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A Photochromic Histidine Kinase Rhodopsin (HKR1) That Is Bimodally Switched by Ultraviolet and Blue Light*

  • Meike Luck
    Footnotes
    Affiliations
    Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany
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  • Tilo Mathes
    Footnotes
    Affiliations
    Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany

    Faculty of Sciences, Department of Physics and Astronomy, Biophysics Group, Vrije Universiteit, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands
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  • Sara Bruun
    Affiliations
    Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany

    Institute of Chemistry, Technische Universität zu Berlin, Sekretariat PC14, Strasse des 17 Juni 135, 10623 Berlin, Germany
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  • Roman Fudim
    Footnotes
    Affiliations
    Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany
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  • Rolf Hagedorn
    Affiliations
    Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany
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  • Tra My Tran Nguyen
    Affiliations
    Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany
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  • Suneel Kateriya
    Affiliations
    Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India
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  • John T.M. Kennis
    Footnotes
    Affiliations
    Faculty of Sciences, Department of Physics and Astronomy, Biophysics Group, Vrije Universiteit, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands
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  • Peter Hildebrandt
    Affiliations
    Institute of Chemistry, Technische Universität zu Berlin, Sekretariat PC14, Strasse des 17 Juni 135, 10623 Berlin, Germany
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  • Peter Hegemann
    Correspondence
    Supported by NWO-DFG Bilateral program. To whom correspondence should be addressed. Tel.: 49-30-2093-8681; Fax: 49-30-2093-8520
    Affiliations
    Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany
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  • Author Footnotes
    * This work was supported by Deutsche Forschungsgemeinschaft Grants FOR1261 (to P. Hegemann), HE3824/19-1 (to S. B. and P. Hildebrandt), and Cluster of Excellence Grant UniCat/BIG-NSE.
    This article contains supplemental Figs. 1 and 2.
    1 Supported by the LaserLab Europe access program LCVU001753.
    2 Supported by the Chemical Sciences Council of The Netherlands Organization for Scientific Research through an ECHO grant (to J. T. M. K.). Supported by NWO-DFG Bilateral program.
    3 Supported by a Chemical Sciences Council of The Netherlands Organization for Scientific Research VICI grant. Supported by NWO-DFG Bilateral program.
Open AccessPublished:October 01, 2012DOI:https://doi.org/10.1074/jbc.M112.401604
      Rhodopsins are light-activated chromoproteins that mediate signaling processes via transducer proteins or promote active or passive ion transport as ion pumps or directly light-activated channels. Here, we provide spectroscopic characterization of a rhodopsin from the Chlamydomonas eyespot. It belongs to a recently discovered but so far uncharacterized family of histidine kinase rhodopsins (HKRs). These are modular proteins consisting of rhodopsin, a histidine kinase, a response regulator, and in some cases an effector domain such as an adenylyl or guanylyl cyclase, all encoded in a single protein as a two-component system. The recombinant rhodopsin fragment, Rh, of HKR1 is a UVA receptor (λmax = 380 nm) that is photoconverted by UV light into a stable blue light-absorbing meta state Rh-Bl (λmax = 490 nm). Rh-Bl is converted back to Rh-UV by blue light. Raman spectroscopy revealed that the Rh-UV chromophore is in an unusual 13-cis,15-anti configuration, which explains why the chromophore is deprotonated. The excited state lifetime of Rh-UV is exceptionally stable, probably caused by a relatively unpolar retinal binding pocket, converting into the photoproduct within about 100 ps, whereas the blue form reacts 100 times faster. We propose that the photochromic HKR1 plays a role in the adaptation of behavioral responses in the presence of UVA light.

