Cyanobacteriochromes - Biology

Cyanobacteriochromes - Biology

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Blue-/Green-Light-Responsive Cyanobacteriochromes Are Cell Shade Sensors in Red-Light Replete Niches

Cyanobacteriochrome (CBCRs) photoreceptors show various photochemical properties, but their ecophysiological functions remain elusive. Here, we report that the blue/green CBCRs SesA/B/C can serve as physiological sensors of cell density. Because cyanobacterial cells show lower transmittance of blue light than green light, higher cell density gives more green-light-enriched irradiance to cells. The cell-density-dependent suppression of cell aggregation under blue-/green-mixed light and white light conditions support this idea. Such a sensing mechanism may provide information about the cell position in cyanobacterial mats in hot springs, the natural habitat of Thermosynechococcus. This cell-position-dependent SesA/B/C-mediated regulation of cellular sessility (aggregation) might be ecophysiologically essential for the reorganization and growth of phototrophic mats. We also report that the green-light-induced dispersion of cell aggregates requires red-light-driven photosynthesis. Blue/green CBCRs might work as shade detectors in a different niche than red/far-red phytochromes, which may be why CBCRs have evolved in cyanobacteria.

Keywords: Biological Sciences Microbiology Sensor.

Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes

Bacterial photoreceptors absorb light energy and transform it into intracellular signals that regulate metabolism. Bacterial phytochrome photoreceptors (BphPs), some cyanobacteriochromes (CBCRs) and allophycocyanins (APCs) possess the near-infrared (NIR) absorbance spectra that make them promising molecular templates to design NIR fluorescent proteins (FPs) and biosensors for studies in mammalian cells and whole animals. Here, we review structures, photochemical properties and molecular functions of several families of bacterial photoreceptors. We next analyze molecular evolution approaches to develop NIR FPs and biosensors. We then discuss phenotypes of current BphP-based NIR FPs and compare them with FPs derived from CBCRs and APCs. Lastly, we overview imaging applications of NIR FPs in live cells and in vivo. Our review provides guidelines for selection of existing NIR FPs, as well as engineering approaches to develop NIR FPs from the novel natural templates such as CBCRs.

Keywords: allophycocyanin bacterial photoreceptor cyanobacteriochrome near-infrared fluorescent protein phytochrome tetrapyrrole.

Conflict of interest statement

The authors declare no conflict of interest.


Chromophores and structures of selected…

Chromophores and structures of selected bacterial photoreceptors. Linear tetrapyrrole chromophores are: ( A…

Structural properties of cyanobacteriochromes (CBCRs):…

Structural properties of cyanobacteriochromes (CBCRs): ( A ) topology diagram of CBCR GAF…

Molecular evolution steps, methods and…

Molecular evolution steps, methods and techniques employed to develop near-infrared fluorescent proteins (NIR…

Applications of near-infrared fluorescent proteins.…

Applications of near-infrared fluorescent proteins. ( A ) Image of iRFP713 transgenic newborn…

Molecular characterization of D X CF cyanobacteriochromes from the cyanobacterium Acaryochloris marina identifies a blue-light power sensor

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors that sense a wide range of wavelengths from ultraviolet to far-red. The primary photoreaction in these reactions is a Z/E isomerization of the double bond between rings C and D. After this isomerization, various color-tuning events establish distinct spectral properties of the CBCRs. Among the various CBCRs, the DXCF CBCR lineage is widely distributed among cyanobacteria. Because the DXCF CBCRs from the cyanobacterium Acaryochloris marina vary widely in sequence, we focused on these CBCRs in this study. We identified seven DXCF CBCRs in A. marina and analyzed them after isolation from Escherichia coli that produces phycocyanobilin, a main chromophore for the CBCRs. We found that six of these CBCRs covalently bound a chromophore and exhibited variable properties, including blue/green, blue/teal, green/teal, and blue/orange reversible photoconversions. Notably, one CBCR, AM1_1870g4, displayed unidirectional photoconversion in response to blue-light illumination, with a rapid dark reversion that was temperature-dependent. Furthermore, the photoconversion took place without Z/E isomerization. This observation indicated that AM1_1870g4 likely functions as a blue-light power sensor, whereas typical CBCRs reversibly sense two light qualities. We also found that AM1_1870g4 possesses a GDCF motif in which the Asp residue is swapped with the next Gly residue within the DXCF motif. Site-directed mutagenesis revealed that this swap is essential for the light power-sensing function of AM1_1870g4. This is the first report of a blue-light power sensor from the CBCR superfamily and of photoperception without Z/E isomerization among the bilin-based photoreceptors.

Keywords: cyanobacteria cyanobacteriochrome photobiology photoreceptor phytochrome site-directed mutagenesis spectroscopy.

