LOV Takes a Pick: Thermodynamic and Structural Aspects of the Flavin-LOV-Interaction of the Blue-Light Sensitive Photoreceptor YtvA from Bacillus subtilis

LOV domains act as versatile photochromic switches servicing multiple effector domains in a variety of blue light sensing photoreceptors abundant in a multitude of organisms from all kingdoms of life. The perception of light is realized by a flavin chromophore that upon illumination reversibly switches from the non-covalently bound dark-state to a covalently linked flavin-LOV adduct. It is usually assumed that most LOV domains preferably bind FMN, but heterologous expression frequently results in the incorporation of all natural occurring flavins, i.e. riboflavin, FMN and FAD. Over recent years, the structures, photochemical properties, activation mechanisms and physiological functions of a multitude of LOV proteins have been studied intensively, but little is known about its affinities to physiologically relevant flavins or the thermodynamics of the flavin-LOV interaction. We have investigated the interaction of the LOV domain of the well characterized bacterial photoreceptor YtvA with riboflavin, FMN and FAD by ITC experiments providing binding constants and thermodynamic profiles of these interactions. For this purpose, we have developed a protocol for the production of the apo forms of YtvA and its isolated LOV domain and we demonstrate that the latter can be used as a molecular probe for free flavins in cell lysates. Furthermore, we show here using NMR spectroscopic techniques and Analytical Ultracentrifugation that the flavin moiety stabilizes the conformation of the LOV domain and that dimerization of YtvA is caused not only by intermolecular LOV-LOV but also by STAS-STAS contacts.     

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.     

Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin

The plant photoreceptor phototropin is an autophosphorylating serine-threonine protein kinase activated by UV-A/blue light. Two domains, LOV1 and LOV2, members of the PAS domain superfamily, mediate light sensing by phototropin. Heterologous expression studies have shown that both domains function as FMN-binding sites. Although three plant blue light photoreceptors, cry1, cry2, and phototropin, have been identified to date, the photochemical reactions underlying photoactivation of these light sensors have not been described so far. Herein, we demonstrate that the LOV domains of Avena sativa phototropin undergo a self-contained photocycle characterized by a loss of blue light absorbance in response to light and a spontaneous recovery of the blue light-absorbing form in the dark. Rate constants and quantum efficiencies for the photoreactions indicate that LOV1 exhibits a lower photosensitivity than LOV2. The spectral properties of the photoproduct produced for both LOV domains are unrelated to those found for photoreduced flavins and flavoproteins, but are consistent with those of a flavin-cysteinyl adduct. Flavin-thiol adducts are generally short-lifetime reaction intermediates formed during the flavoprotein-catalyzed reduction of protein disulfides. By site-directed mutagenesis, we have identified several amino acid residues within the putative chromophore binding site of LOV1 and LOV2 that appear to be important for FMN binding and/or the photochemical reactivity. Among those is Cys39, which plays an important role in the photochemical reaction of the LOV domains. Replacement of Cys39 with Ala abolished the photochemical reactions of both LOV domains. We therefore propose that light sensing by the phototropin LOV domains occurs via the formation of a stable adduct between the FMN chromophore and Cys39.     

A LOV story: the signaling state of the phot1 LOV2 photocycle involves chromophore-triggered protein structure relaxation, as probed by far-UV time-resolved optical rotatory dispersion spectroscopy

Light-, oxygen-, or voltage-regulated (LOV1 and LOV2) domains bind flavin mononucleotide (FMN) and activate the phototropism photoreceptors phototropin 1 (phot1) and phototropin 2 (phot2) by using energy from absorbed blue light. Upon absorption of blue light, chromophore and protein conformational changes trigger the kinase domain for subsequent autophosphorylation and presumed downstream signal transduction. To date, the light-induced photocycle of the phot1 LOV2 protein is known to involve formation of a triplet flavin mononucleotide (FMN) chromophore followed by the appearance of a FMN adduct within 4 micros [Swartz, T. E., Corchnoy, S. B., Christie, J. M., Lewis, J. W., Szundi, I., Briggs, W. R., and Bogomolni, R. A. (2001) J. Biol. Chem. 276, 36493-36500] before thermal decay back to the dark state. To probe the mechanism by which the blue light information is relayed from the chromophore to the protein, nanosecond time-resolved optical rotatory dispersion (TRORD) spectroscopy, which is a direct probe of global secondary structure, was used to study the phot1 LOV2 protein in the far-UV region. These TRORD experiments reveal a previously unobserved intermediate species (tau approximately 90 micros) that is characterized by a FMN adduct chromophore and partially unfolded secondary structure (LOV390(S2)). This intermediate appears shortly after the formation of the FMN adduct. For LOV2, formation of a long-lived species that is ready to interact with a receptor domain for downstream signaling is much faster by comparison with formation of a similar species in other light-sensing proteins.     

