Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein. Proc Natl Acad Sci USA

Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8816, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2011; 108(23):9449-54. DOI: 10.1073/pnas.1100262108
Source: PubMed


Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.

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Available from: Kevin H Gardner
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    • "Those in turn influence a multitude of different cellular light responses in plants (Möglich et al., 2010), bacteria (Herrou and Crosson, 2011), and fungi (Idnurm et al., 2010). In recent years, it became apparent, that adduct formation leads to small-scale structural changes in the vicinity of the FMN chromophore, which are in many cases relayed to the fused effector domains via helical interdomain linkers (termed N-terminal cap or A'α-helix and C-terminal Jα-helix) (Harper et al., 2003, 2004; Halavaty and Moffat, 2007, 2013; Nash et al., 2011; Diensthuber et al., 2013; Herman et al., 2013; Endres et al., 2015; Herman and Kottke, 2015). There is growing experimental evidence that those structural changes in turn result in altered LOV photoreceptor biological activities (Harper et al., 2004; Vaidya et al., 2011; Aihara et al., 2012; Okajima et al., 2014; Kashojiya et al., 2015). "
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    ABSTRACT: The modularity of light, oxygen, voltage (LOV) blue-light photoreceptors has recently been exploited for the design of LOV-based optogenetic tools, which allow the light-dependent control of biological functions. For the understanding of LOV sensory function and hence the optimal design of LOV-based optogentic tools it is essential to gain an in depth atomic-level understanding of the underlying photoactivation and intramolecular signal-relay mechanisms. To address this question we performed molecular dynamics simulations on both the dark- and light-adapted state of PpSB1-LOV, a short dimeric bacterial LOV-photoreceptor protein, recently crystallized under constant illumination. While LOV dimers remained globally stable during the light-state simulation with regard to the Jα coiled-coil, distinct conformational changes for a glutamine in the vicinity of the FMN chromophore are observed. In contrast, multiple Jα-helix conformations are sampled in the dark-state. These changes coincide with a displacement of the Iβ and Hβ strands relative to the light-state structure and result in a correlated rotation of both LOV core domains in the dimer. These global changes are most likely initiated by the reorientation of the conserved glutamine Q116, whose side chain flips between the Aβ (dark state) and Hβ strand (light state), while maintaining two potential hydrogen bonds to FMN-N5 and FMN-O4, respectively. This local Q116-FMN reorientation impacts on an inter-subunit salt-bridge (K117-E96), which is stabilized in the light state, hence accounting for the observed decreased mobility. Based on these findings we propose an alternative mechanism for dimeric LOV photoactivation and intramolecular signal-relay, assigning a distinct structural role for the conserved "flipping" glutamine. The proposed mechanism is discussed in light of universal applicability and its implications for the understanding of LOV-based optogenetic tools.
    Full-text · Article · Oct 2015
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    • "EL222 consists of a LOV domain that is coupled to an N-terminal HTH domain. In the dark state the DNA binding domain is bound to the β-sheet of the LOV domain [39]. This surface is directly interacting with the FMN chromophore and, consequently, illumination releases the HTH domain which is followed by dimerization and DNA binding. "
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    ABSTRACT: Aureochrome 1 from Vaucheria frigida is a recently identified blue-light receptor that acts as a transcription factor. The protein comprises a photosensitive light-, oxygen- and voltage-sensitive (LOV) domain and a basic zipper (bZIP) domain that binds DNA rendering aureochrome 1 a prospective optogenetic tool. Here, we studied the photoreaction of full-length aureochrome 1 by molecular spectroscopy. The kinetics of the decay of the red-shifted triplet state and the blue-shifted signaling state were determined by time-resolved UV/Vis spectroscopy. It is shown that the presence of the bZIP domain further prolongs the lifetime of the LOV390 signaling state in comparison to the isolated LOV domain whereas bound DNA does not influence the photocycle kinetics. The light-dark Fourier transform infrared (FTIR) difference spectrum shows the characteristic features of the flavin mononucleotide chromophore except that the S-H stretching vibration of cysteine 254, which is involved in the formation of the thio-adduct state, is significantly shifted to lower frequencies compared to other LOV domains. The presence of the target DNA influences the light-induced FTIR difference spectrum of aureochrome 1. Vibrational bands that can be assigned to arginine and lysine side chains as well to the phosphate backbone, indicate crucial changes in interactions between transcription factor and DNA.
    Full-text · Article · Jul 2014 · PLoS ONE
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    • "InstructuralstudiesoftheVaucheriafrigidaAure- ochrome1LOVdomain,bothN-andC-terminal extensionswerefoundassociatedwiththeLOVcore, raisingthepossibilitythateffectordomainscouldbe fusedtoeitherendofLOVforphotoregulation(Mitra etal.,2012). Manyofthesestudiesexaminedphotosensorydo- mainsaloneorwithsmallN-orC-terminalextensions,ratherthaninthecontextofthefull-lengthproteins .Structuralstudiesoffull-lengthLOVproteins withdiverseeffectordomains,ashasbeencarriedout withEL222(Nashetal.,2011),canprovidevalu- ablemechanisticinsightsintowaystoengineerLOV- containingproteinsforoptogeneticapplications. "
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    ABSTRACT: Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light-actuated tools allow manipulation of molecular events with ultra-fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub-micrometer precision within cells. While light-actuated chemicals such as photolabile "caged" compounds have been in existence for decades, the use of genetically-encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever-growing optogenetic toolkit.
    Full-text · Article · Nov 2012 · Biology of the Cell
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