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article


The opacity of mammalian tissue to visible light and the strong attenuation of infrared light by water at ≥900 nm have contributed to growing interest in the development of far-red and near-infrared absorbing tools for visualizing and actuating responses within live cells. Here we report the discovery of cyanobacteriochromes (CBCRs) responsive to light in this far-red window. CBCRs are linear tetrapyrrole (bilin)-based light sensors distantly related to plant phytochrome sensors. Our studies reveal far-red (λmax = 725–755 nm)/orange (λmax = 590–600 nm) and far-red/red (λmax = 615–685 nm) photoswitches that are small (<200 amino acids) and can be genetically reconstituted in living cells. Phylogenetic analysis and characterization of additional CBCRs demonstrated that far-red/orange CBCRs evolved after a complex transition from green/red CBCRs known for regulating complementary chromatic acclimation. Incorporation of different bilin chromophores demonstrated that tuning mechanisms responsible for red-shifted chromophore absorption act at the A-, B-, and/or C-rings, whereas photoisomerization occurs at the D-ring. Two such proteins exhibited detectable fluorescence extending well into the near-infrared region. This work extends the spectral window of CBCRs to the edge of the infrared, raising the possibility of using CBCRs in synthetic biology applications in the far-red region of the spectrum.

In describing photocycles, we use a convention whereby the photostate with the 15Z bilin configuration is listed first, followed by the photostate with the 15E configuration. Color definitions in this study are as follows: near UV, 300–394 nm violet, 395–410 nm blue, 411–485 nm teal, 486–514 nm green, 515–569 nm yellow, 570–585 nm orange, 586–614 nm red, 615–685 nm far red, 686–760 nm near infrared, 761–1000 nm.

Cyanobacteriochromes in full color and three dimensions

Sensory photoreceptors occur in all kingdoms of life, eliciting diverse organismal adaptations in response to incident light. The recently identified cyanobacteriochromes (CBCRs) mediate photochromatic and phototactic responses in cyanobacteria (1 ⇓ –3). Great strides toward a molecular understanding of photoreception and signal transduction in this spectrally diverse and exciting photoreceptor family have now been taken by Narikawa et al. (4), who report high-resolution structures of two CBCR photosensor modules in PNAS.

CBCRs are related to plant and bacterial phytochromes (Phys), with which they share the intrinsic ability to form thioether linkages to linear tetrapyrrole (bilin) chromophores via conserved cysteine residues. Moreover, these photoreceptor families use a unifying photochemical mechanism (Fig. 1A): photoisomerization of the chromophore between 15Z and 15E configurations with concomitant rotation of the terminal bilin D-ring (5 ⇓ –7). The 15Z and 15E states differ in their absorption properties (photochromism) and modulate the behavior of output domains and downstream signal transduction pathways. Despite similar chromophores, photochemistry, and self-assembly, Phys and CBCRs differ in several striking ways. Most phytochromes require a three-domain PAS-GAF-PHY architecture [GAF domain, cGMP-phosphodiesterase/adenylate cyclase/FhlA (8) PAS domain, Per/ARNT/Sim PHY domain, phytochrome-specific] for reversible photoconversion (3, 5, 7, 9). CBCRs instead achieve fully reversible photochemistry with a lone chromophore-binding GAF domain. Multiple CBCRs often occur in tandem within a single protein, allowing integration of multiple light signals at a single C-terminal output domain (10). Whereas Phys predominantly respond to the red/far-red spectral region, CBCRs display a rich variety of photocycles spanning the entire visible and near-UV spectrum (2, 11 ⇓ –13). At least four subfamilies of CBCRs can be distinguished on the basis of their underlying photochemistry and primary structure.

Cyanobacteriochrome structure and function. (A) Bilin chromophores and photochemistry of CBCRs. PCB and PVB are shown with their 15,16-double bonds in the configurations revealed by the work of Narikawa et al. (4). Conjugated π systems are outlined, propionate sidechains are indicated by “P,” and selected carbon atoms are labeled by red numbers. (B) Structure of the AnPixJ CBCR dimer showing the six-helix bundle formed by the distal helices. CBCR domains frequently occur in tandem, modeled in white by the duplicate structure. (C) The interdomain linker in tandem-GAF proteins, as measured between the positions highlighted in blue in B, shows a strong preference for discrete lengths, hinting at conserved mechanisms of signal transduction and integration.

Curiously, two of these subfamilies feature opposite photocycles: green/red CBCRs have a green-absorbing 15Z dark (ground) state and red-absorbing 15E photoproduct (2), but red/green CBCRs instead have a red-absorbing 15Z dark state and green-absorbing 15E photoproduct (14). The other two subfamilies, insert-Cys and DXCF CBCRs, both make use of additional conserved cysteine residues and typically exhibit a 15Z dark state sensitive to shorter wavelengths (near-UV to blue) and a 15E photo-product absorbing at longer wavelengths from blue to orange (3, 11, 12, 15). DXCF CBCRs can autocatalytically isomerize the phycocyanobilin (PCB) chromophore of CBCRs into phycoviolobilin (PVB) (Fig. 1A), thereby tuning photoproduct absorbance between teal and orange light (12, 15, 16). Two of these subfamilies and both photostates are represented in the structures described by Narikawa et al. (4).