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.     

Variations in protein-flavin hydrogen bonding in a light, oxygen, voltage domain produce non-Arrhenius kinetics of adduct decay

Light, oxygen, voltage (LOV) domains utilize a conserved blue light-dependent mechanism to control a diverse array of effector domains in biological and engineered proteins. Variations in the kinetics and efficiency of LOV photochemistry fine-tune various aspects of the photic response. Characterization of the kinetics of a key aspect of this photochemical mechanism in EL222, a blue light responsive DNA binding protein from Erythrobacter litoralis HTCC2594, reveals unique non-Arrhenius behavior in the rate of dark-state cleavage of the photochemically generated adduct. Sequence analysis and mutagenesis studies establish that this effect stems from a Gln to Ala mutation unique to EL222 and homologous proteins from marine bacteria. Kinetic and spectroscopic analyses reveal that hydrogen bonding interactions between the FMN N1, O2, and ribityl hydroxyls and the surrounding protein regulate photocycle kinetics and stabilize the LOV active site from temperature-induced alteration in local structure. Substitution of residues interacting with the N1-O2 locus modulates adduct stability, structural flexibility, and sequestration of the active site from bulk solvent without perturbation of light-activated DNA binding. Together, these variants link non-Arrhenius behavior to specific alteration of an H-bonding network, while affording tunability of photocycle kinetics.     

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.     

Comparative investigation of the LOV1 and LOV2 domains in Adiantum phytochrome3

Phototropin (phot) is a blue-light photoreceptor for phototropic responses, relocation of chloroplasts, and stomata opening in plants. Phototropin has two chromophore-binding domains named LOV1 and LOV2 in its N-terminal half, each of which binds a flavin mononucleotide (FMN) noncovalently. The C-terminal half is a Ser/Thr kinase. A transgenic study of Arabidopsis suggested that only LOV2 domain is necessary for the kinase activity, whereas X-ray crystallographic structures of LOV1 and LOV2 domains are almost identical. These facts imply that the detailed structures and/or structural changes are different between LOV1 and LOV2 domains. In this study, we compared light-induced structural changes of the LOV1 and LOV2 domains of a phototropin, Adiantum phytochrome3 (phy3), by means of UV-visible and Fourier transform infrared (FTIR) spectroscopy. Photochemical properties of an adduct formation between FMN and a cysteine are essentially similar between phy3-LOV1 and phy3-LOV2. On the other hand, the S-H group of the reactive cysteine forms a hydrogen bond in phy3-LOV1, which is strengthened at low temperatures. This is possibly correlated with the fact that no adduct formation takes place for phy3-LOV1 at 77 K as revealed by the UV-visible absorption spectra. The most prominent difference was seen in the amide-I vibration that monitors the secondary structure of peptide backbone. Protein structural changes in phy3-LOV2 involve the regions of loops, alpha-helices, and beta-sheets, which differ significantly among various temperatures. Extended protein structural changes are probably correlated with the signal transduction activity of LOV2. In contrast, protein structural changes were very small in phy3-LOV1, and they were almost temperature independent. The photocycle of phy3-LOV1 takes 3.1 h, being more than 100 times longer than that of phy3-LOV2. These facts suggest that Adiantum phy3-LOV1 does not work for light sensing, being consistent with the previous transgenic study of Arabidopsis. It is likely that plants utilize a unique protein architecture (LOV domain) for different functions by regulating their protein structural changes.     

