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.     

Photoreactions of aureochrome-1

Aureochrome is a recently discovered blue light photosensor that controls a light-dependent morphology change. As a photosensor, it has a unique DNA binding domain (bZIP). Although the biological functions of aureochrome have been revealed, the fundamental photochemistry of this protein has not been elucidated. The photochemical reaction dynamics of the LOV (light, oxygen, or voltage) domain of aureochrome-1 (AUREO1-LOV) and the LOV domain with the bZIP domain (AUREO1-ZL) were studied by employing the transient-grating (TG) technique, using size-exclusion chromatography to verify results. For both samples, adduct formation takes place with a time constant of 2.8 μs. Although significant diffusion changes were observed for both AUREO1-LOV and AUREO1-ZL after adduct formation, the origins of these changes were significantly different. The TG signal of AUREO1-LOV was strongly concentration-dependent. From analysis of the signal, it was concluded that AUREO1-LOV exists in equilibrium between the monomer and dimer, and dimerization of the monomer is the main reaction, i.e., irradiation with blue light enhances the strength of the interdomain interaction. On the other hand, the reaction of AUREO1-ZL is independent of concentration, suggesting that an intraprotein conformational change occurs in the bZIP domain with a time constant of 160 ms. These results revealed the different reactions and roles of the two domains; the LOV domain acts as a photosensor, leading to a subsequent conformational change in the bZIP domain, which should change its ability to bind to DNA. A model is proposed that demonstrates how aureochrome uses blue light to control its affinity for DNA.     

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.     

Charting the Signal Trajectory in a Light-Oxygen-Voltage Photoreceptor by Random Mutagenesis and Covariance Analysis

Modular signal receptors empower organisms to process environmental stimuli into adequate physiological responses. At the molecular level, a sensor module receives signals and processes the inherent information into changes of biological activity of an effector module. To better understand the molecular bases underpinning these processes, we analyzed signal reception and processing in the dimeric light-oxygen-voltage (LOV) blue light receptor YF1 that serves as a paradigm for the widespread Per-ARNT-Sim (PAS) signal receptors. Random mutagenesis identifies numerous YF1 variants in which biological activity is retained but where light regulation is abolished or inverted. One group of variants carries mutations within the LOV photosensor that disrupt proper coupling of the flavin-nucleotide chromophore to the protein scaffold. Another larger group bears mutations that cluster at the dyad interface and disrupt signal transmission to two coaxial coiled-coils that connect to the effector. Sequence covariation implies wide conservation of structural and mechanistic motifs, as also borne out by comparison to several PAS domains in which mutations leading to disruption of signal transduction consistently map to confined regions broadly equivalent to those identified in YF1. Not only do these data provide insight into general mechanisms of signal transduction, but also they establish concrete means for customized reprogramming of signal receptors.     

Signaling pathway of a photoactivable Rac1-GTPase in the early stages

In modern life‐ and medical‐sciences major efforts are currently concentrated on creating artificial photoenzymes, consisting of light‐ oxygen‐voltage‐sensitive (LOV) domains fused to a target enzyme. Such protein constructs possess great potential for controlling the cell metabolism as well as gene function upon light stimulus. This has recently been impressively demonstrated by designing a novel artificial fusion protein, connecting the AsLOV2‐Jα‐photosensor from Avena sativa with the Rac1‐GTPase (AsLOV2‐Jα‐Rac1), and by using it, to control the motility of cancer cells from the HeLa‐line. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their signaling pathway after photoexcitation is still in its infancy. Here, we show through computer simulations of the AsLOV2‐Jα‐Rac1‐photoenzyme that the early processes after formation of the Cys450‐FMN‐adduct involve the breakage of a H‐bond between the carbonyl oxygen FMN‐C4?O and the amino group of Gln513, followed by a rotational reorientation of its sidechain. This initial event is followed by successive events including β‐sheet tightening and transmission of torsional stress along the Iβ‐sheet, which leads to the disruption of the Jα‐helix from the N‐terminal end. Finally, this process triggers the detachment of the AsLOV2‐Jα‐photosensor from the Rac1‐GTPase, ultimately enabling the activation of Rac1 via binding of the effector protein PAK1. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Biological photoreceptors of light-dependent regulatory processes

