ring and the D-ring. The isomerization changes the biliverdin from Pr-specific ZZZssa conformation to Pfr-specific ZZEssa conformation. 115–117 This D-ring rotation of about 180° occurs during the formation of the Lumi state. 40,116,118 It is not known how the chromophore-binding pocket reacts to the isomerization. One hypothesis is that this places the chromophore in a non-ideal position, forcing it to slide within its pocket. 78 New interactions with protein side chains within the chromophore-binding pocket would then be formed. 119 The propionate side chains of the B and C-rings swap interactions and the hydrogen-bonding network of the surrounding waters is altered. 76 Interactions between the chromophore, conserved amino acid side chains and water molecules in the chromophorebinding pocket are important for photochemistry. The crystal structures of PAS-GAF fragments in the Pr state (Table 1) have Table 1 Solved crystal structures of phytochrome PAS-GAF fragments. Structures of cyanobacteriochromes are not considered in this review and therefore excluded from the table. Note that the constructs that have significant amount of mutations are referred as their given name (e.g. IFP or miRFP). In these cases, the full set of mutations is available in the referred paper. The I 0 state in the structure 6T3U represents an intermediate en route to the Lumi-R state 95 Name Organism PDB code Release year Resolution (Å) Pr/Pfr Mutations Comment Ref. DrBphP D. radiodurans 1ZTU 2005 2.5 Pr P240T 88 DrBphP D. radiodurans 2O9B 2007 2.15 Pr Y307S 101 DrBphP D. radiodurans 2O9C 2007 1.45 Pr Y307S 101 RpBphP3 R. palustris 2OOL 2007 2.2 Pr 97 DrBphP D. radiodurans 3S7N 2012 2.45 Pr D207H, Y263F Fluorescent 102 DrBphP D. radiodurans 3S7O 2012 1.24 Pr D207H (IFP1.0) Fluorescent 102 DrBphP D. radiodurans 3S7P 2012 1.72 Pr D207H (IFP1.0) Fluorescent 102 DrBphP D. radiodurans 3S7Q 2012 1.75 Pr F145S, D207H, L311E, L314E Fluorescent, monomeric 102 DrBphP D. radiodurans 4CQH 2014 1.14 Pr IFP2.0 Fluorescent 103 RpBphP2 R. palustris 4E04 2012 1.79 Pr 16 mutations Packing mutations 104 DrBphP D. radiodurans 4O8G 2014 1.65 Pr IFP1.4 Fluorescent 105 DrBphP D. radiodurans 4IJG 2012 1.7 Pr F145S, L311E, L314E Monomeric 105 DrBphP D. radiodurans 4Q0H 2014 1.16 Pr Y307S 96 DrBphP D. radiodurans 4Q0I 2014 1.74 Pr Y307S, D207A 96 SaBphP1 S. aurantiaca 4RQ9 2016 2.5 Pr T289H See structure ‘6BAK’— RpBphP1 R. palustris 4XTQ 2015 1.64 Pr BphP1-FP, C20S Fluorescent 19 DrBphP D. radiodurans 4Y3I 2015 1.69 Pr Y307S Low X-ray 106 DrBphP D. radiodurans 4Y5F 2015 1.7 Pr Y307S High X-ray 106 DrBphP D. radiodurans 4Z1W 2015 1.3 Pr D207L, Y263F Fluorescent 107 DrBphP D. radiodurans 4ZRR 2015 1.5 Pr D207L Y263F Fluorescent 107 DrBphP D. radiodurans 5AJG 2016 1.11 Pr IFP1.4 Fluorescent 108 DrBphP D. radiodurans 5K5B 2016 1.35 Pr 109 DrBphP D. radiodurans 5L8M 2016 2.1 Pr SFX 109 DrBphP D. radiodurans 5LBR 2016 2.2 Pr SFX 109 DrBphP D. radiodurans 5MG0 2017 1.65 Pr Y307S SFX 110 DrBphP D. radiodurans 5NFX 2018 1.34 Pr Y263F 111 RpBphP1 R. palustris 5VIK 2017 1.35 Pr miRFP703 Fluorescent, bathy 112 RpBphP1 R. palustris 5VIQ 2017 1.34 Pr miRFP709 Fluorescent, bathy 112 RpBphP1 R. palustris 5VIV 2017 1.33 Pr miRFP670, monomeric Fluorescent, bathy, two BV linkages 112 SaBphP1 S. aurantiaca 6BAF 2017 2.73 Pr 31 SaBphP1 S. aurantiaca 6BAK 2017 2.5 Pr T289H 31 DrBphP D. radiodurans 6FTD 2018 1.4 Pr H290T 113 IsPadC Idiomarina sp. A28L 6SAX 2019 2.4 Pr Monomeric 114 IsPadC Idiomarina sp. A28L 6SAW 2019 3.0 Pfrlike Dimeric 114 DrBphP D. radiodurans 6T3L 2020 2.07 Pr SFX 95 DrBphP D. radiodurans 6T3U 2020 2.21 I 0 SFX, 1 ps after photoexcitation 95 phyB Sorghum bicolor 6TBY 2020 1.80 Pr Plant, with PCB 38 phyB Sorghum bicolor 6TC5 2020 2.10 Pr Plant, with PΦB38 phyA Glycine max 6TC7 2020 2.13 Pr Plant, with PCB 38 Photochemical & Photobiological Sciences Perspective This journal is © The Royal Society of Chemistry and Owner Societies 2020 Photochem. Photobiol. Sci. Open Access Article. Published on 27 October 