Using X-ray diffraction, Narikawa et al. (4) have determined 1.8-Å and 2.0-Å resolution structures of two CBCR photosensor modules from the cyanobacteria Nostoc sp. PCC 7120 (AnPixJ) and Thermosynecchococcus elongatus BP-1 (TePixJ). Both CBCRs adopt the canonical GAF fold and bind their bilin chromophores in a cleft formed by a six-stranded antiparallel β sheet and three proximal α helices three distal helices are situated on the opposite face of the sheet (Fig. 1B). AnPixJ is a red/green CBCR using PCB as chromophore (14), and it was crystallized in the red-absorbing 15Z dark state. TePixJ is a DXCF CBCR containing a mix of PCB and PVB (15), with only the PVB population represented in the crystal structure of the green-absorbing 15E photoproduct (Fig. 1A). In phytochromes, the 15Z configuration is associated with the red-absorbing Pr state, and the 15E conformation is associated with the far-red-absorbing Pfr state (3, 6, 7, 9). A comparison of the CBCR structures to those of bacterial Phys thus grants unprecedented molecular insight into photosensory mechanisms inherent to all bilin-based photoreceptors and into specific mechanisms used by individual CBCR subfamilies.

In phytochromes, crystallography and NMR spectroscopy provide robust evidence for Z/E photoisomerization of the 15,16-double bond (3, 6, 7, 9, 17). A large body of biochemical data implicates the same primary photochemistry in CBCRs (2, 11 ⇓ ⇓ ⇓ ⇓ –16, 18), which is now confirmed by the 15Z dark state and 15E photoproduct seen in the present structures. Excitingly, key protein–chromophore interactions are also conserved between CBCRs and Phys: a conserved histidine or tyrosine residue forms a hydrogen bond to the carbonyl oxygen of the bilin D-ring in the 15Z state (4, 5, 9), and the amide nitrogen of the D-ring is hydrogen-bonded to a conserved aspartate residue in the 15E state (4, 7, 17). Conservation of both primary photochemistry and key chromophore–protein interactions raises the intriguing possibility that transduction of the photochemical signal to the C-terminal output domain will also be conserved.

The CBCR structures also shed light on the diverse panoply of photocycles. CBCRs lack the PAS and PHY domains of Phys, causing the bilin A- and B-rings to be solvent-exposed. In both AnPixJ and the cyanobacterial phytochrome Cph1 (9), the chromophore adopts the 15Z configuration with overall similar geometry. However, the conserved aspartate plays different roles: in Cph1, it interacts with a conserved residue in the PHY domain, but in AnPixJ it directly interacts with the bilin rings A, B, and C (4). The structural basis for formation of the green-absorbing photoproduct of AnPixJ and related proteins remains to be elucidated (10, 13, 14). The case is reversed for TePixJ, in which the green-absorbing photoproduct was crystallized and the blue-absorbing dark state remains to be characterized. Electron density unambiguously identifies the bilin chromophore as a singly linked PVB adduct (4) the critical DXCF cysteine residue (15) is unattached to the chromophore. In PVB, the C5 methine bridge of PCB is saturated, shortening the conjugated π electron system (Fig. 1A). The structure of TePixJ thus elucidates the basis for perception of green light. There are not yet structures for the blue-absorbing dark state, but biochemical and spectroscopic studies provide compelling evidence for a covalent linkage between the DXCF cysteine and the C10 atom of the bilin chromophore in this state (11, 12, 15, 16, 18).

The work by Narikawa et al. now provides a structural backdrop for future spectroscopic and mechanistic studies of CBCRs.

The CBCR structures also offer tantalizing clues about signal propagation from chromophore to output domain. AnPixJ and TePixJ both crystallize as parallel dimers, with the distal α helices of the dimeric partners forming a helical bundle. Highly similar quaternary structural arrangements have been observed for other GAF proteins (8) and phytochromes (5, 7), in which the distal helical bundle has been implicated in the transduction of light signals to downstream output modules (7, 17). On the basis of sequence analysis, Narikawa et al. (4) argue that CBCRs also connect to their output modules via continuous “signaling helices” (19), which propagate the signal toward the C terminus (e.g., via piston, pivot or rotary movements within helical bundles). Interestingly, sequence data further indicate that both tandem CBCR photosensor modules and tandem GAF domains are serially connected by α helices of conserved length (Figs. 1 B and C). Tandem CBCR photosensor modules might thus integrate multiple light signals via a series of helical movements conserved in GAF and other domains (10, 20), implying a wider relevance for the work of Narikawa et al. (4).

In summary, the work by Narikawa et al. (4) now provides a structural backdrop for future spectroscopic and mechanistic studies of CBCRs. Because of their related photochemistry but simpler domain architecture, CBCRs can serve as powerful paradigms for phytochromes. Finally, given their compact size and their ability to sense various light colors and intensities (13), CBCRs are attractive building blocks in the engineering of photoreceptors for use in optogenetics, and the present structures will provide a structural rationale.

Note Added in Proof.

Burgie et al. have recently determined two structures of TePixJ in its blue-absorbing dark state that confirm the presence of a covalent bond between the DXCF cysteine and the C10 atom of the bilin (21).