Progress in understanding primary mechanisms of light reception in photoregulatory processes is achieved through discovering new biological photoreceptors, chiefly the regulatory sensors of blue/UV-A light. Among them are LOV domain-containing proteins and DNA photolyase-like cryptochromes, which constitute two widespread groups of photoreceptors that use flavin cofactors (FMN or FAD) as the photoactive chromophores. Bacterial LOV domain modules are connected in photoreceptor proteins with regulatory domains such as diguanylate cyclases/phosphodiesterases, histidine kinases, and DNA-binding domains that are activated by photoconversions of flavin. Identification of red/far-red light sensors in chemotrophic bacteria (bacteriophytochromes) and crystal structures of their photosensor module with bilin chromophore are significant for decoding the mechanisms of phytochrome receptor photoconversion and early step mechanisms of phytochrome-mediated signaling. The only UV-B regulatory photon sensor, UVR8, recently identified in plants, unlike other photoreceptors functions without a prosthetic chromophore: tryptophans of the unique UVR8 protein structure provide a “UV-B antenna”. Our analysis of new data on photosensory properties of the identified photoreceptors in conjunction with their structure opens insight on the influence of the molecular microenvironment on light-induced chromophore reactions, the mechanisms by which the photoactivated chromophores trigger conformational changes in the surrounding protein structure, and structural bases of propagation of these changes to the interacting effector domains/proteins.     

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）.     

Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of phototropin 1

The phototropins are a family of membrane-associated flavoproteins that function as the primary blue light receptors regulating phototropism, chloroplast movements, stomatal opening, and leaf expansion in plants. Phot1, a member of this family, contains two FMN-binding domains, LOV1 and LOV2, within the N-terminal region and a C-terminal serine-threonine protein kinase domain. Light irradiation of oat phot1 LOV2 produces a cysteinyl adduct (Cys-39) at the flavin C(4a) position, which decays thermally back to the dark state. We measured pH and isotope effects on the photocycle. Between pH 3.7 and 9.5, adduct formation showed minimal pH dependence, and adduct decay showed only slight pH dependence, indicating that the pK values of mechanistically relevant groups are outside this range. LOV2 showed a nearly 5-fold slowing of adduct formation in D(2)O relative to H(2)O, indicating that the rate-limiting step involves proton transfer(s). Light-induced changes in the far UV CD spectrum of LOV2 revealed putative protein structural perturbations. The light minus dark CD difference spectrum resembles an inverted alpha-helix spectrum, suggesting that alpha-helicity is reversibly lost upon light irradiation. Decay kinetics for CD spectral changes in the far UV region occur at the same rate as those in the visible region, indicating synchronous relaxation of protein and chromophore structures.     

A eukaryotic LOV-histidine kinase with circadian clock function in the picoalga Ostreococcus

The marine environment has unique properties of light transmission, with an attenuation of long wavelengths within the first meters of the water column. Marine organisms have therefore evolved specific blue‐light receptors such as aureochromes to absorb shorter‐wavelength light. Here, we identify and characterize a light, oxygen, or voltage sensing (LOV) containing histidine kinase (LOV‐HK) that functions as a new class of eukaryotic blue‐light receptor in the pico‐phytoplanktonic cell Ostreococcus tauri. This LOV‐HK is related to the large family of LOV‐HKs found in prokaryotes. Phylogenetic analysis indicates that the LOV domains from LOV‐HKs, including O. tauri LOV‐HK, and phototropins (phot; plant and green algal LOV serine/threonine kinases) have different evolutionary histories. Photochemical analysis shows that the LOV domain of LOV‐HK binds a flavin cofactor and absorbs blue light with a fast photocycle compared with its prokaryotic counterparts. Ostreococcus tauri LOV‐HK expression is induced by blue light and is under circadian control. Further, both overexpression and downregulation of LOV‐HK result in arrhythmia of the circadian reporter CCA1:Luc under constant blue light. In contrast, photochemical inactivation of O. tauri LOV‐HK is without effect, demonstrating its importance for function of the circadian clock under blue light. Overexpression/downregulation of O. tauriLOV‐HK alters CCA1 rhythmicity under constant red light, irrespective of LOV‐HK’s photochemical reactivity, suggesting that O. tauri LOV‐HK also participates in regulation of the circadian clock independent of its blue‐light‐sensing property. Molecular characterization of O. tauri LOV‐HK demonstrates that this type of photoreceptor family is not limited to prokaryotes.     