2020. Downloaded on 10/28/2020 10:23:41 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online provided structural basis for understanding these interactions (Fig. 3b). PAS-GAF crystal structures, 88 which now have reached a resolution close to 1 Å, 96,109 show that the D-ring of the biliverdin has substantially more space than the other pyrrole rings and thereby greater freedom to move within the chromophore-binding pocket. A number of conserved residues surround the chromophore. Mutational investigations show that most of them are important for proper photo-switching, but that only a few of them completely disrupt photo-conversion. 73,90,102,120,121 We note that the majority of the results here have evaluated the mutations against photoconversion as measured by optical spectroscopy. This may be appropriate as long as the investigated residue is located close to the chromophore. It would be useful to repeat some of the studies in the future by probing the direct structural changes or biochemical activity of the phytochromes. 59,92 The incorporation of the chromophore appears to be relatively robust against single mutations. Next, we name a few prominent amino acids that interact with the chromophore (see Fig. 3b): Two residues, an aspartate and a histidine, are shown to be crucial for proper phytochrome photochemistry. 73 The aspartate (Asp207 in DrBphP) is part of the highly conserved DIP motif and resides at close proximity of pyrrole rings A and D. When this aspartate was removed in plant phytochrome B, phytochrome failed to photoconvert and instead became fluorescent. 122 The same mutation in DrBphP has been used to generate fluorescent phytochrome variants. 102 The histidine (His260 in DrBphP) lies face-to-face on the Band C-rings and forms a hydrogen network with the pyrrole nitrogens of the biliverdin. The histidine is also important for buffering the deprotonation and protonation events of the chromophore in different states of the photocycle. 123,124 The aromatic character of a conserved tyrosine (Tyr263 in DrBphP) has been demonstrated to be important for Pfr formation in Cph1 and its absence affects the position of the PHY domain and its interactions. 125 In DrBphP, the absence of the hydroxyl group of Tyr263 destabilizes the β-sheet conformation of the PHY-tongue in the Pr state and makes the protein more prone to adopt an α-helical structure regardless of the chromophore configuration. 111 One of the two phytochromes in Stigmatella aurantiaca,SaBphP1, has a threonine instead of the highly conserved histidine (His290 in DrBphP) that is in H-binding distance to the D-ring carbonyl. In terms of absorption spectra, this protein shows an incomplete photoconversion but an unusually high quantum yield of reaching the first intermediate state. In this case, normal photoconversion can be obtained when the threonine is mutated to a histidine. 59,69,113 It is noteworthy that regardless of whether a histidine or threonine occupies this position, the structural changes of the PSM (PHY separation, see Fig. 4) remain similar to other bacteriophytochromes in the Pr-to-Pfr transition. 59 The crystal structures of phytochromes reveal various consistent waters in close contact to the biliverdin chromophore. The waters are thought to be involved in the structural mechanism upon photoactivation as they act as fast structural mediators between biliverdin and surrounding amino acids. Although their potential role in the photoactivation mechanism has been recognized, the detailed role of these waters remains unclear. The most prominent water is the so-called pyrrole water 88 (Fig. 3b). The pyrrole water is located in the center of the bilin and in hydrogen bond distance to the nitrogens of pyrrole rings A, B and C. It also resides at the hydrogen bond distance to the backbone carbonyl group of the aspartate in the DIP motif (Asp207 in DrBphP). There are two highly ordered waters forming a hydrogen-bonding network between the D-ring carbonyl, the C-ring