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 characterization 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 photocycle 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 dependent 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, suggesting 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 cyanobacteria (44). The complete protein (705 residues) was purified 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 phosphorylation 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. 2B). the dramatic green/red absorption change. This protochromic Denaturation analysis after pH titration confirmed that changes in photocycle relies on three residues conserved in the green/red bilin configuration due to pH change were not significant (Fig. S3 subfamily. Our work shows that, like two-Cys CBCRs, green/red A and B). Furthermore, difference spectra resulting from pH CBCRs also combine photoisomerization with a subsequent change alone were distinct from difference spectra resulting from ground-state chemical reaction to generate the different absorptions a combination of pH and denaturant (Fig. S3 C and D), con- of the two photostates. firming that the spectral changes were not a simple artifact of 15Z denaturation at extreme pH. Therefore, we conclude that Pg Results 15Z 15E 15E converted into Pr, and not Pr, at acidic pH, whereas Pr Photoconversion and in Vitro Kinase Activity of RcaE. To charac- 15E fi converted to Pg at alkaline pH. Similar results were observed terize the spectral properties of RcaE, we rst expressed the for Synechocystis CcaS (Fig S3 E and F), implicating pH-de- CBCR GAF domain (159 amino acids) as an isolated truncation Escherichia coli pendent spectral shifts as a general phenomenon in the green/red in cells genetically engineered to produce the CBCR subfamily. cyanobacterial bilin chromophore phycocyanobilin (PCB) (41). fi 15Z λ = Analysis of pH-dependent absorption changes at selected Puri ed holoprotein reversibly converted between Pg ( max 532 wavelengths demonstrated that the transitions were well de- 15E λ = B nm) and Pr ( max 661 nm) upon illumination (Fig. 1 ). An scribed by models with one or two titrating groups (Fig. 2 and identical photocycle was obtained when the same truncated protein Fig. S3; equations shown in SI Data Analysis). The derived pK fi A fi a was puri ed from cyanobacterial cells (Fig. S2 ), con rming that differed significantly for the two photostates, with an apparent PCB is the authentic chromophore. RcaE spectra taken under de- pKa of 5.6 for 15Z and 7.9 for 15E (Fig. 2 C and D and Table 1). naturing conditions confirmed the expected 15Z configuration for Synechocystis 15Z 15E This difference also was seen for native CcaS (Fig. Pg and 15E configuration for Pr (Fig. S2B). In darkness, slow 15E 15Z 15Z S3) but was not seen under denaturing conditions, in which both conversion from Pr to Pg was observed (Fig. S2C). Both Pg 15Z 15E – 15E and states exhibited a single pKa value of 6.1 6.4 (Fig. and Pr exhibited negative CD on the long-wavelength band (Fig. S4 and Table 1). This denatured value is in reasonable agree- C α 15Z 15E 1 ), indicative of the -facial bilin D-ring in both and ment with a pKa of 5.7 measured for the conjugated π system of states (25, 42) seen in many CBCRs (19, 23, 24). These features of free biliverdin (45), implicating the conjugated electronic system RcaE are very similar to those previously described for CcaS (16, of the bilin as the titrating moiety in native green/red CBCRs. 40), implicating a conserved green/red photocycle in known For the titration of the native 15E state of RcaE, a second CCA photoreceptors. 15Z transition with a pKa value of 8.9 also was observed (Table 1). In vivo analyses have implicated Pg as the physiologically This transition was not seen in native or denatured CcaS, native active signaling state of RcaE, with red light triggering auto- 15Z RcaE, or denatured RcaE, nor was it associated with a change phosphorylation and subsequent phosphotransfer (35). To test this in peak wavelength (Fig. 2B). This suggests that this second hypothesis, we expressed the previously annotated full-length transition arises through titration of an amino acid residue, or RcaE protein [655 amino acids (43)] in Synechocystis. Although even of the bilin propionate moiety, rather than of the bilin small amounts of protein could be partially purified (Fig. S2D) π system.