Exploring the multiscale signaling behavior of phototropin1 from Chlamydomonas reinhardtii using a full‐residue space kinetic Monte Carlo molecular dynamics technique

Devising analysis tools for elucidating the regulatory mechanism of complex enzymes has been a challenging task for many decades. It generally requires the determination of the structural‐dynamical information of protein solvent systems far from equilibrium over multiple length and time scales, which is still difficult both theoretically and experimentally. To cope with the problem, we introduce a full‐residue space multiscale simulation method based on a combination of the kinetic Monte Carlo and molecular dynamics techniques, in which the rates of the rate‐determining processes are evaluated from a biomolecular forcefield on the fly during the simulation run by taking into account the full space of residues. To demonstrate its reliability and efficiency, we explore the light‐induced functional behavior of the full‐length phototropin1 from Chlamydomonas reinhardtii (Cr‐phot1) and its various subdomains. Our results demonstrate that in the dark state the light oxygen voltage‐2‐Jα (LOV2‐Jα) photoswitch inhibits the enzymatic activity of the kinase, whereas the LOV1‐Jα photoswitch controls the dimerization with the LOV2 domain. This leads to the repulsion of the LOV1‐LOV2 linker out of the interface region between both LOV domains, which results in a positively charged surface suitable for cell–membrane interaction. By contrast, in the light state, we observe that the distance between both LOV domains is increased and the LOV1‐LOV2 linker forms a helix–turn–helix (HTH) motif, which enables gene control through nucleotide binding. Finally, we find that the kinase is activated through the disruption of the Jα‐helix from the LOV2 domain, which is followed by a stretching of the activation loop (A‐loop) and broadening of the catalytic cleft of the kinase. Proteins 2014; 82:2018–2040. © 2014 Wiley Periodicals, Inc.     

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.     

Light-induced Conformational Changes of LOV1 (Light Oxygen Voltage-sensing Domain 1) and LOV2 Relative to the Kinase Domain and Regulation of Kinase Activity in Chlamydomonas Phototropin

Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.     

Functional variations among LOV domains as revealed by FT-IR difference spectroscopy.

The two LOV domains, LOV1 and LOV2, from Chlamydomonas reinhardtii were investigated by light-induced FT-IR difference spectroscopy and compared to the LOV domain of Bacillus subtilis (YtvA-LOV). It is shown that the two S-H conformations of the reactive LOV1 cysteine C57(1) are exposed to environments of different hydrogen bonding strength. Thus, the two rotamer configurations of C57 might be related to the fact that the triplet state decays bi-exponentially into the LOV1-390 photoproduct. Exchange of the two other cysteines of LOV1 (C32S and C83S) does not alter the S-H stretching band providing evidence that this band feature arises solely from C57. The reactive cysteine of LOV2 from Chlamydomonas reinhardtii (C250) and of YtvA-LOV (C62) exhibit both a homogenous S-H stretching vibrational band which suggests a single conformer of the amino acid side chain. Finally, the FT-IR difference spectrum of YtvA from Bacillus subtilis comprising the light absorbing LOV domain and the putative signaling STAS (sulfate transporter/antisigma-factor antagonist) domain, reveals conformational changes in the latter after blue-light excitation.     

Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch

The phototropins are flavoprotein kinases that control phototropic bending, light-induced chloroplast movement, and stomatal opening in plants. Two flavin mononucleotide binding light, oxygen, or voltage (LOV) domains are the sites for initial photochemistry in these blue light photoreceptors. We have determined the steady state, photoexcited crystal structure of a flavin-bound LOV domain. The structure reveals a unique photochemical switch in the flavin binding pocket in which the absorption of light drives the formation of a reversible covalent bond between a highly conserved Cys residue and the flavin cofactor. This provides a molecular picture of a cysteinyl-flavin covalent adduct, the presumed signaling species that leads to phototropin kinase activation and subsequent signal transduction. We identify closely related LOV domains in two eubacterial proteins that suggests the light-induced conformational change evident in this structure is an ancient biomolecular response to light, arising before the appearance of plants.     

Disruption of the LOV-Jalpha helix interaction activates phototropin kinase activity

Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.     

TULIPs: tunable, light-controlled interacting protein tags for cell biology

Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.     

