Structure of a novel photoreceptor, the BLUF domain of AppA from Rhodobacter sphaeroides

The flavin-binding BLUF domain of AppA represents a new class of blue light photoreceptors that are present in a number of bacterial and algal species. The dark state X-ray structure of this domain was determined at 2.3 A resolution. The domain demonstrates a new function for the common ferredoxin-like fold; two long alpha-helices flank the flavin, which is bound with its isoalloxazine ring perpendicular to a five-stranded beta-sheet. The hydrogen bond network and the overall protein topology of the BLUF domain (but not its sequence) bear some resemblance to LOV domains, a subset of PAS domains widely involved in signaling. Nearly all residues conserved in BLUF domains surround the flavin chromophore, many of which are involved in an intricate hydrogen bond network. Photoactivation may induce a rearrangement in this network via reorientation of the Gln63 side chain to form a new hydrogen bond to the flavin O4 position. This shift would also break a hydrogen bond to the Trp104 side chain, which may be critical in induction of global structural change in AppA.     

Short LOV Proteins in Methylocystis Reveal Insight into LOV Domain Photocycle Mechanisms

Light Oxygen Voltage (LOV) proteins are widely used in optogenetic devices, however universal signal transduction pathways and photocycle mechanisms remain elusive. In particular, short-LOV (sLOV) proteins have been discovered in bacteria and fungi, containing only the photoresponsive LOV element without any obvious signal transduction domains. These sLOV proteins may be ideal models for LOV domain function due to their ease of study as full-length proteins. Unfortunately, characterization of such proteins remains limited to select systems. Herein, we identify a family of bacterial sLOV proteins present in Methylocystis. Sequence analysis of Methylocystis LOV proteins (McLOV) demonstrates conservation with sLOV proteins from fungal systems that employ competitive dimerization as a signaling mechanism. Cloning and characterization of McLOV proteins confirms functional dimer formation and reveal unexpected photocycle mechanisms. Specifically, some McLOV photocycles are insensitive to external bases such as imidazole, in contrast to previously characterized LOV proteins. Mutational analysis identifies a key residue that imparts insensitivity to imidazole in two McLOV homologs and affects adduct decay by two orders of magnitude. The resultant data identifies a new family of LOV proteins that indicate a universal photocycle mechanism may not be present in LOV proteins.     

The amino-terminal helix modulates light-activated conformational changes in AsLOV2

The mechanism of light-triggered conformational change and signaling in light-oxygen-voltage (LOV) domains remains elusive in spite of extensive investigation and their use in optogenetic studies. The LOV2 domain of Avenasativa phototropin 1 (AsLOV2), a member of the Per-Arnt-Sim (PAS) family, contains a flavin mononucleotide chromophore that forms a covalent bond with a cysteine upon illumination. This event leads to the release of the carboxy-terminal Jα helix, the biological output signal. Using mutational analysis, circular dichroism, and NMR, we find that the largely ignored amino-terminal helix is a control element in AsLOV2's light-activated conformational change. We further identify a direct amino-to-carboxy-terminal "input-output" signaling pathway. These findings provide a framework to rationalize the LOV domain architecture, as well as the signaling mechanisms in both isolated and tandem arrangements of PAS domains. This knowledge can be applied in engineering LOV-based photoswitches, opening up new design strategies and improving existing ones.     

Crucial role in light signal transduction for the conserved Met93 of the BLUF protein PixD/Slr1694

PixD/Slr1694 from the cyanobacterium Synechocystis sp. PCC6803 is a member of a new class of flavin-containing blue-light sensory proteins containing a BLUF (blue light using flavin) domain. The photocycle reaction mechanism of BLUF is unique because only small structural changes of a bound chromophore are accompanied by a few hydrogen bond rearrangements in the chromophore-binding site. Here, we show that in PixD, Met93, the residue conserved in all BLUF domains, is crucial for light-dependent signal transduction. Specifically, the light-insensitive M93A mutant of PixD revealed biochemical and physiological activities compatible with those of the light-adapted wild-type PixD. However, the W91A mutant of PixD retained light sensitivity and biological function, although the corresponding mutant of another BLUF protein, AppA, has been reported to be locked in the light signaling state. These observations suggest that the pathway through which the light signal is transformed into apoprotein structural changes has been modified in BLUF proteins for their respective functions.     

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered,light-switchable anti-CRISPR protein

Optogenetic control of CRISPR–Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications. Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs.     

Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa

The recent success of channelrhodopsin in optogenetics has also caused increasing interest in enzymes that are directly activated by light. We have identified in the genome of the bacterium Beggiatoa a DNA sequence encoding an adenylyl cyclase directly linked to a BLUF (blue light receptor using FAD) type light sensor domain. In Escherichia coli and Xenopus oocytes, this photoactivated adenylyl cyclase (bPAC) showed cyclase activity that is low in darkness but increased 300-fold in the light. This enzymatic activity decays thermally within 20 s in parallel with the red-shifted BLUF photointermediate. bPAC is well expressed in pyramidal neurons and, in combination with cyclic nucleotide gated channels, causes efficient light-induced depolarization. In the Drosophila central nervous system, bPAC mediates light-dependent cAMP increase and behavioral changes in freely moving animals. bPAC seems a perfect optogenetic tool for light modulation of cAMP in neuronal cells and tissues and for studying cAMP-dependent processes in live animals.     

