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Green/Red Cyanobacteriochromes Regulate Complementary Chromatic Acclimation Via a Protochromic Photocycle

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Green/Red Cyanobacteriochromes Regulate Complementary Chromatic Acclimation Via a Protochromic Photocycle Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle Yuu Hirosea, Nathan C. Rockwellb, Kaori Nishiyamac, Rei Narikawad, Yutaka Ukajic, Katsuhiko Inomatac, J. Clark Lagariasb,1, and Masahiko Ikeuchid,1 aElectronics-Inspired Interdisciplinary Research Institute, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan; bDepartment of Molecular and Cellular Biology, University of California, Davis, CA 95616; cDivision of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan; and dDepartment of Life Sciences (Biology), The University of Tokyo, Meguro, Tokyo 153-8902, Japan Contributed by J. Clark Lagarias, February 13, 2013 (sent for review January 24, 2013) Cyanobacteriochromes (CBCRs) are cyanobacterial members of the spectral diversity, with peak absorptions ranging from 330 to 680 phytochrome superfamily of photosensors. Like phytochromes, nm and hence spanning the entire visible spectrum and near UV CBCRs convert between two photostates by photoisomerization (13, 15–24). CBCR subfamilies that sense light in the near-UV to of a covalently bound linear tetrapyrrole (bilin) chromophore. blue region (330–470 nm) have been studied extensively. Such Although phytochromes are red/far-red sensors, CBCRs exhibit CBCRs combine bilin photoisomerization with subsequent for- diverse photocycles spanning the visible spectrum and the near- mation or elimination of a second thioether linkage to the bilin UV (330–680 nm). Two CBCR subfamilies detect near-UV to blue light C10 atom, shortening the conjugated π system to induce a re- (330–450 nm) via a “two-Cys photocycle” that couples bilin 15Z/15E markable spectral shift (19, 21, 23, 25–29). In such “two-Cys photoisomerization with formation or elimination of a second bilin– photocycles,” the reactive thiol group is supplied by a second cysteine adduct. On the other hand, mechanisms for tuning the conserved Cys residue within the GAF domain. Two such Cys absorption between the green and red regions of the spectrum residues have been identified in different CBCR subfamilies have not been elucidated as of yet. CcaS and RcaE are members (Fig. S1C) (19, 25). Such a second Cys is not found in CBCR of a CBCR subfamily that regulates complementary chromatic accli- subfamilies sensing green to red light (520–670 nm) (16, 17), BIOCHEMISTRY mation, in which cyanobacteria optimize light-harvesting antennae implicating other mechanisms allowing perception of green light in response to green or red ambient light. CcaS has been shown to by the intrinsically red-absorbing bilin chromophore. undergo a green/red photocycle: reversible photoconversion be- Physiologically, CBCRs are implicated in regulation of pho- 15Z 15Z tween a green-absorbing state ( Pg) and a red-absorbing totaxis (30–34), but the best-understood CBCR function is the 15E 15E Fremyella diplosi- state ( Pr). We demonstrate that RcaE from regulation of complementary chromatic acclimation (CCA). In phon undergoes the same photocycle and exhibits light-regulated CCA, cyanobacteria optimize the composition of their photo- kinase activity. In both RcaE and CcaS, the bilin chromophore is synthetic pigments in response to the availability of green and 15Z 15E deprotonated as Pg but protonated as Pr. This change of bilin red light (35). RcaE was the first genetically isolated CBCR, protonation state is modulated by three key residues that are con- identified as a regulator of CCA in Fremyella diplosiphon (35, served in green/red CBCRs. We therefore designate the photocycle 36). F. diplosiphon regulates both red-absorbing phycocyanin and of green/red CBCRs a “protochromic photocycle,” in which the dra- green-absorbing phycoerythrin (type III CCA, Fig. 1A). In vivo matic change from green to red absorption is not induced by initial genetic studies demonstrated that RcaE functions in a three- bilin photoisomerization but by a subsequent change in bilin component phosphorelay pathway to regulate expression of protonation state. phycocyanin and phycoerythrin genes, but in vitro characteriza- tion of spectral properties or kinase activity of RcaE has proved light sensing | phycobiliprotein | signal transduction | spectral tuning | challenging (37–39). Homologous CcaS CBCRs from Synecho- two-component signaling cystis sp. PCC 6803 and Nostoc punctiforme ATCC 29133 re- cently were characterized (16, 40). CcaS proteins contain a CBCR GAF domain closely related to that of RcaE, photo- hytochrome photosensors initially were discovered in plants 15Z convert between a green-absorbing dark state ( Pg) and a red- Pand later found in cyanobacteria, nonoxygenic photosynthetic 15E bacteria, nonphotosynthetic bacteria, fungi, and algae (1, 2). These absorbing photoproduct ( Pr), and exhibit green-stimulated photoreceptors bind linear tetrapyrrole (bilin) chromophores histidine kinase activity (16). CcaS functions in a two-component within a conserved