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

ABSTRACT 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, Sep 29, 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.
    PLoS ONE 07/2014; 9(7):e103307. DOI:10.1371/journal.pone.0103307 · 3.23 Impact Factor
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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.
    Biology of the Cell 11/2012; 105(2). DOI:10.1111/boc.201200056 · 3.51 Impact Factor
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    • "However, the mechanism is different: here, light promotes dimerization (Morgan et al., 2010). A similar phenomenon is observed in the natural photoreceptor EL222 LOV-HTH, where the fully-folded HTH effector domain binds to the dimerization interface of LOV and renders monomeric LOV-HTH unable to bind DNA in the dark state (Nash et al., 2011). Light decouples this sensor-effector complex, exposes the dimer interface of both the LOV and HTH domains, promotes formation of dimeric HTH and thus DNA binding. "
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    ABSTRACT: Aureochrome1, a signaling photoreceptor from a eukaryotic photosynthetic stramenopile, confers blue-light-regulated DNA binding on the organism. Its topology, in which a C-terminal LOV sensor domain is linked to an N-terminal DNA-binding bZIP effector domain, contrasts with the reverse sensor-effector topology in most other known LOV-photoreceptors. How, then, is signal transmitted in Aureochrome1? The dark- and light-state crystal structures of Aureochrome1 LOV domain (AuLOV) show that its helical N- and C-terminal flanking regions are packed against the external surface of the core β sheet, opposite to the FMN chromophore on the internal surface. Light-induced conformational changes occur in the quaternary structure of the AuLOV dimer and in Phe298 of the Hβ strand in the core. The properties of AuLOV extend the applicability of LOV domains as versatile design modules that permit fusion to effector domains via either the N- or C-termini to confer blue-light sensitivity.
    Structure 04/2012; 20(4):698-706. DOI:10.1016/j.str.2012.02.016 · 5.62 Impact Factor
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