Cyanobacteriochromes: a new superfamily of tetrapyrrole -binding photoreceptors in cyanobacteria

M. Ikeuchi and T. Ishizuka, Photochem. Photobiol. Sci., 2008, 7, 1159 DOI: 10.1039/B802660M

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Structural uniqueness of the green- and red-light sensing photosensor in cyanobacteria

RcaE senses green and red lights and regulates the absorptive maxima of light-harvesting antenna supercomplex (phycobilisome, inset figure) in cyanobacteria. Credit: Toyohashi University Of Technology

Certain cyanobacteria can change the absorbing light colors for photosynthesis using a green- and red-light sensing photosensor protein. A Japanese research group elucidated the molecular structure of RcaE, a representative member of the photosensors. They revealed the unique conformation of the bilin chromophore and the unique protein structure that potentially functions as a proton transfer route to bilin. They also demonstrated that RcaE undergoes protonation and deprotonation of the bilin chromophore during the green and red photoconversion. These results provide insights into how cyanobacteria evolved photosensors with diverse spectral sensitivities and contribute to the development of new photoswitches of gene expression.

Certain cyanobacteria can utilize both green and red lights for photosynthesis by using their light-harvesting antenna supercomplex called phycobilisome. They can control the absorptive maxima of phycobilisome, which results in remarkable changes in cell color. This phenomenon is regulated by RcaE that belongs to cyanobacteriochrome family of photosensors. RcaE harbors a bilin chromophore and photoconverts green- and red-absorbing states to sense ambient light colors. Although the green and red photoconversion is involved in bilin photoisomerization and subsequent change in bilin protonation state, the structural basis of this photoconversion remains unknown.

The research group comprised Takayuki Nagae (Nagoya University), Masaki Mishima (Tokyo University of Pharmacy and Life Science), Yuu Hirose (Toyohashi University of Technology), Masashi Unno (Saga University), Kei Wada (Miyazaki University), and Yutaka Itoh (Tokyo City University). They determined the high-resolution structure of RcaE in its red-absorbing state via X-ray crystallography. The bilin chromophore showed a conformation with co-planar A-C rings, wherein the nitrogen atoms were facing inward the nitrogen of the tilted D-ring was facing outward (classified as C15-E,syn structure). Additionally, they identified a porous cavity containing water molecules in the bilin-binding pocket of RcaE. The clustered water molecules were connected with the nitrogen atoms of bilin A-C rings by a hydrogen bond network through the conserved acidic residue, Glu217.

  • Unique leaky bucket structure of RcaE. Clustered water molecules behind the pick loop are shown as red balls. Credit: Toyohashi University Of Technology.
  • A proposed model of the green and red photoconversion of RcaE. Credit: Toyohashi University Of Technology.

The research group demonstrated by molecular dynamic simulations that the water molecules in the cavity were exchanged with the solvent water. They also demonstrated by 15N NMR spectroscopy that four pyrrole nitrogen atoms of bilin are fully protonated in the red-absorbing state, whereas one nitrogen atom is deprotonated in the green-absorbing state. They assume that the unique porous cavity functioned as a proton exit or inlet pathway during the green and red photoconversion. Considering previous study reports on Raman spectroscopy of RcaE, they proposed that bilin deprotonation occurred in the B-ring nitrogen with the C15-Z,anti structure. They are currently working on the crystallization of the green-absorbing state of RcaE to confirm this model.

Elucidating the structure and spectral tuning mechanisms of RcaE provides insights into how cyanobacteria have evolved diverse cyanobacterial subfamilies to acclimate to different light environments. Green and red light-sensing cyanobacteriochromes have been utilized in synthetic biology as sophisticated photoswitches that control gene expression. Amino acid residue modification based on RcaE structure will contribute to the development of new photoswitches with desirable photosensitivities.


Phylogenetic and Spectroscopic Analyses Define a Previously Uncharacterized frCBCR Lineage.

In previous work, we noted that recombinant JSC1_58120g3 exhibited an unusually red-shifted far-red/red photocycle [712/654 nm (45)]. This CBCR is one of three arranged in tandem in a CBCR-regulated methyl-accepting chemotaxis protein (cMCP), an architecture consistent with known phototaxis sensors (53, 54). Neither of the adjacent domains, JSC1_58120g2 and JSC1_58120g4, exhibited similar behavior. Instead, these CBCRs exhibited UV/blue and red/green photocycles with phycoviolobilin (PVB) and PCB chromophores, respectively (SI Appendix, Fig. S1C). BLAST searches (55) using JSC1_58120g3 as a query identified a number of CBCR sequences with characteristic amino acid substitutions relative to canonical red/green CBCRs, with other residues conserved among the two groups (SI Appendix, Fig. S2A). Phylogenetic analysis established that JSC1_58120g3 and related proteins comprise a clade that arose within the XRG lineage. These proteins are distinct from the AmBV4 family (Fig. 1A and SI Appendix, Fig. S2B). As members of the XRG lineage, JSC1_58120g3 and other members of this lineage are also not closely related to the previously identified PCB-binding FRoGGR cluster that evolved within the greater green/red (GGR) lineage of CBCRs (52).

