Blue light activates the sigmaB-dependent stress response of Bacillus subtilis via YtvA

Here we present evidence for a physiologically relevant light response mediated by the LOV domain-containing protein YtvA in the soil bacterium Bacillus subtilis. The loss and overproduction of YtvA abolish and enhance, respectively, the increase in sigma(B)-controlled ctc promoter activity at moderate light intensities. These effects were absent in the dark and in red light but present under blue-light illumination. Thus, activation of the general stress response in B. subtilis is modulated by blue light.     

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.     

Differentiation of function among the RsbR paralogs in the general stress response of Bacillus subtilis with regard to light perception

The general stress response of Bacillus subtilis can be activated by a wide range of signals, including low intensities of visible light. It is regulated by a dedicated σ factor via a complex signal transduction pathway that makes use of stressosomes: hetero-oligomeric complexes that include one or more of the RsbR proteins (RsbRA, RsbRB, RsbRC, and RsbRD). The response to blue light is mediated by the photoreceptor YtvA. We show here which of the four RsbR proteins are necessary for the activation of the σ(B) response by blue light. Experiments performed with single-, double-, and triple-deletion strains in the rsbR genes show that RsbRB and RsbRA function antagonistically, with the former being a negative regulator and the latter a positive regulator of the YtvA-dependent light activation of the stress response. A strain with RsbRB as the only RsbR protein is unable to respond to light-activation of σ(B). Furthermore, RsbRC and RsbRD can replace RsbRA's function only in the absence of RsbRB. This differentiation of function is confined to light stress, since strains with RsbRA or RsbRB as the only RsbR protein behave similarly in our experimental conditions in response to physicochemical stresses. Interestingly, RsbRB's absence is sufficient to result in light activation of the general stress response at wild-type expression levels of ytvA, while it was previously reported that YtvA could only activate σ(B) when overproduced, or when cells are supplemented with an additional environmental stress.     

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.     

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.     

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.     

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.     

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.     

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.     

In Vivo Mutational Analysis of YtvA from Bacillus subtilis: MECHANISM OF LIGHT ACTIVATION OF THE GENERAL STRESS RESPONSE

The general stress response of Bacillus subtilis can be activated by stimuli such as the addition of salt or ethanol and with blue light. In the latter response, YtvA activates σ^B through a cascade of Rsb proteins, organized in stressosomes. YtvA is composed of an N-terminal LOV (light, oxygen, and voltage) domain and a C-terminal STAS (sulfate transporter and anti-sigma factor) domain and shows light-modulated GTP binding in vitro. Here, we examine the mechanism of YtvA-mediated activation of σ^B in vivo with site-directed mutagenesis. Constitutive off and constitutive on mutations have been identified. Disruption of GTP binding in the STAS domain eliminates light activation of σ^B. In contrast, modification of sites relevant for phosphorylation of STAS domains does not affect the stress response significantly. The data obtained are integrated into a model for the structure of full-length YtvA, which presumably functions as a dimer.LOV² domains (1), members of the superfamily of PAS domains (2, 3), are abundant in all domains of life and were first identified in plant phototropins (4). These photoreceptors regulate stomatal opening, phototropism, etc. and contain two N-terminal LOV domains that confer light regulation on the C-terminal Ser/Thr kinase domain (4). They also occur in bacteria, in which YtvA from Bacillus subtilis has been best characterized (for a review, see e.g. Ref. 5). Its N-terminal LOV domain binds FMN and shows the typical LOV photochemistry (6, 7): covalent adduct formation between a cysteine and the FMN chromophore. A linker helix, denoted Jα (7), connects the LOV domain to a STAS domain. The latter domain is present in many regulators of the general stress response of B. subtilis (8, 9). Stress via the addition of salt or ethanol (for a review, see Ref. 10) and blue light (11, 12) activates the general stress response via the environmental pathway, which integrates various signals via a large multiprotein complex, called the stressosome (13, 14). YtvA, which mediates light activation of σ^B (11, 12, 15), co-purifies with other STAS domain proteins in the stressosomes (16).When cells are stressed, STAS domains of several stressosome proteins (e.g. RsbS and RsbR) are phosphorylated by another intrinsic stressosome component, the serine/threonine kinase RsbT (9, 14, 17, 18). Next, RsbT is released from the complex to trigger RsbU, a protein phosphatase, thus (indirectly) activating σ^B (19). Phosphorylation of YtvA, however, has never been detected. Rather, it has been demonstrated in vitro that YtvA shows light-dependent GTP binding, presumably at its NTP-binding site in the STAS domain (20).Little is known about the mechanism of signal transmission in and by YtvA, except that in the C62A mutant, photochemistry in vitro (12) and light activation of σ^B in vivo (12, 15) are abolished. More detailed information is available for LOV domains of phototropins. A conserved glutamine, which is in hydrogen-bonding contact with the isoalloxazine ring of FMN, rotates its side chain by 180° upon covalent adduct formation (21). Replacement of this residue by leucine in the LOV2 domain of Phy3 from Adiantum results in a considerable reduction of the light-induced structural change (22). The corresponding mutation in phototropin 1 from Arabidopsis impairs autophosphorylation activity (23). The signal generated in the LOV2 domain is transmitted to the downstream kinase domain of phototropin 1 of Avena sativa through disruption of the interaction between its central β-sheet and the C-terminal linker region, the Jα-helix (24).Here, we study the mechanism of activation of YtvA in vivo, i.e. light-induced activation of the σ^B response, with site-directed mutagenesis. We focus on three regions of the protein, the flavin-binding pocket, the β-sheet of the LOV domain, and the GTP-binding site, and on potential phosphorylation sites of the STAS domain. We demonstrate that light-activated GTP binding is crucial for functional YtvA. A computational approach was used to model the structure of full-length YtvA. The model suggests that light modulates accessibility of the GTP-binding site of the STAS domain of YtvA.     