Signals of LOV1: a computer simulation study on the wildtype LOV1-domain of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and its mutants

Phototropins are photoreceptors regulating the blue-light response in plants and bacteria. They consist of two LOV (light oxygen voltage sensitive) domains each containing a non-covalently bound flavin-mononucleotide (FMN) chromophore, which are connected to a serine/threonine-kinase. Upon illumination, the LOV-domains undergo conformational changes, triggering a signal cascade in the organism through kinase activation. Here, we present results from molecular dynamics simulations in which we investigate the signal transduction pathway of the wildtype LOV1-domain of Chlamydomonas reinhardtii and a methyl-mercaptan (MM) adduct of its Cys57Gly-mutant at the molecular level. In particular, we analyzed the effect of covalent-bond formation between the reactive cysteine Cys57 and the FMN-reaction center, as well as the subsequent charge redistribution, on the spatio-dynamical behavior of the LOV1-domain. We compare the calculation results with experimental data and demonstrate that these adduct state characteristics have an important influence on the response of this photosensor. The light-induced changes implicate primarily an alteration of the surface charge distribution through rearrangement of the highly flexible Cα-, Dα- and Eα-helices including the Glu51-Lys91-salt bridge on the hydrophilic side of the protein domain and a β-sheet tightening process via coupling of the Aβ- and Bβ-strands. Our findings confirm the aptitude of the LOV1-domain to function as a dimerization partner, allowing the green alga to adapt its reproduction and growth speed to the environmental conditions.     

Chromophore exchange in the LOV2 domain of the plant photoreceptor phototropin1 from oat

Phototropins are a family of plant photoreceptors mediating blue light responses such as phototropism, leaf expansion, chloroplast relocation, and stomatal opening. Characteristic for phototropins are two LOV domains which, when expressed in heterologous systems, each carry a single flavin mononucleotide (FMN) chromophore. Here we describe removal of FMN from the LOV2 domain of Avena sativa using a hydrophobic matrix and successful incorporation of flavin adenine dinucleotide (FAD), riboflavin, and 5'-malonyl-riboflavin into the resulting apoprotein; 5-deaza-FMN was not incorporated under the applied conditions. The chromoproteins reconstituted with the various flavins showed absorption spectra and photocycle almost identical to those of the native LOV2 domain and that reconstituted with FMN except for the kinetics: LOV2-riboflavin and LOV2-5'-malonyl-riboflavin showed more rapid regeneration in the dark. LOV2-FAD can be hydrolyzed to LOV2-FMN with phosphodiesterase, indicating that the adenosine part extrudes from the protein. Together with the data from the X-ray structure (Crosson, S., and Moffat, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2995-3000), the results allow us to decide which of the chromophore-protein interactions are essential for the reconstitution process.     

Structural basis for light-dependent signaling in the dimeric LOV domain of the photosensor YtvA

The photosensor YtvA binds flavin mononucleotide and regulates the general stress reaction in Bacillus subtilis in response to blue light illumination. It belongs to the family of light-oxygen-voltage (LOV) proteins that were first described in plant phototropins and form a subgroup of the Per-Arnt-Sim (PAS) superfamily. Here, we report the three-dimensional structure of the LOV domain of YtvA in its dark and light states. The protein assumes the global fold common to all PAS domains and dimerizes via a hydrophobic interface. Directly C-terminal to the core of the LOV domain, an alpha-helix extends into the solvent. Light absorption causes formation of a covalent bond between a conserved cysteine residue and atom C(4a) of the FMN ring, which triggers rearrangements throughout the LOV domain. Concomitantly, in the dark and light structures, the two subunits of the dimeric protein rotate relative to each other by 5 degrees . This small quaternary structural change is presumably a component of the mechanism by which the activity of YtvA is regulated in response to light. In terms of both structure and signaling mechanism, YtvA differs from plant phototropins and more closely resembles prokaryotic heme-binding PAS domains.     

A critical element of the light‐induced quaternary structural changes in YtvA‐LOV

YtvA, a photosensory LOV (light‐oxygen‐voltage) protein from Bacillus subtilis, exists as a dimer that previously appeared to undergo surprisingly small structural changes after light illumination compared with other light‐sensing proteins. However, we now report that light induces significant structural perturbations in a series of YtvA‐LOV domain derivatives in which the Jα helix has been truncated or replaced. Results from native gel analysis showed significant mobility changes in these derivatives after light illumination; YtvA‐LOV without the Jα helix dimerized in the dark state but existed as a monomer in the light state. The absence of the Jα helix also affected the dark regeneration kinetics and the stability of the flavin mononucleotide (FMN) binding to its binding site. Our results demonstrate an alternative way of photo‐induced signal propagation that leads to a bigger functional response through dimer/monomer conversions of the YtvA‐LOV than the local disruption of Jα helix in the As‐LOV domain.     