A newly identified lineage of far-red photosensors. (A) A partially collapsed phylogenetic tree is shown for the DPYLoar lineage described in this study. Parentheses next to collapsed branches indicate the number of CBCRs found on that branch, as well as a characterized CBCR from the branch, if available. Full tree available in SI Appendix, Fig. S2B. (BD) Dark-adapted (blue, 15Z) and photoproduct (orange, 15E) absorption spectra are shown for JSC1_58120g3 (B), Mic7113_1903g4 (C), and AFZ15460g4 (D) expressed with PCB biosynthetic enzymes. (EG) Spectra are shown as in BD for proteins expressed with PΦB biosynthetic enzymes.

We next expressed two additional proteins within this lineage in Escherichia coli cells engineered for PCB or PΦB synthesis. Both Mic7113_1903g4 and AFZ15460g4 yielded photoactive CBCRs with FR/R photocycles similar to those of JSC1_58120g3 (Fig. 1 BG and SI Appendix, Table S1). Dark-state spectra for each CBCR obtained from PΦB-producing cell lines were red-shifted by ∼10 nm relative to those obtained from PCB expression lines (Fig. 1: compare BD with EG). However, this bilin-dependent shift was considerably less for the photoproduct states. Taken together, these data indicate that this clade encompasses a previously uncharacterized lineage of FR/R sensors.

Members of This Lineage of frCBCRs Possess Atypical Chromophores.

To determine the molecular basis of spectral tuning in this family, we first measured the absorption spectra of the three FR/R CBCRs following denaturation in 6 M guanidinium-HCl (SI Appendix, Fig. S3A). The denatured far-red–absorbing dark state of JSC1_58120g3 was photochemically inactive, while the denatured red-absorbing photoproduct was significantly blue shifted and was still photoactive (SI Appendix, Fig. S3B). Based on the known behavior of bilins under denatured conditions (56, 57), these results establish 15Z and 15E configurations for the chromophore in the far-red–absorbing and red-absorbing states, respectively. Typically, chromophore-binding CBCRs expressed in PCB- or PΦB-producing cells retain PCB or PΦB adducts or carry out isomerization at the C5 methine bridge (31, 33). Absorption maxima of the three CBCRs in this lineage under denaturing conditions were red shifted relative to those of red/green CBCRs incorporating PCB or PΦB used as standards (NpR6012g4 and NpR5113g2: SI Appendix, Fig. S3 BF). Unexpectedly, denatured spectra for all three frCBCRs from PΦB-producing cells resembled spectra for BV-bound AM1_C0023g2 (47), while those from PCB-producing cells were intermediate between PCB- and BV-adduct standards, similar to a recently reported population with bound 18 1 ,18 2 –DHBV (46). Denaturation disrupts bilin-protein interactions responsible for spectral tuning of the bilin chromophore in the native protein context (30, 31, 58), so these results indicate that the bilin chromophores of this new frCBCR lineage differ from those of the PCB and PΦB adducts of similarly expressed CBCR standards (32, 59).

Members of This Lineage of frCBCRs Bind Verdins Rather than A-Ring–Reduced Phycobilins.

We were fortunate to obtain well-diffracting crystals of JSC1_58120g3 in the far-red–absorbing state isolated from cells engineered to produce either PCB (PDB ID 6XHH) or PΦB (PDB ID 6XHG). For the PCB-expressed protein, crystals adopted P1 symmetry with two monomers per asymmetric unit. The crystal structure was determined at 1.5-Å resolution by molecular replacement using a polyalanine model of the red-absorbing dark state of AnPixJg2 (PDB ID 3W2Z), which refined to R-factor/R-free of 19.4/23.1% (SI Appendix, Table S2). For the PΦB-expressed protein, crystals also adopted P1 symmetry with two monomers per asymmetric unit. This structure was determined at 2.3-Å resolution by molecular replacement using the PCB-expressed protein as search model and was refined to R-factor/R-free of 23.1/26.4% (SI Appendix, Table S2).

As expected, both JSC1_58120g3 structures adopted GAF folds similar to those of other XRG CBCRs in the 15Z state, such as AnPixJg2 (PDB ID 3W2Z) (60, 61), AnPixJg2_BV4 (5ZOH) (37), NpR6012g4 (6BHN) (62), and slr1393g3 (5DFX) (63). In all these structures, the bilin chromophore is sandwiched between the central β-sheet (strands β1–β6) and helices α3 and α4, with the “backside” helices α1, α2, and α5 bundled on the opposite side of the central β-sheet (SI Appendix, Fig. S4A). JSC1_58120g3 contains a 16-residue insertion between strand β4 and helix α4 that takes on a random coil structure (brick red arrow, SI Appendix, Fig. S4A) and packs against strand β4 and helix α5, but this loop feature is not conserved in the new frCBCR lineage. JSC1_58120g3 also lacks a small loop immediately preceding strand β3 (teal arrow, SI Appendix, Fig. S4A) that is present in other known XRG CBCR structures.