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1302909110 Hirose et al. Downloaded by guest on September 27, 2021 “Protochromic Triad” Modulating the Bilin pKa in Green/Red CBCRs. To identify the residue(s) that cause the significant shift in bilin pKa upon photoisomerization, we chose RcaE residues for site- directed mutagenesis by reference to the known crystal structure of cyanobacterial phytochrome Cph1 (Fig. S1 B and C) (6). Cys259 of Cph1 and Cys248 of RcaE are the conserved Cys residues covalently attached to the bilin. Tyr176 and His290 of Cph1, forming part of the pocket for the photoactive bilin D- ring, are conserved in RcaE as Tyr187 and His285. Asp207 and His260 of Cph1 form hydrogen bonds with the bilin A-, B-, and C-rings, maintaining a high bilin pKa value of 9–11 (6, 12). Se- quence homology in the vicinity of Asp207 is poor between Cph1 and RcaE, but Glu217 of RcaE in this region is highly conserved among the green/red CBCRs. By contrast, His260 of Cph1 clearly corresponds to Leu249 of RcaE. Phe216 and Ser272 of Cph1, interacting with the bilin C-ring and/or C12-propionate, correspond to Tyr227 and Lys261 of RcaE, respectively. We mutated these residues, particularly seeking to identify potential proton donors, and assayed absorption maxima, photoconversion, and bilin pKa values in the 15Z and 15E states. The results of these experiments are summarized in Table 1. As expected, all engineered RcaE proteins covalently in- corporated bilin except C248A, confirming Cys248 as the bilin- binding residue (Fig. 3A). We found that Y227F RcaE had no major defect in bilin photoisomerization and pKa values (Fig. S5 and Table 1). In H285Q, forward 15Z→15E photoisomerization efficiency was reduced, but both 15Z and 15E states exhibited BIOCHEMISTRY normal absorption spectra and pKa values (Fig. S5 and Table 1). Fig. 2. Protochromic absorption changes of RcaE. (A) Absorbance spectra of In Y187F, the 15Z state exhibited normal absorption maxima the RcaE 15Z state are shown for pH values between 5.0 (red) and 11.0 (blue) A in 0.5 pH unit increments. The composite inset shows the colors of the RcaE (Table 1) and underwent full photoisomerization (Fig. S6 ), but solutions between pH 5.5 (left) and 11.0 (right). (B) Absorbance spectra and the 15E state exhibited an abnormal spectrum similar to that of a composite inset are shown for the 15E state of RcaE at the same pH values the wild type between pH 8.5 and pH 9.0 (Figs. 2B and 3B). as in A.(C) Absorbance values at the indicated wavelengths were plotted vs. Titration experiments showed that the pKa of the Y187F 15E pH for the RcaE 15Z state. Data were fit to a model having one titrating state was one pH unit lower than that of the wild type, with little group (Eq. S6). (D) A similar analysis was performed for the RcaE 15E state, effect on the 15Z state (Table 1 and Fig. S6). using a model having two titrating groups (Eq. S9). Data for 593 nm (gray) were magnified 15× for clarity; the axis runs from 0.1 to 0.12. (E) RcaE is shown after in vitro reconstitution with PCB or 15Za-PCB. Proteins were analyzed by SDS/PAGE followed by staining with Coomassie Brilliant Blue Table 1. Peak wavelengths and pKa values (CBB, Upper) or zinc blotting (Lower). The asterisk indicates a contaminating 15Z peak, 15E peak, protein found in apoprotein preparations and derived from E. coli cells. (F) Protein nm 15Z pKa nm 15E pKa Photocycle of holo-RcaE reconstituted with 15Za-PCB (black) or PCB (green and red) in vitro. No change in absorption was observed after prolonged SyCcaS 538 5.8 ± 0.1 672 8.6 ± 0.2, — irradiation with green light for 15Za-PCB–reconstituted RcaE. RcaE 531 5.6 ± 0.1 662 7.9 ± 0.2, 8.9 ± 0.5 RcaE* 662 6.4 ± 0.2 598 6.1 ± 0.2 † NpR6012g4 662 6.0 ± 0.3 598 5.8 ± 0.2 To examine the role of bilin photoisomerization, we used the RcaE Y F 533 5.6 ± 0.1 663 7.8 ± 0.1, 8.6 ± 0.5 15Za 227 PCB analog, -PCB, in which the bilin C- and D-rings are RcaE H Q 533 5.7 ± 0.1 663 