Structural basis for the slow dark recovery of a full-length LOV protein from Pseudomonas putida

Blue-light photoreceptors containing light–oxygen–voltage (LOV) domains regulate a myriad of different physiological responses in both eukaryotes and prokaryotes. Their light sensitivity is intricately linked to the photochemistry of the non-covalently bound flavin mononucleotide (FMN) chromophore that forms a covalent adduct with a conserved cysteine residue in the LOV domain upon illumination with blue light. All LOV domains undergo the same primary photochemistry leading to adduct formation; however, considerable variation is found in the lifetime of the adduct state that varies from seconds to several hours. The molecular mechanism underlying this variation among the structurally conserved LOV protein family is not well understood. Here, we describe the structural characterization of PpSB1-LOV, a very slow cycling full-length LOV protein from the Gram-negative bacterium Pseudomonas putida KT2440. Its crystal structure reveals a novel dimer interface that is mediated by N- and C-terminal auxiliary structural elements and a unique cluster of four arginine residues coordinating with the FMN-phosphate moiety. Site-directed mutagenesis of two arginines (R61 and R66) in PpSB1-LOV resulted in acceleration of the dark recovery reaction approximately by a factor of 280. The presented structural and biochemical data suggest a direct link between structural features and the slow dark recovery observed for PpSB1-LOV. The overall structural arrangement of PpSB1-LOV, together with a complementary phylogenetic analysis, highlights a common ancestry of bacterial LOV photoreceptors and Per-ARNT-Sim chemosensors.     

Blue light perception in plants. Detection and characterization of a light-induced neutral flavin radical in a C450A mutant of phototropin

The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420-480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.     

Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains, designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis and is necessary to activate phot1 kinase activity through light-induced structural changes within a conserved alpha-helix situated C-terminal to LOV2. Here we have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln(575)) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN binding. These findings, in combination with double mutant analyses, indicate that Gln(575) plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain.     

Role of Phe1010 in light-induced structural changes of the neo1-LOV2 domain of Adiantum

Phototropin (phot) is a blue-light sensor protein that elicits several photo responses in plants. Phototropin has two flavin mononucleotide (FMN)-binding domains, LOV1 and LOV2, in its N-terminal half. The C-terminal half is a blue-light-regulated Ser/Thr kinase. Various functional studies have reported that only LOV2 is responsible for the kinase activity, whereas the X-ray crystallographic structures of the LOV1 and LOV2 domains are almost identical. How does such a functional difference emerge? Our previous FTIR study of the LOV domains of Adiantum neochrome1 (neo1) showed that light-induced protein structural changes are small and temperature independent for neo1-LOV1, whereas the structural changes are large and highly temperature dependent for neo1-LOV2, which involve loops, alpha-helices, and beta-sheets. These observations successfully explained the different functions in terms of protein structural changes. They also suggested the presence of some crucial amino acids responsible for greater protein structural changes in the LOV2 domain. Here, we focused on phenylalanine-1010 (Phe1010) in neo1-LOV2, where FMN is sandwiched between Phe1010 and the reactive cysteine. Phenylalanine at this position is conserved for LOV2 domains, while the corresponding amino acid is leucine for LOV1 domains in almost all plant phototropins. We observed that unlike wild-type LOV2, the FTIR spectra of F1010L LOV2 exhibited no temperature dependence in the alpha-helical and beta-sheet regions and that spectral changes in amide-I of these regions were significantly reduced, which was similar to LOV1. Thus, the replacement of phenylalanine with leucine converts neo1-LOV2 into neo1-LOV1 in terms of protein structural changes that must be related to the different functions. We will discuss the roles of phenylalanine and leucine in the LOV2 and LOV1 domains, respectively.     

Light Signal Transduction Pathway from Flavin Chromophore to the Jα Helix of Arabidopsis Phototropin1

In the plant blue-light sensor phototropin, illumination of the chromophoric LOV domains causes activation of the serine/threonine kinase domain. Flavin mononucleotide (FMN) is a chromophore molecule in the two LOV domains (LOV1 and LOV2), but only LOV2 is responsible for kinase activation. Previous studies reported an important role of an additional helix connected to the C-terminal of LOV2 (Jα helix) for the function of phototropin; however, it remains unclear how the Jα helix affects light-induced structural changes in LOV2. In this study we compared light-induced protein structural changes of the LOV2 domain of Arabidopsis phot1 in the absence (LOV2-core) and presence (LOV2-Jα) of the Jα helix by Fourier-transform infrared spectroscopy. Prominent peaks were observed only in the amide-I region (1650 (−)/1625 (+) cm⁻¹) of LOV2-Jα at physiological temperatures (≥260 K), corresponding to structural perturbation of the α-helix. The peaks were diminished by point mutation of functionally important amino acids such as Phe-556 between FMN and the β-sheet, Gln-575 being hydrogen-bonded with FMN, and Ile-608 on the Jα helix. We thus conclude that a light signal is relayed from FMN through these amino acids and eventually changes the interaction between LOV2-core and the Jα helix in Arabidopsis phot1.     