It also is notable that the chromophore moieties in both JSC1_58120g3 structures exhibit altered electron density relative to that of AnPixJg2 in a manner consistent with trigonal planar geometry at C2 and C3 of the A pyrrole ring and the presence of a 2,3–double bond (Fig. 2 A and B). This contrasts with a tetrahedral geometry in the PCB adduct of AnPixJg2 (Fig. 2 C and K). Electron density also shows that the chromophore is attached to Cys636 through a thioether linkage to the C3 1 carbon in both structures (Fig. 2 A, B, I, and J). This is in contrast with the C3 2 thioether attachment to BV seen in the AnPixJg2_BV4 structure (Fig. 2 D and L) (37) and in most structures of BphPs and CBCRs with BV chromophores (64 ⇓ ⇓ ⇓ ⇓ –69). Known exceptions include an engineered fluorescent variant of NpR3784 that exhibits C3 1 attachment of BV with a C3 1 -C3 2 double bond and a similar variant of Rhodopseudomonas palustris BphP1 that possesses thioether linkages to both C3 1 and C3 2 of BV (24, 38).

JSC1_58120g3 crystal structures. (AD) Chromophore A-ring models and electron densities for JSC1_58120g3 coexpressed with PcyA (A) or with HY2 (B), PCB adduct of AnPixJg2 (C), and BV adduct of AnPixJg2_BV4 (D). (EH) Chromophore D-ring models and electron densities are shown as in AD. (Insets in EH) C19-C18-C18 1 -C18 2 dihedral angles. (IL) Chemical representations of chromophore adducts as determined from AH. (M) Alignments depicting helix α4 and chromophore position shift. Protein mainchain is depicted in ribbon view with Pro591 and Thr292 sidechains shown as space-filling spheres. JSC1_58120g3-DHBV is brick red, AnPixJg2 teal, JSC1_58120g3-BV magenta, and AnPixJg2_BV4 charcoal. DPYLoar-conserved residues are colored by atom with carbon in salmon. XRG-conserved residues are colored by atom with carbon in light gray. (N) Orientation of BV chromophore D-ring with XRG-conserved β6 residues (Left) or DPYLoar-conserved β6 residues (Right). (O) Chromophore arrangement and protein contacts in AnPixJg2_BV4. Colored as in M, and AmBV4-conserved residues are colored by atom with carbon in violet. Water molecules depicted as red spheres. (P) Chromophore arrangement and equivalent protein contacts in JSC1_58120g3-DHBV.

The two JSC1_58120g3 structures exhibit differences in the geometry of their C18 side chains. For the PCB-expressed protein, the C19-C18-C18 1 -C18 2 dihedral angle is 76.1° (Fig. 2E), indicating the presence of an ethyl group at the C18 position like that of the AnPixJg2 chromophore (Fig. 2G). The C19-C18-C18 1 -C18 2 dihedral angle in the crystal obtained from the PΦB expression system measures just 28.4° (Fig. 2F), consistent with the presence of a vinyl substituent at the C18 position like that of the AnPixJg2_BV4 chromophore (Fig. 2H). Taken together with the A-ring geometry, these data support the conclusion that 18 1 ,18 2 –DHBV and BV were the precursors of the respective covalent bilin adducts in JSC1_58120g3 rather than PCB and PΦB (Fig. 2 IL). This result is surprising, because the more oxidized verdins 18 1 ,18 2 –DHBV and BV are expected to be present only in small amounts relative to the phycobilin products PCB and PΦB because 18 1 ,18 2 –DHBV is a transient intermediate in the conversion of BV to PCB by PcyA (50, 51) and because BV remains tightly bound to heme oxygenase until it is converted to PΦB by HY2 (SI Appendix, Fig. S1A). Thus, JSC1_58120g3 discriminates against PCB and PΦB, preferring their verdin biosynthetic precursors. The similarity between denatured spectra of JSC1_58120g3, Mic7113_1903g4, and AFZ15460g4 (SI Appendix, Fig. S3 E and F) demonstrates that this behavior is a conserved feature of this frCBCR lineage.

Conserved Sequence Elements Directly Interact with the Chromophore.