Phototropin Influence on Eyespot Development and Regulation of Phototactic Behavior in Chlamydomonas reinhardtii

The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity–dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.     

Structural basis of the LOV1 dimerization of Arabidopsis phototropins 1 and 2

Phototropin (phot) is a blue-light receptor protein that triggers phototropic responses, chloroplast relocation, and stomata opening to maximize the efficiency of photosynthesis in higher plants. Phot is composed of three functional domains. The N-terminal half folds into two light-oxygen-voltage-sensing domains called LOV1 and LOV2, each binding a flavin mononucleotide to absorb blue light. The C-terminal half is a serine/threonine kinase domain that causes light-dependent autophosphorylation leading to cellular signaling cascades. LOV2 domain is primarily responsible for activation of the kinase, and LOV1 domain is thought to act as a dimerization site and to regulate sensitivity to activation by blue light. Here we show the crystal structures of LOV1 domains of Arabidopsis phot1 and phot2 in the dark at resolutions of 2.1 Å and 2.0 Å, respectively. Either LOV1 domain forms a dimer through face-to-face association of β-scaffolds in the crystallographic asymmetric unit. Three types of interactions stabilizing the dimer structures found are as follows: contacts of side chains in their β-scaffolds, hydrophobic interactions of a short helix found in the N-terminus of a subunit with the β-scaffolds of both subunits, and hydrogen bonds mediated by hydration water molecules filling the dimer interface. The critical residues for dimerization are Cys261, forming a disulfide bridge between subunits in phot1-LOV1 domain, and Thr217 and Met232 in phot2-LOV1. The topology in homodimeric associations of the LOV1 domains is discussed when referring to those of homodimers or heterodimers of light-oxygen-voltage-sensing or Per-ARNT-Sim domains. The present results also provide clues to understanding structural basis in dimeric interactions of Per-ARNT-Sim protein modules in cellular signaling.     

The β-scaffold of the LOV domain of the Brucella light-activated histidine kinase is a key element for signal transduction

Light-oxygen-voltage (LOV) domains are blue-light-activated signaling modules present in a wide range of sensory proteins. Among them, the histidine kinases are the largest group in prokaryotes (LOV-HK). Light modulates the virulence of the pathogenic bacteria Brucella abortus through LOV-HK. One of the striking characteristic of Brucella LOV-HK is the fact that the protein remains activated upon light sensing, without recovering the basal state in the darkness. In contrast, the light state of the isolated LOV domain slowly returns to the dark state. To gain insight into the light activation mechanism, we have characterized by X-ray crystallography and solution NMR spectroscopy the structure of the LOV domain of LOV-HK in the dark state and explored its light-induced conformational changes. The LOV domain adopts the α/β PAS (PER-ARNT-SIM) domain fold and binds the FMN cofactor within a conserved pocket. The domain dimerizes through the hydrophobic β-scaffold in an antiparallel way. Our results point to the β-scaffold as a key element in the light activation, validating a conserved structural basis for light-to-signal propagation in LOV proteins.     

Crystal structures and molecular mechanism of a light-induced signaling switch: The Phot-LOV1 domain from Chlamydomonas reinhardtii

Phot proteins (phototropins and homologs) are blue-light photoreceptors that control mechanical processes like phototropism, chloroplast relocation, or guard-cell opening in plants. Phot receptors consist of two flavin mononucleotide (FMN)-binding light, oxygen, or voltage (LOV) domains and a C-terminal serine/threonine kinase domain. We determined crystal structures of the LOV1 domain of Phot1 from the green alga Chlamydomonas reinhardtii in the dark and illuminated state to 1.9 A and 2.8 A resolution, respectively. The structure resembles that of LOV2 from Adiantum (Crosson, S. and K. Moffat. 2001. PROC: Natl. Acad. Sci. USA. 98:2995-3000). In the resting dark state of LOV1, the reactive Cys-57 is present in two conformations. Blue-light absorption causes formation of a proposed active signaling state that is characterized by a covalent bond between the flavin C4a and the thiol of Cys-57. There are differences around the FMN chromophore but no large overall conformational changes. Quantum chemical calculations based on the crystal structures revealed the electronic distribution in the active site during the photocycle. The results suggest trajectories for electrons, protons, and the active site cysteine and offer an interpretation of the reaction mechanism.