propionate and the conserved histidine (His290 in DrBphP). If the His290 position is occupied by a threonine, this water molecule network is Fig. 3 Overview structure and chromophore binding of the BphP PAS-GAF fragment. (a) The structural features of a PAS-GAF dimer from Deinococcus radiodurans phytochrome (DrBphP, PDB id 6T3L 95 ). Subunit 1 with its PAS and GAF domain is colored in different shades of green whereas subunit 2 is grey. N-Terminal end of the PAS domain as well as biliverdin chromophore are colored in orange. The biliverdin position and knot region are indicated. (b) Zoom in on the biliverdin and selected surrounding amino acid residues. Residue numbers are from DrBphP, and the ring names (A–D) are indicated. The so-called pyrrole water (PW) and two well-defined waters between His290, biliverdin D-ring, and C-ring propionate group are also shown as red spheres (PDB id: 4Q0H 96 ). Perspective Photochemical & Photobiological Sciences Photochem. Photobiol. Sci. This journal is © The Royal Society of Chemistry and Owner Societies 2020 Open Access Article. Published on 27 October 2020. Downloaded on 10/28/2020 10:23:41 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online extended. 113 Furthermore, a water positioned between the pyrrole water and the tyrosine (Tyr263 in DrBphP) has been identified to exchange rapidly at room temperature. 109 In addition, a dynamic water molecule has been observed by molecular dynamics simulations of a simulated Lumi-R state close to the D-ring carbonyl. 126 Crystal structures of the same phytochrome in Pr and Pfr state are now available, in which the position of the D-ring is clearly resolved. 78,96,100,119 This has revealed new chromophore interactions showing that the bilin slides in the chromophore pocket and the propionate side chains of ring B and C forms new interactions with the adjacent amino acid in the Pfr state compared to the Pr state. A crystallographic study of the bathy phytochrome PaBphP resolved the structure of three intermediates between Pfr and Pr by cryo-trapping. 78 It was found that in the first intermediate, the D-ring twists around the C15-methine bridge, the twist is partially released, the C-ring adjusts in the second intermediate. In addition, the Cand B-ring propionates were found to detach from the surrounding protein pocket in the second and third intermediate, respectively. Some surrounding residues were found to move in accordance with these structural changes, but overall the protein structural changes were small. This may be an artifact of the confinement of the protein by the crystal packing. Effects of the chromophore-binding pocket configuration can also be observed in the early photocycle. The evolution from the Pr to Lumi-R state appears much slower than what would have been the case for free isomerization in solution. 68 This is consistent with that the chromophore is tightly held in the binding pocket and that structural changes in the binding pocket have to occur before the isomerization reaction can proceed. For example, a study with mid-infrared spectroscopy of RpBphP2 and RpBphP3 showed that additional hydrogen bonds with the D-ring in Pr state prolong the excited-state lifetime. 127 This trend was also observed for SaBphP1 69 but not for a DrBphP variant. 113 Contrary to these results, the formation of the Lumi-F state from Pfr in a bathy phytochrome was found to take place already within femtoseconds, 128 potentially because the protein structure does not have to adjust as much as in the Pr to Lumi-R transition. Tier two: the photosensory module Structures of photosensory modules. Among the first PSM structures, the structure from Cph1 adopted an antiparallel dimer arrangement, 89 whereas in the PaBphP structure the subunits align in a parallel head-to-head arrangement. 