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.     

Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light-dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light-dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light-induced phosphorylation. These results demonstrate that blue light-dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation.     

Blue news update: BODIPY-GTP binds to the blue-light receptor YtvA while GTP does not

Light is an important environmental factor for almost all organisms. It is mainly used as an energy source but it is also a key factor for the regulation of multiple cellular functions. Light as the extracellular stimulus is thereby converted into an intracellular signal by photoreceptors that act as signal transducers. The blue-light receptor YtvA, a bacterial counterpart of plant phototropins, is involved in the stress response of Bacillus subtilis. The mechanism behind its activation, however, remains unknown. It was suggested based on fluorescence spectroscopic studies that YtvA function involves GTP binding and that this interaction is altered by absorption of light. We have investigated this interaction by several biophysical methods and show here using fluorescence spectroscopy, ITC titrations, and three NMR spectroscopic assays that while YtvA interacts with BODIPY-GTP as a fluorescent GTP analogue originally used for the detection of GTP binding, it does not bind GTP.     

Crystallization and initial X-ray diffraction analysis of the multi-domain Brucella blue light-activated histidine kinase LOV-HK in its illuminated state

The pathogenic bacterium Brucella abortus codes for a multi-domain dimeric cytoplasmic histidine kinase called LOV-HK, which is a key blue light-activated virulence factor in this microorganism. The structural basis of the light activation mechanism of this protein remains unclear. In this work, full-length LOV-HK was cloned, expressed and purified. The protein was activated by light and crystallized under a controlled illumination environment. The merge of 14 individual native data sets collected on a single crystal resulted in a complete X-ray diffraction data set to a resolution of 3.70 Å with over 2 million reflections. Crystals belong to space group P2₁2₁2₁, with unit-cell parameters a = 95.96, b = 105.30, c = 164.49 Å with a dimer in the asymmetric unit. Molecular replacement with Phaser using the individual domains as search models allowed for the reconstruction of almost the whole protein. Very recently, improved LOV-HK crystals led to a 3.25-Å resolution dataset. Refinement and model building is underway. This crystal model will represent one of the very few examples of a multi-domain histidine kinase with known structure.     

Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.     

The Mitosis and Neurodevelopment Proteins NDE1 and NDEL1 Form Dimers, Tetramers, and Polymers with a Folded Back Structure in Solution

Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8–167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.     

PAS domain receptor photoactive yellow protein is converted to a molten globule state upon activation

Biological signaling generally involves the activation of a receptor protein by an external stimulus followed by protein-protein interactions between the activated receptor and its downstream signal transducer. The current paradigm for the relay of signals along a signal transduction chain is that it occurs by highly specific interactions between fully folded proteins. However, recent results indicate that many regulatory proteins are intrinsically unstructured, providing a serious challenge to this paradigm and to the nature of structure-function relationships in signaling. Here we study the structural changes that occur upon activation of the blue light receptor photoactive yellow protein (PYP). Activation greatly reduces the tertiary structure of PYP but leaves the level secondary structure largely unperturbed. In addition, activated PYP exposes previously buried hydrophobic patches and allows significant solvent penetration into the core of the protein. These traits are the distinguishing hallmarks of molten globule states, which have been intensively studied for their role in protein folding. Our results show that receptor activation by light converts PYP to a molten globule and indicate stimulus-induced unfolding to a partially unstructured molten globule as a novel theme in signaling.