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.     

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.     

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.     

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.     

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.     

First evidence for phototropin-related blue-light receptors in prokaryotes

A prokaryotic protein, YtvA from Bacillus subtilis, was found to possess a light, oxygen, voltage (LOV) domain sharing high homology with the photoactive, flavin mononucleotide (FMN)-binding LOV domains of phototropins (phot), blue-light photoreceptors for phototropism in higher plants. Computer-based three-dimensional modeling suggests that YtvA-LOV binds FMN in a similar pocket as phot-LOVs. Recombinant YtvA indeed exhibits the same spectroscopical features and blue-light-induced photochemistry as phot-LOVs, with the reversible formation of a blue-shifted photoproduct, assigned to an FMN-cysteine thiol adduct (Thio383). By means of laser-flash photolysis and time-resolved optoacoustic experiments, we measured the quantum yield of formation for Thio383, Phi(Thio) = 0.49, and the enthalpy change, DeltaH(Thio) = 135 kJ/mol, with respect to the parent state. The formation of Thio383 is accompanied by a considerable volume contraction, DeltaV(Thio) = -13.5 ml/mol. Similar to phot-LOVs, Thio383 is formed from the decay of a red-shifted transient species, T650, within 2 micros. In both YtvA and free FMN, this transient has an enthalpy content of approximately 200 kJ/mol, and its formation is accompanied by a small contraction, DeltaV(T) approximately -1.5 ml/mol, supporting the assignment of T650 to the FMN triplet state, as suggested by spectroscopical evidences. These are the first studies indicating that phototropin-related, blue-light receptors may exist also in prokaryotes, besides constituting a steadily growing family in plants.     

Conformational analysis of the blue-light sensing protein YtvA reveals a competitive interface for LOV-LOV dimerization and interdomain interactions.

The Bacillus subtilis protein YtvA is related to plant phototropins in that it senses UVA-blue-light by means of the flavin binding LOV domain, linked to a nucleotide-binding STAS domain. The structural basis for interdomain interactions and functional regulation are not known. Here we report the conformational analysis of three YtvA constructs, by means of size exclusion chromatography, circular dichroism (CD) and molecular docking simulations. The isolated YtvA-LOV domain (YLOV, aa 25-126) has a strong tendency to dimerize, prevented in full-length YtvA, but still observed in YLOV carrying the N-terminal extension (N-YLOV, aa 1-126). The analysis of CD data shows that both the N-terminal cap and the linker region (aa 127-147) between the LOV and the STAS domain are helical and that the central beta-scaffold is distorted in the LOV domains dimers. The involvement of the central beta-scaffold in dimerization is supported by docking simulation of the YLOV dimer and the importance of this region is highlighted by light-induced conformational changes, emerging from the CD data analysis. In YtvA, the beta-strand fraction is notably less distorted and distinct light-driven changes in the loops/turn fraction are detected. The data uncover a common surface for LOV-LOV and intraprotein interaction, involving the central beta-scaffold, and offer hints to investigate the molecular basis of light-activation and regulation in LOV proteins.     

Enhancement of a sigma(B)-dependent stress response in Bacillus subtilis by light via YtvA photoreceptor

YtvA of Bacillus subtilis consists of light, oxygen or voltage (LOV) domain and sulfate transporter and anti-sigma antagonist (STAS) domain, and was reported to act as a photoreceptor, sensing light signals through the LOV domain, like a plant blue light receptor, phototropin. At the same time, YtvA was reported to act as a positive regulator for stress responsive-gene expression regulated by sigma(B) factor. Here we indicate that, like phototropins, the conserved Cys residue among the LOV domains is required for light-sensing in YtvA in vitro, possibly by the photoadduct formation, and YtvA forms a homodimer via its LOV domain, independently to light signal. We also indicate that, when ytvA expression is in normal level, light itself does not trigger sigma(B) activation, but a photo-enhancement of sigma(B) activity, activated by salt stress, occurs only in the presence of ytvA. The conserved Cys residue in the LOV domain and the STAS domain seem to be responsible for light-sensing and signal-transmission to the sigma(B) regulatory network, respectively.     

NTP-binding properties of the blue-light receptor YtvA and effects of the E105L mutation

YtvA is a blue-light-sensing protein from Bacillus subtilis related to plant phototropins. It carries a LOV (light, oxygen and voltage) domain, binding FMN (flavin mononucleotide) as chromophore, and a STAS (sulphate transporters and antisigma-factor antagonists) domain with poorly characterized function. We have recently shown that YtvA binds triphosphate nucleotides (NTP) and highlighted a structural similarity between the STAS domain and small GTP-binding proteins. In this work we further investigated the NTP-binding properties of YtvA, employing a fluorescent derivative of GTP (GTP_TR) and mutagenesis experiments. The main results are as follows: (a) competition experiments indicate that the affinity of YtvA for GTP is much higher than that for GDP and GMP. (b) Blue-light-induced structural changes are transmitted from the LOV core to the NTP-binding cavity, establishing a possible intraprotein signal-transduction pathway. (c) A mutation in the central β-scaffold of the LOV core, E105L, impairs the light-driven spectroscopic changes of bound GTP_TR. This result is supported by circular dichroism data, in that YtvA-E105L does not show the light-induced conformational change in the turn fraction that characterizes YtvA, implying that E105 is functionally important. (d) In the structural model of the LOV-STAS complex, based on docking algorithms, the interface includes the Iβ–Hβ loop on the LOV core, as well as parts of the central β-scaffold. E105 is predicted to interact with the LOV-STAS linker region, suggested to play a role in phototropin signaling. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

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.     