90 Later, most PSM structures arranged in the parallel dimerization scheme, 31,37,38,90,98–100,110,119,125 which is consistent with the supposed output activity of these phytochromes. The core of the PHY domain shares the same structural motif as the PAS and GAF domains. In addition, a long helix of 18 rotations (72 Å) connects the PHY and GAF domains, and another helix connects the PHY to the C-terminal output domains (Fig. 4). Here, we call these helices as “GAF-PHY helix”and “PHY-OPM helix”, respectively, that together form the phytochrome-spanning helical spine. In the Pr state of canonical BphPs, the GAF-PHY helix has a kinked or slightly bent structure resulting in a left-handed twist with respect to its sister subunit. 37,96,99,100,129 Based on the DrBphP structures, the PHY domains may form a cavity in the center of the dimer with hardly any dimer contacts. See Table 2 for the phytochrome PSM crystal structures that are solved to date. The PHY domain connects back to the chromophorebinding pocket via the so-called PHY tongue (Fig. 4), whose length varies between different species. The interactions of the PHY tongue and the chromophore occur through bridge residues, most notably the conserved Asp residue of the DIP motif (Asp207 in DrBphP). The addition of the PHY domain to PAS-GAF fragment makes the chromophore-binding pocket slightly tighter and shows a small displacement of the D-ring of the chromophore. 96 In some structures, the angle of the helical bundle at the dimer interface of the PAS-GAF domains becomes tilted by the addition of the PHY domain. 31 The presence of the PHY domain appears important for formation of the Pfr state in bacteriophytochromes. 51 For these proteins, the photoactivated state is less stable when lacking the PHY domain, which results a roughly 100 times faster thermal back reversion of the PAS-GAF domain compared to the PSM and full-length phytochrome. 96,130 Moreover, the PHY tongue changes its secondary structure when transforming from Pr (β-sheet) to Pfr (α-helix). 100,131 We will discuss this in detail in the next section. Structural changes by the chromophore are relayed to the PHY tongue. The structural comparison between Pr and Pfr states was initially possible through only a few structures of PSMs in their resting state: a Pfr-state structure of a bathy phytochorme PaBphP 78,90,132 and a Pr-state structure of a canonical phytochrome Cph1. 89 In both structures, the PHY tongue interacts with the DIP motif in GAF through the conserved PRxSF motif. However, it was challenging to distinguish whether the differences originated from their state or from overall structural differences between species. The most prominent difference was that the PHY tongue adopted a β-sheet structure in the Pr state but an α-helical fold in the Pfr state. Further aspects of the tongue refolding were proposed with the release of the Pr structure from the PAS-less Cph2 from Synechocystis sp. and called the “tryptophan switch”. 133 In the proposal, two conserved tryptophans flanking the PRxSF motif in the tongue were suggested to switch places upon tongue refolding and the 180° rotation of the PHY tongue around its own axis. An extensive comparison and clustering of the tongue fold can be found in a review by Burgie and Vierstra. 44 The crystal structures of DrBphP PSM both in its dark (Pr) and illuminated (mixed Pfr/Pr) form 100 directly confirmed that the PHY tongue refolds upon photoconversion from β-sheet to α-helical conformation. The refolding occurs every time the phytochrome photoswitches and is an integral part of the phytochrome photoconversion mechanism. Two years after the initial discovery, a structure of a DrBphP F469W mutant was presented, which had a high Pfr content (87%). 