7.2 ± 0.2 15Z anti fi 285 locked in , con guration by an additional covalent RcaE Y187F 533 5.5 ± 0.1 670 6.9 ± 0.1, 9.4 ± 0.3 A bridge (Fig S1 ) (46, 47). In vitro reconstitution of apo-RcaE RcaE L249H 632 7.5 ± 0.2 566 6.4 ± 0.2, 10.1 ± 0.4 with 15Za-PCB resulted in covalent chromophore incor- RcaE F252H 535 5.2 ± 0.3 626 7.1 ± 0.6 ± ± ± poration, with formation of a green-absorbing peak with RcaE E217D 537 5.5 0.2 640 8.1 0.2, 9.1 0.2 ± — ± a maximum at 580 nm and a shoulder at 535 nm (Fig. 2 E and RcaE E217A 533 5.5 0.5 540 , 9.8 0.4 15E ± — ± F). This peak did not convert into P , even after prolonged RcaE E217Q 533 5.7 0.6 540 ,10.0 0.2 r — ± ± irradiation with green light (Fig. S3I). In contrast, recon- RcaE K261M533 533 5.8 0.7, 6.9 0.8 RcaE K A527 — 527 5.3 ± 0.6, — stitution of apoprotein with PCB yielded a holoprotein with 261 fi fi a normal green/red photocycle (Fig. 2G). These results dem- To derive pKa values, absorption data at at least ve wavelengths were t onstrate that bilin photoisomerization precedes proton transfer as a function of pH to models for one or two titrating groups as described in fi in the green/red photocycle. Taken together, these pH titration SI Data Analysis. Errors are reported as SDs about the mean for the tted values. Cases in which a transition was not seen are indicated with a dash. and reconstitution studies indicate that RcaE and CcaS use Values were measured under native conditions unless otherwise stated, with photoisomerization of the bilin and a subsequent bilin pKa shift native peak wavelengths reported for pH 7.5. to drive proton transfer, resulting in a shift between green and *RcaE also was examined under denaturing conditions to measure the pKa red absorption. We designate this process a “protochromic of the covalent PCB adduct in the absence of native protein structure. Peak wavelengths are reported at pH 2.0. ” † photocycle to underscore the importance of proton transfer in The red/green CBCR NpR6012g4, belonging to a different subfamily, also conferring spectrally distinct absorption maxima on the was examined under denaturing conditions to provide an independent mea- two photostates. surement. Peak wavelengths are reported at pH 2.0.

Hirose et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 15Z Pr state produced a bleached, orange-absorbing 15E state with an apparent bilin pKa one unit lower than that of wild type (Fig. 3B and Table 1). Further irradiation with orange light did not cause 15E-to-15Z conversion in L249H, but dark reversion of L249H RcaE was much faster than wild type, with a t½ of 2 h (Fig S2C). Phe252 is predicted to be close to Leu249, but F252H did not have significant effects on the photocycle or on bilin pKa values (Table 1 and Fig. S5). Glu217 is predicted to lie on the bilin β-face, approximately similar to the position of Asp207 in Cph1. The 15Z state of 15Z E217A exhibits a normal Pg spectrum, but red absorption was absent in the 15E photoproduct (Fig. 3B), despite efficient forward photoisomerization as judged by denaturation (Fig. S7A). The resulting green/green photocycle is spectrally similar to the deprotonated 15Z and 15E states observed for wild-type RcaE at pH 10 (Fig. 3B). Titration of E217A RcaE showed that the 15Z state exhibited a normal pKa value, but the 15E state underwent 15E only slight absorption changes and did not exhibit Pr formation (Fig. 3C and Table 1). Similar results were obtained for E217Q(Fig. S7 and Table 1). On the other hand, the conservative E217D substitution had no significant effect on the absorption spectra or pKa values of either photostate (Fig. 3B and Table 1). Lys261 of RcaE corresponds to Ser272 of Cph1 and hence is predicted to interact with the bilin C12-propionate. Like proteins containing substitutions at Glu217, K261M RcaE exhibited 15Z 15E a normal Pg spectrum but did not yield Pr (Fig. 3B). It displayed only modest forward photoisomerization (Fig. S8A). Strikingly, pH titration of K261M did not result in a protonated 15Z state even at pH 5.0 (Fig. 3C and Table 1). The pKa of 15E K261M RcaE also was substantially decreased, but protonated 15E bilin was formed at low pH (Fig. 3C and Table 1). Similar results