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.     

The Switch that Does Not Flip: The Blue-Light Receptor YtvA from Bacillus subtilis Adopts an Elongated Dimer Conformation Independent of the Activation State as Revealed by a Combined AUC and SAXS Study

Photoreceptors play an important role in plants and bacteria by converting extracellular stimuli into intracellular signals. One distinct class are the blue-light-sensitive phototropins harboring a light-oxygen-voltage (LOV) domain coupled to various effector domains. Photon absorption by the chromophore within the LOV domain results in an activation of the output domain via mechanisms that are hitherto not well understood. The photoreceptor YtvA from Bacillus subtilis is a bacterial analog of phototropins, consists of an LOV and a sulfate transporter/anti-sigma factor antagonist domain, and is involved in the response of the bacterium to environmental stress. We present here analytical ultracentrifugation studies and small-angle X-ray scattering experiments, showing that YtvA is a dimer. On the basis of these results, we present a low-resolution model of the dimer in the dark and the lit state of the protein. In addition, we show that YtvA does not change its oligomerization state or its overall shape upon light activation.     

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.     

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.     

Phot-LOV1: Photocycle of a Blue-Light Receptor Domain from the Green Alga Chlamydomonas reinhardtii

The “Phot” protein family comprises blue-light photoreceptors that consist of two flavin mononucleotide (FMN)-binding LOV (light, oxygen, and voltage) domains and a serine/threonine kinase domain. We have investigated the LOV1 domain of Phot1 from Chlamydomonas reinhardtii by time-resolved absorption spectroscopy. Photoexcitation of the dark form, LOV1-447, causes transient bleaching and formation of two spectrally similar red-shifted intermediates that are both assigned to triplet states of the FMN. The triplet states decay with time constants of 800 ns and 4 μs with an efficiency of >90% into a blue-shifted intermediate, LOV1-390, that is attributed to a thiol adduct of cysteine 57 to FMN C(4a). LOV1-390 reverts to the dark form in hundreds of seconds, the time constant being dependent on pH and salt concentration. In the mutant C57S, where the thiol adduct cannot be formed, the triplet state displays an oxygen-dependent decay directly to the dark form. We present here a spectroscopic characterization of an algal sensory photoreceptor in general and of a LOV1 domain photocycle in particular. The results are discussed with respect to the behavior of the homologous LOV2 domain from oat.     

Blue news: NTP binding properties of the blue-light sensitive YtvA protein from Bacillus subtilis

The blue-light sensitive protein YtvA from Bacillus subtilis is built of a photoactive, flavin-binding LOV (Light, Oxygen and Voltage) domain and a STAS domain with unknown function. Here we show that YtvA binds a fluorescent derivative of guanosine triphosphate (GTPTR) that can be displaced by both GTP or ATP. Unspecific NTP (N=G or A) binding is supported by the molecular model of YtvA-STAS. Blue-light activation of YtvA results in small and dark-reversible spectroscopic changes for GTPTR, suggesting that light-driven conformational changes are transmitted from the LOV core to the GTPTR binding site. These results support the idea that STAS domains may have a general NTP binding role and open a way to investigate the molecular functionality of YtvA-STAS.     

Phototropins and Their LOV Domains：Versatile Plant Blue-Light Receptors

The phototropins phot1 and phot2 are plant blue-light receptors that mediate phototropism, chloroplast movements, stomatal opening, leaf expansion, the rapid Inhibition of hypocotyl growth in etiolated seedlings, and possibly solar tracking by leaves in those species in which It occurs. The phototroplns are plasma membrane-associated hydrophilic proteins with two chromophore domains （designated LOV1 and LOV2 for their resemblance to domains In other signaling proteins that detect light, oxygen, or voltage） in their Nterminal half and a classic serine/threonlne kinase domain in their C-terminal half. Both chromophore domains bind flavin mononucleotide （FMN） and both undergo light-activated formation of a covalent bond between a nearby cystelne and the C（4a） carbon of the FMN to form the signaling state. LOV2-cystelnyl adduct formation leads to the release downstream of a tightly bound amphlpathlc α-helix, a step required for activation of the klnase function. This cysteinyl adduct then slowly decays over a matter of seconds or minutes to return the photoreceptor chromophore modules to their ground state. Functional LOV2 is required for light-activated phosphorylation and for various blue-light responses mediated by the phototroplns. The function of LOV1 is still unknown, although It may serve to modulate the signal generated by LOV2. The LOV domain Is an ancient chromophore module found In a wide range of otherwise unrelated proteins In fungi and prokaryotes, the latter Including cyanobacterla, eubacterla, and archaea. Further general reviews on the phototropins are those by Celaya and Liscum （2005） and Christie and Briggs （2005）.