      Introduction

      The main sensory photoreceptor of the animal kingdom is rhodopsin. The respective spectral sensitivity of rhodopsins may cover an extremely broad range of the sunlight spectrum, from 358 nm (near ultraviolet, UVA) to 630 nm (near infrared) (
      • Kleinschmidt J.
      • Harosi F.I.
      Anion sensitivity spectral tuning of cone visual pigments in situ.
      ,
      • Imai H.
      • Hirano T.
      • Terakita A.
      • Shichida Y.
      • Muthyala R.S.
      • Chen R.L.
      • Colmenares L.U.
      • Liu R.S.
      Probing for the threshold energy for visual transduction: red-shifted visual pigment analogs from 3-methoxy-3-dehydroretinal and related compounds.
      ). Rhodopsins with absorption at the red edge of the spectrum (XL-iodopsins) are found in flies, mollusks, fish, and in some birds. At this spectral range, thermal isomerization increases, producing significant background at high rhodopsin concentrations (
      • Luo D.G.
      • Yue W.W.
      • Ala-Laurila P.
      • Yau K.W.
      Activation of visual pigments by light and heat.
      ). On the other end of the spectrum, the UVA-absorbing pigment is of limited use because UV-transparent lenses promote damage of the retina. Even so, UV-sensitive rhodopsins are widely distributed in flies, birds, and nocturnal rodents (
      • Kusnetzow A.K.
      • Dukkipati A.
      • Babu K.R.
      • Ramos L.
      • Knox B.E.
      • Birge R.R.
      Vertebrate ultraviolet visual pigments: protonation of the retinylidene Schiff base and a counterion switch during photoactivation.
      ,
      • Tarttelin E.E.
      • Bellingham J.
      • Hankins M.W.
      • Foster R.G.
      • Lucas R.J.
      Neuropsin (Opn5): a novel opsin identified in mammalian neural tissue.
      ). Action and absorption spectra have been recorded for rhodopsins at both edges of the visible spectrum, but further spectroscopic studies based on the structure and dynamics of the photoreceptors are missing because of difficulties in purifying these proteins in sufficient amounts. In prokaryotes like archaea or eubacteria, rhodopsins serve as light-driven H+ or Cl pumps or mediate signal transduction via a two-component signaling system (
      • Gao R.
      • Stock A.M.
      Biological insights from structures of two-component proteins.
      ). Another branch that is rich in rhodopsin-related proteins is freshwater algae. Phototaxis of motile species like Chlamydomonas reinhardtii is mainly mediated by the light-gated ion channel channelrhodopsin, which is currently widely used in the new field of optogenetics to activate individual cells or cell types in brain slices or live animals to understand neuronal networks (
      • Zhang F.
      • Vierock J.
      • Yizhar O.
      • Fenno L.E.
      • Tsunoda S.
      • Kianianmomeni A.
      • Prigge M.
      • Berndt A.
      • Cushman J.
      • Polle J.
      • Magnuson J.
      • Hegemann P.
      • Deisseroth K.
      The microbial opsin family of optogenetic tools.
      ). Recently, a previously undescribed class of rhodopsin sequences has been found in several algal genomes including C. reinhardtii (
      • Kateriya S.
      • Nagel G.
      • Bamberg E.
      • Hegemann P.
      “Vision” in single-celled algae.
      ). These rhodopsins are directly connected via the C terminus to a histidine kinase and a response regulator, defining a novel rhodopsin subfamily of histidine-kinase rhodopsins (HKRs).
      The abbreviations used are: HKR
      histidine kinase rhodopsin
      Bl
      blue
      BR
      bacteriorhodopsin
      EADS
      evolution-associated difference spectra
      ESA
      excited state absorption
      GSB
      ground state bleaching
      LED
      light-emitting diode
      Rh
      rhodopsin
      RR
      response regulator.
      C. reinhardtii contains four HKR sequences, two of them with an additional cyclase domain (Fig. 1, A and B). They are believed to constitute functional two-component systems. It has been suggested that these HKRs are involved in cell cycle regulation and circadian rhythmicity in the marine microalga Ostreococcus, but molecular characterization is still lacking (
      • Troein C.
      • Corellou F.
      • Dixon L.E.
      • van Ooijen G.
      • O'Neill J.S.
      • Bouget F.Y.
      • Millar A.J.
      Multiple light inputs to a simple clock circuit allow complex biological rhythms.
      ).
      Figure thumbnail gr1
      FIGURE 1Basic features of HKR1. A, general modular assembly of HKRs. B, specific modular composition of HKR1 and HKR2 of C. reinhardtii (Rhod, rhodopsin; Kin, histidine kinase; RR, response regulator; Cyc, cyclase). C, localization of HKR1 in the Chlamydomonas eye using antibodies against Rh (red) in comparison with Nomarsky images (left two panels) and against the response regulator RR (red; right two panels) combined with an α-tubulin labeling (green). Scale bars, 4 μm. D, absorption spectra of recombinant Rh-UV and Rh-Bl. E, stability of Rh-Bl (red line) and Rh-UV (black line). Absorbance changes were recorded during 10-min illumination and 50 min in darkness.