FMN Binding and Photochemical Properties of Plant Putative Photoreceptors Containing Two LOV Domains,LOV/LOV Proteins

LOV domains function as blue light-sensing modules in various photoreceptors in plants, fungi, algae, and bacteria. A LOV/LOV protein (LLP) has been found from Arabidopsis thaliana (AtLLP) as a two LOV domain-containing protein. However, its function remains unknown. We isolated cDNA clones coding for an LLP homolog from tomato (Solanum lycopersicum) and two homologs from the moss Physcomitrella patens. The tomato LLP (SlLLP) contains two LOV domains (LOV1 and LOV2 domains), as in AtLLP. Most of the amino acids required for association with chromophore are conserved in both LOV domains, except that the amino acid at the position equivalent to the cysteine essential for cysteinyl adduct formation is glycine in the LOV1 domain as in AtLLP. When expressed in Escherichia coli, SlLLP binds FMN and undergoes a self-contained photocycle upon irradiation of blue light. Analyses using mutant SlLLPs revealed that SlLLP binds FMN in both LOV domains, although the LOV1 domain does not show spectral changes on irradiation. However, when Gly⁶⁶ in the LOV1 domain, which is located at the position equivalent to the essential cysteine of LOV domains, is replaced by cysteine, the mutated LOV1 domain shows light-induced spectral changes. In addition, all four LOV domains of P. patens LLPs (PpLLP1 and PpLLP2) show the typical features of LOV domains, including the reactive cysteine in each. This study shows that plants have a new LOV domain-containing protein family with the typical biochemical and photochemical properties of other LOV domain-containing proteins such as the phototropins.     

A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization

Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.     

Structural Refinement of a Key Tryptophan Residue in the BLUF Photoreceptor AppA by Ultraviolet Resonance Raman Spectroscopy

The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. The BLUF domain is of special interest because it uses a rigid flavin rather than an isomerizable chromophore, such as a rhodopsin or phytochrome, for its light-activation process. Crystal and solution structures of several BLUF domains were recently obtained, and their overall structures are consistent. However, there is a key ambiguity regarding the position of a conserved tryptophan (Trp-104 in AppA), in that this residue was found either close to flavin (Trp_in conformation) or exposed to the solvent (Trp_out conformation). The location of Trp-104 is a crucial factor in understanding the photocycle mechanism of BLUF domains, because this residue has been shown to play an essential role in the activation of AppA. In this study, we demonstrated a Trp_in conformation for the BLUF domain of AppA through direct observation of the vibrational spectrum of Trp-104 by ultraviolet resonance Raman spectroscopy, and also observed light-induced conformational and environmental changes in Trp-104. This study provides a structural basis for future investigations of the photocycle mechanism of BLUF proteins.     

13C Isotopologue editing of FMN bound to phototropin domains

The plant blue light receptor phototropin comprises a protein kinase domain and two FMN-binding LOV domains (LOV1 and LOV2). Blue light irradiation of recombinant LOV domains is conducive to the addition of a cysteinyl thiolate group to carbon 4a of the FMN chromophore, and spontaneous cleavage of that photoadduct completes the photocycle of the receptor. The present study is based on (13)C NMR signal modulation observed after reconstitution of LOV domains of different origins with random libraries of (13)C-labeled FMN isotopologues. Using this approach, all (13)C signals of FMN bound to LOV1 and LOV2 domains of Avena sativa and to the LOV2 domain of the fern, Adiantum capillus-veneris, could be unequivocally assigned under dark and under blue light irradiation conditions. (13)C Chemical shifts of FMN are shown to be differently modulated by complexation with the LOV domains under study, indicating slight differences in the binding interactions of FMN and the apoproteins.     

Spatio‐temporally precise activation of engineered receptor tyrosine kinases by light

Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low‐intensity blue light. We screened light‐oxygen‐voltage (LOV)‐sensing domains for their ability to activate RTKs by light‐activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto‐RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour.     

Irreversible photoreduction of flavin in a mutated Phot-LOV1 domain

Phot photoreceptors make up an important protein family regulating biological processes in response to blue light. They contain two light, oxygen, and voltage sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN). Upon absorption of blue light, the LOV domains undergo a photocycle, transiently forming a covalent adduct of a cysteine residue and the FMN (LOV-390). The mechanism of formation of this flavin-thiol adduct is still unclear. We studied a mutant of the LOV1 domain from the green alga Chlamydomonas reinhardtii with a methionine replacing the reactive cysteine 57 (C57M). As in the wild type, irradiation leads to formation of a photoadduct, which, however, is irreversibly converted into a red absorbing species, C57M-675. On the basis of spectroscopic results and the 2.1 A resolution crystal structure, this highly unusual FMN species was assigned to a neutral flavin radical covalently attached to the apoprotein at the N(5) position. In contrast to other flavoprotein neutral radicals, C57M-675 is stable even under aerobic or denaturing conditions. Pathways for the photoinduced formation of the adduct are discussed for the C57M mutant as well as the wild-type LOV1 domain.     