Conserved residues known to be important for chromophore positioning in XRG CBCRs, including W588, D590, and H637 (61), all maintain typical interactions with the chromophore in both JSC1_58120g3 structures (SI Appendix, Fig. S5 A and B). The conserved β6 tyrosine of XRG CBCRs is replaced in JSC1_58120g3 by Phe667. The tyrosine phenolic moiety normally interacts with the D-ring carbonyl (37, 61 ⇓ –63), but instead Ser665 of JSC1_58120g3 supplies a similar interaction from the other face of the D-ring (Fig. 2N). Ser665 and Phe667 are conserved in the JSC1_58120g3 cluster (SI Appendix, Fig. S2A). Helix α4 contains the canonical chromophore-binding residue Cys636. Both this helix and the covalently attached chromophore are substantially shifted in both JSC1_58120g3 structures relative to AnPixJg2, AnPixJg2_BV4 (Fig. 2M), and slr1393g3 (SI Appendix, Fig. S4 A and B). This shift causes the JSC1_58120g3 chromophores to sit at an angle in the binding pocket relative to the chromophores in AnPixJg2 and AnPixJg2_BV4, and to a lesser extent relative to that in slr1393g3. Relative to AnPixJg2, the chromophore in both JSC1_58120g3 structures is shifted such that the D-ring is closer to the central β-sheet, similar to the chromophores in AnPixJg2_BV4 and slr1393g3 (SI Appendix, Fig. S4B). Valine (JSC1_58120g3, AnPixJg2_BV4) or threonine (slr1393g3) residues on β5 appear to accommodate this shift toward the β-sheet (Fig. 2 O and P and SI Appendix, Fig. S5H), compared to a bulkier isoleucine in AnPixJg2 and many other XRG CBCRs. The overall shift in chromophore and helix α4 appears to correlate with another conserved residue in this cluster, Pro591. Structural superpositions indicate that Pro591 would clash with the chromophore A-rings were it present in AnPixJg2 and AnPixJg2_BV4 (Fig. 2M). Similarly, aligning the C3 2 -linked BV from AnPixJg2_BV4 into JSC1_58120g3 would result in a clash between Pro591 and the A-ring as well as clashes between β5 and β6 residues and the D-ring (SI Appendix, Fig. S4 CF).

Closer inspection of Pro591 reveals that it contributes to a reduced binding pocket width surrounding the A-ring. In JSC1_58120g3 structures, there is a 7.1- or 7.4-Å gap, compared with 8.6 Å in AnPixJg2 with PCB adduct or 7.9 Å in AnPixJg2_BV4 (Fig. 3 AD). We reasoned that such a restricted binding pocket could provide a structural basis for preventing phycobilin chromophore incorporation due to the bulkier A-rings of PCB and PΦB caused by tetrahedral geometry at C2 and C3 (Fig. 2K). To test this, we used site-directed mutagenesis to replace Pro591 with Thr, the equivalent residue in AnPixJg2. The P591T variant of JSC1_58120g3 readily incorporated chromophore when expressed with PcyA and exhibited a substantially blue-shifted R/O photocycle compared to wild type (WT) (Fig. 3E). Denatured difference spectra for this variant closely resembled PCB and were distinct from WT JSC1_58120g3 expressed with PcyA (Fig. 3F). Likewise, P591T JSC1_58120g3 expressed with HY2 exhibited a blue-shifted R/O photocycle relative to wild-type and denatured spectra resembled PΦB (Fig. 3 F and G). Both P591T preparations retained FR shoulders in their native spectra (Fig. 3 E and G), probably indicating residual verdin chromophore(s). In P591T expressed with HY2, the FR shoulder is substantial enough to extract separate PΦB and BV populations from the native difference spectra (Fig. 3H). Together, these results indicate that Pro591 is necessary for phycobilin exclusion in this lineage, but not for verdin affinity. Based on the Asp-Pro-Tyr-Leu consensus sequence of the motif containing this proline and the preference of these CBCRs for verdin chromophores with “oxidized A-rings (oar),” we designate this lineage “DPYLoar.”

Phycobilin exclusion by Pro591. (AD) View of the A-ring–binding pocket and nearby residues with opening width labeled for JSC1_58120g3-DHBV (A), JSC1_58120g3-BV (B), AnPixJg2 (C), and AnPixJg2_BV4 (D). Mainchain is depicted in ribbon view with Pro591/Thr292, chromophore, and primary cysteine represented as space-filling spheres. Coloring as in Fig. 2. (E) Dark-adapted 15Z (blue) and photoproduct 15E (orange) absorbance spectra are shown for P591T JSC1_58120g3 expressed with PcyA. Arrowhead denotes FR shoulder. (F) Stacked 15Z-15E difference spectra for denatured proteins compare P591T and WT JSC1_58120g3 to standards for PCB adduct (NpR6012g4) and PΦB adduct (NpR5113g2). (G) Spectra are shown for P591T JSC1_58120g3 expressed with HY2 as in E. (H) Native 15Z-15E difference spectra for P591T-PCB (violet) and estimated P591T-PΦB (bronze) and P591T-BV (pink) populations.

Distinct Mechanisms for BV Binding in AmBV4 and DPYLoar Families.

Four residues conserved within the AmBV4 lineage are sufficient to confer BV affinity to multiple XRG CBCRs (37). The full set of four residues (Tyr293, Thr308, Tyr318, Val336 in AnPixJg2_BV4, Fig. 2O) was determined to be essential for efficient incorporation of BV into a model red/green CBCR, yet DPYLoar CBCRs retain only two of these residues (Tyr592 and Val651 in JSC1_58120g3: Fig. 2P). The other residues are equivalent to Phe607 and Leu633. Introduction of Val336 or Val651 relative to canonical red/green CBCRs replaces larger Ile/Leu residues, apparently accommodating the shift of the chromophore toward the β-sheet in both lineages. However, the remaining three positions appear to perform divergent roles in DPYLoar and AmBV4 CBCRs. Tyr293 in AnPixJg2_BV4 forms only hydrophobic interactions with the chromophore B-ring, while Tyr592 in JSC1_58120g3 forms a water-mediated polar contact with the C-ring propionate.