were obtained for K261A RcaE (Fig. S8 and Table 1). Taken together, our results demonstrate that Leu249, Glu217, and Lys261 are essential for the green/red photocycle. Thus, we designate these three residues as a protochromic triad that tunes the bilin pKa and defines the spectral properties of the green/red CBCR subfamily. Discussion We have elucidated the mechanistic basis for the protochromic photocycle in CCA sensors: the bilin chromophore of RcaE is 15Z 15E deprotonated in Pg but protonated in Pr. RcaE thus combines bilin photoisomerization with a subsequent shift in the bilin pKa, using a protochromic triad of three key residues to drive the proton transfer that causes the shift between green and red absorption. Our results show that CBCRs need not share the be- havior of phytochromes and phycobiliproteins, in which the bilin Fig. 3. Identification of a protochromic triad regulating the bilin pKa.(A) RcaE proteins were analyzed by SDS/PAGE, followed by CBB staining (Upper) chromophore is always protonated under static conditions (10–12).

or zinc blotting (Lower). (B) Absorption spectra are shown for Y187F, L249H, The three members of the protochromic triad possess distinct E217Q, E217D, and K261M RcaE proteins in the 15Z state (green) and 15E state roles in tuning the bilin pKa. Leu249 stabilizes the deprotonated 15Z 15E (red) at pH 7.5. Absorption spectra are shown for wild-type RcaE at pH 10.0 Pg state. Glu217 specifically stabilizes the protonated Pr in the same color scheme for comparison. The percentage of total hol- state, thereby inducing the pKa shift upon photoconversion. oprotein converted to the 15Z and 15E states was estimated by acid de- Lys261 favors protonation of the bilin π system in both photo- naturation followed by absorption spectroscopy and is indicated for each states, perhaps by positioning the C12-propionate moiety. No state. (C) pH titrations of E217A and K261M RcaE were performed as described fi for wild-type RcaE in Fig. 2. other residues were identi ed as essential for protonation of the 15E state, suggesting that either Glu217 or Lys261 is the proton donor for the protochromic photocycle. Glu217 is part of Engineered substitutions in Leu249, Glu217, and Lys261 had a conserved EVFP sequence motif of green/red CBCRs that more drastic effects. Leu249, corresponding to His260 in Cph1, corresponds to the DIP motif of phytochromes and the DXCF C is predicted to be located on the bilin α-face. L D and L A motif of one subfamily of two-Cys CBCRs (Fig. S1 ). In both 249 249 these motifs, the Asp side chain specifically and directly interacts RcaE proteins exhibited complex 15Z spectra with little to no with the 15E bilin (9, 28). Moreover, typical pK values for Glu photoconversion (Fig. S5), although neither protein could be a residues are closer to the intrinsic bilin pKa [∼6 (45)] than are obtained in substantial amounts. Interestingly, the L249H variant Lys pK values. We therefore favor Glu217 as the proton donor/ 15Z B a exhibited a red-absorbing state (Fig. 3 ) with an apparent acceptor in the protochromic photocycle. pKa almost two units higher than that of wild type (Table 1 and We propose the mechanistic model for the protochromic pho- 15Z Fig. S6), consistent with direct proton donation by the in- tocycle shown in Fig. 4A. The deprotonated Pg bilin is troduced His249 imidazole group. Red illumination of the L249H within a hydrophobic pocket formed by Leu249 and less conserved

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1302909110 Hirose et al. Downloaded by guest on September 27, 2021 physiological cues and to provide complete coverage of the entire visible spectrum with a single chromophore precursor and a small, soluble protein sensor. Materials and Methods Cloning of RcaE. We used genomic DNA from Tolypothrix sp. PCC 7601/1, astrainequivalenttoF. diplosiphon (51). Inverse PCR for full-length RcaE was performed using the RcaE–IVF/IVR primer pair with HpaI-digested genomic DNA as the template (Table S1). The sequence of full-length RcaE (705 residues) has been deposited in the DNA Data Base of Japan (accession no. AB710467). Constructs for the isolated GAF domain, the 655-residue protein Fig. 