GAF (cGMP phosphodiesterase/adenylyl phosphorelay pathway to regulate CCA in N. punctiforme (40), cyclase/FhlA) domain via a covalent thioether linkage to a con- with phycoerythrin being regulated (type II CCA) (36). served Cys residue (Fig. S1A)(3–6). Upon illumination, phyto- In this study, we establish that full-length RcaE also exhibits chromes reversibly convert between a red-absorbing dark state a green/red photocycle and light-regulated kinase activity in and a far-red–absorbing photoproduct. This red/far-red photo- cycle is triggered by photoisomerization of the bilin 15,16-double bond between the 15Z and 15E configurations (7, 8), with 15Z Author contributions: Y.H. and N.C.R. designed research; Y.H. performed research; Y.H., giving red absorption and 15E far-red absorption (4, 6, 9). In K.N., R.N., Y.U., and K.I. contributed new reagents/analytic tools; Y.H. and N.C.R. analyzed data; and Y.H., N.C.R., J.C.L., and M.I. wrote the paper. phytochromes, the conjugated π system of the bilin is protonated The authors declare no conflict of interest. in both photostates, and this protonation is necessary to maintain – Data deposition: The RcaE locus reported in this paper has been deposited in DNA Data the red and far-red absorption (10 12). Conserved GAF residues Base of Japan (DDBJ) and the Genbank database (accession no. AB710467). supply a hydrogen bond network to tune the chemical and 1 B To whom correspondence may be addressed. E-mail: [email protected] or mikeuchi@ spectral properties of the bilin (Fig. S1 ).* bio.c.u-tokyo.ac.jp. Cyanobacteriochromes (CBCRs) are widespread cyanobacte- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rial photosensors with phytochrome-related GAF domains (1, 2, 1073/pnas.1302909110/-/DCSupplemental. 13, 14). Although CBCRs also convert between two photostates *Throughout this report, we designate photocycles with the 15Z photostate followed by 15Z 15E via bilin photoisomerization at C15, they exhibit much more the 15E photostate, so a green/red photocycle has Pg and Pr photostates. www.pnas.org/cgi/doi/10.1073/pnas.1302909110 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 with modest but detectable green/red photoconversion (Fig. S2A), autophosphorylation activity of this preparation was not de- pendent on photostate (Fig. S2E). However, we noted that the N-terminal sequence of this construct is homologous to the C-terminal portion of a PAS (PER/ARNT/SIM) domain, sug- gesting that additional protein sequence is encoded upstream of the annotated ATG start codon. We therefore performed PCR amplification of F. diplosiphon genomic DNA and sequenced the upstream region (primers are listed in Table S1). We found that the ORF of RcaE extended upstream for another 50 residues to an apparent GTG start codon, completing the PAS domain (Fig. 1A and Fig. S2F). There is a stop codon immediately upstream of this GTG (Fig. S2F), and GTG is a known start codon in cya- nobacteria (44). The complete protein (705 residues) was puri- fied readily from E. coli cells and exhibits both a robust green/red photocycle and light-regulated autophosphorylation activity (Fig. 1D and Fig. S2). This kinase activity was about threefold 15Z 15E greater in Pg than in Pr (Fig. 1D), consistent with in vivo studies of red-activated phosphorylation in the RcaE pathway (35) but with reversed polarity relative to the green-activated phos- phorylation of CcaS (16). These studies establish RcaE as a func- Fig. 1. RcaE from F. diplosiphon is a light-regulated protein kinase. (A, Upper) tional light-regulated histidine kinase, properties consistent with its Cells of F. diplosiphon that were fully acclimated to green light (GL) or red knownfunctioninregulatingtypeIIICCA. light (RL). (Lower) Domain architecture of full-length RcaE (705 residues). (B) 15Z 15E Absorption and (C) CD spectra of the Pg (green lines) and Pr (red lines) Green/Red CBCR Photocycle Uses a Protochromic Absorption Change. forms of the truncated GAF domain from RcaE at pH 7.5. (B, Insets)Colorsof We focused on the isolated GAF domain of RcaE for mechanistic 15Z 15E the Pg and Pr forms of the GAF domain in solution. (D)Autophosphor- characterization of the green/red photocycle. After adventitiously 15Z 15E 32 ylation of full-length RcaE in its Pg and Pr forms. Incorporation of Pwas noting that slight changes in buffer pH resulted in substantial color fi quanti ed with a PhosphorImager. (Inset) Original autoradiograph. changes, we systematically examined changes in absorption as a function of pH for both photostates. The absorption peak of 15Z Pg was unchanged at alkaline pH. However, at acidic pH, the vitro, confirming its role as the photosensor for type III CCA. 15Z Moreover, we elucidate the basis for sensitivity to green or red Pg peak disappeared and a distinct red-absorbing peak appeared at 680 nm (Fig. 2A). In contrast, the absorption peak of light in green/red CBCRs: RcaE combines bilin photoisomerization 15E with a subsequent change in bilin protonation state to induce Pr was unchanged at acidic pH but disappeared at alkaline pH, and a distinct green-absorbing peak appeared at 545 nm (Fig.
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