      DISCUSSION

      The first spectroscopically characterized UV-rhodopsin belongs to a yet-uncharacterized class of HKRs (supplemental Fig. 2). The localization of HKR1 within the eyespot of C. reinhardtii originally surprised us because phototaxis was thought to be mediated exclusively by the channelrhodopsins ChR1 and ChR2 (
      • Berthold P.
      • Tsunoda S.P.
      • Ernst O.P.
      • Mages W.
      • Gradmann D.
      • Hegemann P.
      Channelrhodopsin-1 initiates phototaxis and photophobic responses in Chlamydomonas by immediate light-induced depolarization.
      ,
      • Sineshchekov O.A.
      • Jung K.H.
      • Spudich J.L.
      Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii.
      ), and control of photobehavior has been considered the major if not exclusive role of the eye.
      However, because the proposed photoreceptor Chlamyopsin-2 (Cop2), which is involved in the assembling of photosystem-1 (
      • Ozawa S.
      • Nield J.
      • Terao A.
      • Stauber E.J.
      • Hippler M.
      • Koike H.
      • Rochaix J.D.
      • Takahashi Y.
      Biochemical and structural studies of the large Ycf4-photosystem I assembly complex of the green alga Chlamydomonas reinhardtii.
      ), is also localized in the eye, it appears to be that algal eyes are more general sensory organelles. Furthermore, the fact that the dark-adapted Rh of HKR1 absorbs in the UV range is surprising because, to our knowledge, no specific UV response has ever been described for C. reinhardtii, suggesting that HKR1 mediates more general UV avoidance reactions or simply UV stress responses that modify sensory processes in the presence of UVA light. We have so far been unable to functionally reconstitute the complete HKR1 in E. coli or Xenopus oocytes as we have done previously for the small photoactivated cyclase bPAC (
      • Stierl M.
      • Stumpf P.
      • Udwari D.
      • Gueta R.
      • Hagedorn R.
      • Losi A.
      • Gärtner W.
      • Petereit L.
      • Efetova M.
      • Schwarzel M.
      • Oertner T.G.
      • Nagel G.
      • Hegemann P.
      Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa.
      ); therefore, we do not know whether HKR1 is a guanylyl cyclase or an adenylyl cyclase. cAMP has been suggested as an abiotic stress metabolite in C. reinhardtii whereas the role of cGMP is unknown. Intraflagellar cAMP increases upon sexual agglutination to reduce flagellar motility after the mating partners come in contact (
      • Saito T.
      • Small L.
      • Goodenough U.W.
      Activation of adenylyl-cyclase in Chlamydomonas reinhardtii by adhesion and by heat.
      ). A regulatory role of cAMP within the eyespot organelle is conceivable. UVA-sensitive microbial rhodopsins have not been described so far but are now expected to be present in many algae. Functions for this “new” rhodopsin subfamily await further elucidation. C. reinhardtii additionally contains a UVB receptor, whose homolog, UVR8, has been recently characterized in Arabidopsis (
      • Christie J.M.
      • Arvai A.S.
      • Baxter K.J.
      • Heilmann M.
      • Pratt A.J.
      • O'Hara A.
      • Kelly S.M.
      • Hothorn M.
      • Smith B.O.
      • Hitomi K.
      • Jenkins G.I.
      • Getzoff E.D.
      Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges.
      ). This protein uses tryptophan side chains arranged in an excitonically coupled cluster at the dimer interface as the sole intrinsic receptor pigment. Excitation with 280 nm leads to monomerization of the protein and binding to COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1), resulting in a general photoprotection response.
      The large photochromic shifts as found for HKR1 and the great stability of Rh-UV are unprecedented findings for a microbial rhodopsin. Such photochromism is commonly found in fly rhodopsins or in retinochromes (
      • Furutani Y.
      • Terakita A.
      • Shichida Y.
      • Kandori H.
      FTIR studies of the photoactivation processes in squid retinochrome.