Structural tuning of the fluorescent protein iLOV for improved photostability

Fluorescent proteins derived from light, oxygen, or voltage (LOV) domains offer advantages over green fluorescent protein (GFP) from their small size and efficacy under anaerobic conditions. The flavoprotein improved LOV (iLOV) was engineered from the blue light receptor phototropin as a reporter of viral infection. To inform the molecular basis for the improved, photoreversible, fluorescent properties of iLOV, we employed directed evolution and determined five LOV crystallographic structures. Comparative structural analyses between iLOV and its progenitors reveal mutation-induced constraints in the environment of the flavin mononucleotide (FMN) chromophore; in iLOV, the methyl group of Thr-394 "crowds" the FMN isoalloxazine ring, Leu-470 triggers side chain "flipping" of Leu-472, and the terminal FMN phosphate shows increased anchoring. We further engineered iLOV variants that are readily detectable in bacterial and mammalian cells due to order-of-magnitude photostability increases. Structure determination of a resulting representative photostable iLOV (phiLOV) variant reveals additional constraints on the chromophore. Aromatic residues Tyr-401 and Phe-485 in phiLOV sandwich the FMN isoalloxazine ring from both sides, whereas Ser-390 anchors the side chain of FMN-interacting Gln-489 Our combined structural and mutational results reveal that constraining the FMN fluorophore yields improved photochemical properties for iLOV and its new photostable derivative. These findings provide a framework for structural fine-tuning of LOV scaffold proteins to maximize their potential as oxygen-independent fluorescent reporters.     

Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis,rice, and Chlamydomonas reinhardtii

Phototropins (phot1 and phot2, formerly designated nph1 and npl1) are blue-light receptors that mediate phototropism, blue light-induced chloroplast relocation, and blue light-induced stomatal opening in Arabidopsis. Phototropins contain two light, oxygen, or voltage (LOV) domains at their N termini (LOV1 and LOV2), each a binding site for the chromophore flavin mononucleotide (FMN). Their C termini contain a serine/threonine protein kinase domain. Here, we examine the kinetic properties of the LOV domains of Arabidopsis phot1 and phot2, rice (Oryza sativa) phot1 and phot2, and Chlamydomonas reinhardtii phot. When expressed in Escherichia coli, purified LOV domains from all phototropins examined bind FMN tightly and undergo a self-contained photocycle, characterized by fluorescence and absorption changes induced by blue light (T. Sakai, T. Kagawa, M. Kasahara, T.E. Swartz, J.M. Christie, W.R. Briggs, M. Wada, K. Okada [2001] Proc Natl Acad Sci USA 98: 6969-6974; M. Salomon, J.M. Christie, E. Knieb, U. Lempert, W.R. Briggs [2000] Biochemistry 39: 9401-9410). The photocycle involves the light-induced formation of a cysteinyl adduct to the C(4a) carbon of the FMN chromophore, which subsequently breaks down in darkness. In each case, the relative quantum efficiencies for the photoreaction and the rate constants for dark recovery of LOV1, LOV2, and peptides containing both LOV domains are presented. Moreover, the data obtained from full-length Arabidopsis phot1 and phot2 expressed in insect cells closely resemble those obtained for the tandem LOV-domain fusion proteins expressed in E. coli. For both Arabidopsis and rice phototropins, the LOV domains of phot1 differ from those of phot2 in their reaction kinetic properties and relative quantum efficiencies. Thus, in addition to differing in amino acid sequence, the phototropins can be distinguished on the basis of the photochemical cycles of their LOV domains. The LOV domains of C. reinhardtii phot also undergo light-activated spectral changes consistent with cysteinyl adduct formation. Thus, the phototropin family extends over a wide evolutionary range from unicellular algae to higher plants.     

Domain Swapping to Assess the Mechanistic Basis of Arabidopsis Phototropin 1 Receptor Kinase Activation and Endocytosis by Blue Light

Phototropins (phot1 and phot2) are plasma membrane–associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.