119 Due to this, Photochemical & Photobiological Sciences Perspective This journal is © The Royal Society of Chemistry and Owner Societies 2020 Photochem. Photobiol. Sci. Open Access Article. Published on 27 October 2020. Downloaded on 10/28/2020 10:23:41 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online the electron density of the chromophore and surrounding residues stabilizing the Pfr state were clearer. This structure confirmed the PHY tongue refolding mechanism, and revealed some more details on the interactions between biliverdin and the surrounding amino acids in the Pfr state. The tongue region contains conserved motifs, with PRxSF being the most prominent one. It makes close contacts with the GAF domain close to the chromophore. In the Pr state, the arginine in this motif (Arg466 in DrBphP) forms a salt bridge with the aspartate in the DIP motif of the GAF domain (Asp207 in DrBphP), which stabilizes the β-sheet arrangement of the PHY tongue. 133 Upon photoactivation, the salt bridge between the aspartate and the arginine breaks. This leads to the release of the tongue and the formation of new interaction between of the DIP aspartate and the PRxSF serine (Ser468 in DrBphP) in the Pfr state. The importance of this conserved PRxSF motif is supported by mutational studies. In DrBphP, many mutations in the motif cause incomplete photoconversion and alter dark reversion rates, especially in the case of serine (Ser468 in DrBphP). 96 Proline forms packing interactions with the biliverdin A-ring in the Pr state and stabilizes the Pfr state in Agp1. 99 RpBphP3 has a threonine instead of proline in this site, which leads to a formation of a near-red-absorbing Pnr state instead of a Pfr state. 98 Finally, a phenylalanine in the motif (Phe469 in DrBphP) stabilizes the Pfr state by impeding the thermal dark reversion. 73 Structural changes of the PHY domain. The first crystal structures of a same BphP PSM in both Pr and Pfr showed that the refolding of the PHY tongue seemingly pulls the PHY domain closer to the GAF domain in Pfr. 100 This movement leads to an increase in separation between the PHY domains Table 2 Solved crystal structures of phytochrome photosensory modules (PSM) Name Organism PDB code Release year Resolution (Å) Pr/Pfr Mutations Comment Ref. Cph1 Synechocystis sp. PCC6803 2VEA 2008 2.21 Pr Antiparallel 89 PaBphP P. aeruginosa 3C2W 2008 2.9 Pfr Bathy 90 PaBphP P. aeruginosa 3G6O 2009 2.85 Mixed Q188L Bathy 132 PaBphP P. aeruginosa 3IBR 2009 2.97 Mixed Q188L Bathy 132 PaBphP P. aeruginosa 3NHQ 2011 2.55 Pfr Bathy 78 PaBphP P. aeruginosa 3NOP 2011 2.8 L1 Bathy 78 PaBphP P. aeruginosa 3NOT 2011 2.7 L2 Bathy 78 PaBphP P. aeruginosa 3NOU 2011 3 L3 Bathy 78 Cph1 Synechocystis sp. PCC6803 3ZQ5 2011 1.95 Pr Y263F Antiparallel 125 Cph2 Synechocystis sp. PCC6803 4BWI 2013 2.6 Pr PAS-less 133 DrBphP D. radiodurans 4O01 2014 3.24 Pr/Pfr Illuminated 100 DrBphP D. radiodurans 4O0P 2014 3.8 Pr Dark 100 phyB A. thaliana 4OUR 2014 3.4 Pr Plant 37 DrBphP D. radiodurans 4Q0J 2014 2.75 Pr 96 RpBphP2 R. palustris 4R6L/ 4S21 2015 3.39 Pr 98 RpBphP3 R. palustris 4R70 2015 2.85 Pr Antiparallel 98 DrBphP D. radiodurans 5C5K 2016 3.31 Pfr F469W 119 Agp1 A. fabrum 5HSQ 2016 1.85 Pr E86A, E87A, E336A, K337A Antiparallel 99 Agp1 A. fabrum 5I5L 2016 2.7 Pr 99 IsPadC Idiomarina sp. A28L 5LLX 2017 2.8 Pr With GTP 92 IsPadC Idiomarina sp. A28L 5LLY 2017 2.4 Pr 92 DrBphP D. radiodurans 5MG1 2017 3.3 Pr Y307S SFX 110 DrBphP D. radiodurans 5NM3 2018 3.3 Pr/Pfr Y263F BV in Pr/Pfr, protein Pfr 111 DrBphP D. radiodurans 5NWN 2018 3.6 Pr/Pfr Y263F BV in Pr, protein Pfr 111 RpBphP1 R. palustris 5OY5 2019 2.6 Pfr Monomeric — SaBphP1 S. aurantiaca 6BAO 2017 2.5 Pr 31 SaBphP1 S. aurantiaca 6BAP 2017 2.5 Pr T289H 31 SaBphP1 S. aurantiaca 6BAY 2017 2.5 Pr T289H SFX 31 Agp2 A. fabrum 6G1Y 2018 2.5 Pfr Bathy 80 Agp2 A. fabrum 6G1Z 2018 2.03 Pfr PAiRFP2, 24 mutations Fluorescent bathy, antiparallel 80 Agp2 A. fabrum 6G20 2018 2.16 MetaFPAiRFP2, 24 mutations Fluorescent bathy, antiparallel 80 SaBphP2 S. aurantiaca 6PTQ 2019 2.1 Pr SFX 134 SaBphP2 S. aurantiaca 6PTX 2019 1.65 Pr 134 SaBphP2 S. aurantiaca 6PU2 2019 2.2 Pr H275T 134 Agp1 A. fabrum 6R26 2020 3.11 Pr-like Synthetic chromophore — Agp1 A. fabrum 6R27 2020 3.40 Pr-like Synthetic chromophore — phyB Glycine max 6TL4 2020 2.90 Pr Plant 38 Perspective Photochemical & Photobiological Sciences Photochem. 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