Reshaping the optical dimension in optogenetics

Optogenetics has been revolutionizing circuit neuroscience in the last few years. Optical methods combined with genetics and molecular techniques have provided new tools for stimulation of neurons, which hold great promise to provide a solution to the circuit mapping problem and more generally provide us with the ability to artificially control the natural stimulus space. Nevertheless, until very recently almost all applications of optogenetics have been based on relatively simple optical schemes mainly used for inducing population activity in neuronal assembles. In this context, alternative optical schemes that enhance the spatial or temporal resolution of excitation and allow for flexible and arbitrary generation of light patterns have all synergetic impact on the development of new optogenetic actuators. In the following we discuss and compare the main new optical techniques that have become available in the recent years. Their respective strengths and limitations as well as their application to different biological contexts are illustrated.     

The photochemistry of the light-, oxygen-, and voltage-sensitive domains in the algal blue light receptor phot

Phot proteins are blue light photoreceptors in plants and algae that mainly regulate photomovement responses. They contain two light-, oxygen-, and voltage-sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN) as chromophore. Upon blue light illumination, the LOV domains undergo a photocycle, transiently forming a covalent adduct of the FMN moiety with a nearby cysteine residue. The presence of two light-sensitive domains in the photoreceptor raises the question about the differences in properties and function between LOV1 and LOV2. As a model system, the photocycles of the LOV1 and LOV2 domains from phot of the green alga Chlamydomonas reinhardtii have been studied in detail, both separately and in a tandem construct. Here we give an overview about the results on the individual behavior of the domains and their interaction. Furthermore, the current status in the understanding of the role of LOV1 in phot in general is presented.     

The LOV domain family: photoresponsive signaling modules coupled to diverse output domains

For single-cell and multicellular systems to survive, they must accurately sense and respond to their cellular and extracellular environment. Light is a nearly ubiquitous environmental factor, and many species have evolved the capability to respond to this extracellular stimulus. Numerous photoreceptors underlie the activation of light-sensitive signal transduction cascades controlling these responses. Here, we review the properties of the light, oxygen, or voltage (LOV) family of blue-light photoreceptor domains, a subset of the Per-ARNT-Sim (PAS) superfamily. These flavin-binding domains, first identified in the higher-plant phototropins, are now shown to be present in plants, fungi, and bacteria. Notably, LOV domains are coupled to a wide array of other domains, including kinases, phosphodiesterases, F-box domains, STAS domains, and zinc fingers, which suggests that the absorption of blue light by LOV domains regulates the activity of these structurally and functionally diverse domains. LOV domains contain a conserved molecular volume extending from the flavin cofactor, which is the locus for light-driven structural change, to the molecular surface. We discuss the role of this conserved volume of structure in LOV-regulated processes.     

Ultrafast spectroscopy of biological photoreceptors

We review recent new insights on reaction dynamics of photoreceptors proteins gained from ultrafast spectroscopy. In Blue Light sensing Using FAD (BLUF) domains, a hydrogen-bond rearrangement around the flavin chromophore proceeds through a radical-pair mechanism, by which light-induced electron and proton transfer from the protein to flavin result in rotation of a conserved glutamine that switches the hydrogen bond network. Femtosecond infrared spectroscopy has shown that in photoactive yellow protein (PYP), breaking of a hydrogen bond that connects the p-coumaric acid chromophore to the backbone is crucial for trans-cis isomerization and successful entry into the photocycle. Furthermore, isomerization reactions of phycocyanobilin in phytochrome and retinal in the rhodopsins have been revealed in detail through application of femtosecond infrared and femtosecond-stimulated Raman spectroscopy.     

The optogenetic (r)evolution

Optogenetics is a rapidly evolving field of technology that allows optical control of genetically targeted biological systems at high temporal and spatial resolution. By heterologous expression of light-sensitive microbial membrane proteins, opsins, cell type-specific depolarization or silencing can be optically induced on a millisecond time scale. What started in a petri dish is applicable today to more complex systems, ranging from the dissection of brain circuitries in vitro to behavioral analyses in freely moving animals. Persistent technical improvement has focused on the identification of new opsins, suitable for optogenetic purposes and genetic engineering of existing ones. Optical stimulation can be combined with various readouts defined by the desired resolution of the experimental setup. Although recent developments in optogenetics have largely focused on neuroscience it has lately been extended to other targets, including stem cell research and regenerative medicine. Further development of optogenetic approaches will not only highly increase our insight into health and disease states but might also pave the way for a future use in therapeutic applications.