The C-ring propionate in both JSC1_58120g3 structures maintains an orientation orthogonal to the C-ring plane, as in BV in AnPixJg2_BV4, but the propionate is positioned on the α-face rather than the β-face of the chromophore. The C-ring propionate positioning in JSC1_58120g3 is tethered by direct and water-mediated polar contacts to both sidechain and backbone of Arg600 and also by a water-mediated polar contact to the sidechain of Tyr592 (Fig. 2P) the resulting conformation removes a potential clash with Phe607 or interaction with Leu633, negating the apparent roles of Thr308 and Tyr318 in AnPixJg2_BV4 (Fig. 2O). Phe607 is shifted toward the chromophore relative to the position of Phe308 in AnPixJg2 and occupies some of the space vacated by the C-ring propionate (SI Appendix, Fig. S5 CF). This movement may be influenced by a water molecule that inserts between β5 and the loop preceding β4. The backbone of Ala653, conserved in DPYLoars and found on β5, coordinates this water and replaces a proline conserved in XRG CBCRs (SI Appendix, Fig. S5 CF). This alters the position of β4, pushing Phe607 closer to the chromophore. Two nearby Tyr residues on α3 and β5 that are broadly conserved in XRG CBCRs are replaced in DPYLoar CBCRs with Phe601 and Cys649, perhaps to alleviate clashes with repositioned Phe607. Notably, the arrangement of the chromophore and some surrounding residues in JSC1_58120g3 structures appears to be most similar to the red-absorbing 15Z state structure of slr1393g3 (SI Appendix, Figs. S4B and S5 G and H). Given that slr1393g3 is intermediate between AnPixJg2 and DPYLoars in the current phylogeny (SI Appendix, Fig. S2B), this suggests that some of the observed structural rearrangements relative to AnPixJg2 were acquired before the transition to phycobilin exclusion.

An additional set of conserved DPYLoar residues is distal to the chromophore, but they apparently form a lineage-specific interaction network. Arg654, Asp566, Tyr567, and Asp570 (on strands β1–2) and Glu589, Asn594, and Gln602 (preceding and within helix α3) form a network of polar contacts that link the two regions together in a manner not seen in other CBCR structures (SI Appendix, Fig. S6 A and B). These interactions pack around Phe568, another conserved DPYLoar residue that replaces smaller Ser/Thr residues commonly found in XRG CBCRs. This locks Phe568 in a strained backbone configuration and orients the sidechain as a prop against the phycobilin-excluding Pro591 residue. The conserved nature of many of these residues and their positioning in proximity to Pro591 implicate a potential supporting role in phycobilin exclusion or in chromophore incorporation generally.

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Research output : Contribution to journal › Review article › peer-review

T1 - Bacterial phytochromes, cyanobacteriochromes and allophycocyanins as a source of near-infrared fluorescent probes

AU - Verkhusha, Vladislav V.

N1 - Funding Information: This work was supported by grants R35 GM122567, U01 NS099573 and U01 NS103573 from the US National Institutes of Health ERC-2013-ADG-340233 from the EU FP7 program and 263371 and 266992 from the Academy of Finland. Publisher Copyright: © 2017 by the authors. Licensee MDPI, Basel, Switzerland.

N2 - Bacterial photoreceptors absorb light energy and transform it into intracellular signals that regulate metabolism. Bacterial phytochrome photoreceptors (BphPs), some cyanobacteriochromes (CBCRs) and allophycocyanins (APCs) possess the near-infrared (NIR) absorbance spectra that make them promising molecular templates to design NIR fluorescent proteins (FPs) and biosensors for studies in mammalian cells and whole animals. Here, we review structures, photochemical properties and molecular functions of several families of bacterial photoreceptors. We next analyze molecular evolution approaches to develop NIR FPs and biosensors. We then discuss phenotypes of current BphP-based NIR FPs and compare them with FPs derived from CBCRs and APCs. Lastly, we overview imaging applications of NIR FPs in live cells and in vivo. Our review provides guidelines for selection of existing NIR FPs, as well as engineering approaches to develop NIR FPs from the novel natural templates such as CBCRs.

AB - Bacterial photoreceptors absorb light energy and transform it into intracellular signals that regulate metabolism. Bacterial phytochrome photoreceptors (BphPs), some cyanobacteriochromes (CBCRs) and allophycocyanins (APCs) possess the near-infrared (NIR) absorbance spectra that make them promising molecular templates to design NIR fluorescent proteins (FPs) and biosensors for studies in mammalian cells and whole animals. Here, we review structures, photochemical properties and molecular functions of several families of bacterial photoreceptors. We next analyze molecular evolution approaches to develop NIR FPs and biosensors. We then discuss phenotypes of current BphP-based NIR FPs and compare them with FPs derived from CBCRs and APCs. Lastly, we overview imaging applications of NIR FPs in live cells and in vivo. Our review provides guidelines for selection of existing NIR FPs, as well as engineering approaches to develop NIR FPs from the novel natural templates such as CBCRs.