      ). Small fully reversible photochromic shifts have been reported for Anabaena sensory rhodopsin where orange illumination of the dark state (λmax = 549 nm) photoisomerizes all-trans retinal into 13-cis light-adapted state (λmax = 537 nm) that is stable for a few hundred minutes (
      • Vogeley L.
      • Sineshchekov O.A.
      • Trivedi V.D.
      • Sasaki J.
      • Spudich J.L.
      • Luecke H.
      Anabaena sensory rhodopsin: a photochromic color 0 sensor at 2.0 angstrom.
      ). Phototactic signaling in archaea by sensory rhodopsin I is mediated via a bimodal photochemical switching between the dark state absorbing at 590 and the signaling state with deprotonated chromophore absorbing at 370 nm (P370) (
      • Spudich J.L.
      • Bogomolni R.A.
      Mechanism of color discrimination by a bacterial sensory rhodopsin.
      ). However, the thermal life time for the signaling state is a half-second and not hours as in the case of HKR1. From the molecular aspect, it is very interesting to know why the chromophore is deprotonated in its most stable dark state. Because the resonance Raman spectra are sensitive to both 13-cis/trans and 15-syn/anti isomerization (Fig. 5), the excellent agreement between the Raman spectra of Rh-UV and the BR-M410 photocycle intermediate points to a 13-cis configuration of the RSB with an anti conformation of the C = N bond (13-cis,15-anti) (
      • Curry B.
      • Palings I.
      • Broek A.D.
      • Pardoen J.A.
      • Lugtenburg J.
      • Mathies R.
      Vibrational analysis of the retinal isomers.
      ,
      • Fodor S.P.
      • Ames J.B.
      • Gebhard R.
      • van den Berg E.M.M.
      • Stoeckenius W.
      • Lugtenburg J.
      • Mathies R.A.
      Chromophore structure in bacteriorhodopsins-N intermediate: implications for the proton-pumping mechanism.
      ). There are no indications for contributions by an all-trans chromophore because the respective characteristic marker bands, such as a distinct band at 1153 cm−1, are not detectable in the Raman spectrum (
      • Curry B.
      • Palings I.
      • Broek A.D.
      • Pardoen J.A.
      • Lugtenburg J.
      • Mathies R.
      Vibrational analysis of the retinal isomers.
      ). Homology modeling with structures of other microbial rhodopsins as bacteriorhodopsin, halorhodopsin, SRII, and channelrhodopsin C1C2 (Fig. 6), for which three-dimensional structures are available (
      • Schobert B.
      • Lanyi J.K.
      • Spudich E.N.
      • Spudich J.L.
      Crystal structure of sensory rhodopsin II at 2.4 angstroms: insights into color tuning and transducer interaction.
      ,
      • Kolbe M.
      • Besir H.
      • Essen L.O.
      • Oesterhelt D.
      Structure of the light-driven chloride pump halorhodopsin at 1.8 angstrom resolution.
      ,
      • Kato H.E.
      • Zhang F.
      • Yizhar O.
      • Ramakrishnan C.
      • Nishizawa T.
      • Hirata K.
      • Ito J.
      • Aita Y.
      • Tsukazaki T.
      • Hayashi S.
      • Hegemann P.
      • Maturana A.D.
      • Ishitani R.
      • Deisseroth K.
      • Nureki O.
      Crystal structure of the channelrhodopsin light-gated cation channel.
      ), led to the assumption that in a 13-cis,15-anti configuration (Fig. 5A) the free electron pair of the Schiff base nitrogen is facing away from the bona fide counterion Asp-239. This configuration makes protonation of the nitrogen highly unfavorable as in the BR-M state, where the pKa of the Schiff base is 7 units lower compared with the dark state. In contrast, for Rh-Bl we suggest that the electron pair of the Schiff base nitrogen is facing toward Asp-239, thereby stabilizing the protonated state. In this case, two states, 13-cis,15-syn and 13-trans,15-anti, could accomplish this condition (Fig. 5, B and C). Consistent with the Raman data, both such states could exist in slow thermal equilibrium in the Rh-Bl state. This is not unlikely because exactly these two states do coexist in dark-adapted BR, as shown most clearly by nuclear magnetic resonance (NMR) spectroscopy (
      • Schobert B.
      • Lanyi J.K.
      • Spudich E.N.
      • Spudich J.L.
      Crystal structure of sensory rhodopsin II at 2.4 angstroms: insights into color tuning and transducer interaction.
      ). We challenged the claim that Asp-239 is the counterion of the retinal Schiff base by substituting Asp-239 by Glu and Asn. D239N did not form a chromophore whereas D239E-Rh-Bl did (Fig. 6B), and the absorption of the blue state was hypsochromically shifted by 13 nm (Fig. 6C). Photoconversion from Rh-UV to Rh-Bl involves a branched reaction pathway leading to two chromophore isomers with protonated Schiff bases, attributed to an all-trans and 13-cis configuration, respectively. A possible explanation may be that photoexcitation of Rh-UV leads to an electronically excited state that allows for both a rotation around the C(13) = C(14) double bond (Fig. 5, blue arrow) and for an anti/syn isomerization of the C(15) = N Schiff base in principle (Fig. 5, green arrow). This dual option of molecular events may be a tribute to the unusually long lifetime of the electronically excited state of ∼60 ps in case of Rh-UV. A sole anti/syn isomerization around the C(15) = N bond has been described for the photochemical transition from the Meta-II to Meta-III states of rod bovine rhodopsin (
      • Ritter E.
      • Zimmermann K.
      • Heck M.
      • Hofmann K.P.
      • Bartl F.J.
      Transition of rhodopsin into the active metarhodopsin II state opens a new light-induced pathway linked to Schiff base isomerization.
      ). In contrast to the unusual photochemistry of Rh-UV, Rh-Bl shows typical rhodopsin photochemistry as observed in BR and channelrhodopsin. Therefore, it might be conceivable that the two excited state deactivation pathways originate from the two proposed isomers, which are not necessarily both productive for the Rh-UV state. Due to the high stability of the Rh-UV state and thermal equilibrium between both proposed Rh-Bl retinal conformations, it is nevertheless possible to create Rh-UV quantitatively by continuous illumination. In conclusion, the rhodopsin part of HKR1 can switch between two isoforms Rh-UV and Rh-Bl that are both thermally stable in darkness but are efficiently interconverted by UV and blue light. Rh-UV is unusual in the sense that the excited state lifetime is exceptionally long and the early photoproducts are hardly seen as changes in the visible spectrum. The underlying processes await further elucidation, for example by infrared studies on an ultrafast time scale.
      Figure thumbnail gr5
      FIGURE 5A–C, schematic representation of the 13 = 14 and 15 = N conformers of the retinal Schiff base. Possible isomerizations are indicated by colored arrows. We conclude that the conformation of the Rh-UV state is 13-cis,15-anti (A), whereas Rh-Bl represents an equilibrium of 13-cis,15-syn (B) and 13-trans,15-anti (all-trans) (C). The two Rh-Bl conformers thermally equilibrate by double isomerizations (red+blue or red+green). D, proposed photocycle summarizing interconversion of the light-induced reaction intermediates as observed after 380-nm excitation (purple arrow) and 500-nm excitation (green arrow) in ultrafast and flash photolysis spectroscopic experiments.
      Figure thumbnail gr6
      FIGURE 6Active site. A, homology model of HKR1 based on BR. The model shows a seven-transmembrane structure and a lysine to bind the chromophore. The retinal and conserved residues in the retinal binding pocket (HKR1, black; BR, blue), are highlighted in color. B and C, normalized absorbance spectra of the HKR mutant D239E (solid lines) compared with wild-type (WT) HKR1. The WT and D239E Rh-UV forms have an absorbance maximum at 380 nm (B). The aggregation tendency of the purified mutant causes increased scattering. The absorbance maximum of D239E-Rh-Bl shows a 13-nm shift to lower wavelengths and more scattering in the UVA-region (C).

      Acknowledgments

      We thank Christina Mrosek and Margrit Michalsky for excellent technical assistance and Eglof Ritter and Patrick Piwowarski for support with the HPLC.

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