4. A mechanism for the protochromic photocycle. Bilin photo- originally annotated as full length, and the 705-residue full-length protein isomerization does not cause the spectral shift between green and red ab- were amplified using RcaE-5/RcaE-6, RcaE-1/RcaE-2 and RcaE-12/RcaE-2 primer sorption, but instead modulates the bilin pK to trigger proton transfer to or a pairs, respectively. The PCR products were cloned individually into pPCR-Script from Glu217. Protonated 15ZP and deprotonated 15EP intermediates iden- r g (Agilent) and confirmed by DNA sequencing. The constructs then were excised tified in our pH titration study and the protochromic triad identified by site- by NdeI and BamHI digestion and subcloned into the N-terminally hex- directed mutagenesis are shown. P, propionate. ahistidine-tagged vectors pET28a (Merck) and pTCH2031v (15) for expression in E. coli or in Synechocystis cells, respectively. Site-directed mutagenesis was 15Z performed according to the manufacturer’s instructions using QuikChange kit residues. Irradiation of Pg with green light causes 15Z-to-15E 15E reagents (Agilent) and the primer sets shown in Table S1. photoisomerization, yielding a Pg intermediate also seen at high pH and in proteins carrying substitutions for Glu217 or Lys261. Protein Expression, Protein Purification, and Kinase Assay. Expression and Subsequent movement of the bilin and/or Glu217 allows proton fi 15E puri cation of recombinant RcaE and CcaS holoproteins from PCB-producing transfer from Glu217 to the bilin π system, leading to Pr for- E. coli or from Synechocystis sp. PCC 6803 were performed as described mation. For the reverse reaction, red irradiation of the protonated previously (16), with slight modifications. The E. coli strain C41 (52) was used 15E 15Z Pr causes photoisomerization to 15Z, yielding the Pr in- for protein expression with FeCl3 omitted from the LB culture medium. E. coli cells were disrupted in 20 mM Hepes·NaOH, pH 7.5; 0.1 M NaCl, and termediate seen at low pH and in L249H RcaE. Structural changes 10% (wt/vol) glycerol using a microfluidizer (M-110Y, Microfluidics) for five associated with photoisomerization favor proton transfer from the fi fi bilin to Glu217, which then moves away from the bilin to form passages at 15,000 psi. The His-tagged protein was puri ed by af nity 15Z π chromatography using Talon resin (Clontech); elution was performed with BIOCHEMISTRY Pg. In both photostates, protonation of the bilin system is a linear concentration gradient of imidazole from 30 mM to 430 mM. Apo- maintained by appropriate positioning of the propionate C12, RcaE was purified in the same way after expression in C41 cells that lacked which is stabilized by Lys261. the PCB biosynthetic plasmid pKT271 (41). Expression of the 655-residue Our study of the protochromic photocycle and previous analyses RcaE construct in E. coli failed to yield detectable protein, whereas the 705- of two-Cys photocycles (21, 23, 25, 26, 29) illustrate a common residue RcaE construct was expressed readily in E. coli. For kinase activity molecular theme for CBCRs: bilin photoisomerization does not experiments, the 705 amino-residue construct was purified further by size- itself induce a spectral change, but rather triggers a subsequent exclusion column chromatography (Sephadex G-25) in disruption buffer light-independent chemical reaction that induces a dramatic supplemented with 1 M NaCl to prevent protein aggregation. Kinase assays were performed as described previously (16). NpR6012g4 was expressed in spectral shift. Interestingly, the secondary chemical reaction E. coli and purified as described (53). requires only a few residues within the GAF domain (e.g., the protochromic triad or the second Cys), providing a powerful Absorption and CD Spectroscopy. Absorption spectra were acquired at 25 °C mechanism for evolution of CBCRs with distinct spectral prop- using a Cary 50 spectrophotometer modified as described (42). Band-pass erties. This work also provides a potential explanation for the filters used for triggering saturating photochemistry were 535 nm center/ spectral sensitivity of members of the red/green CBCR subfamily, 70 nm width and 670 nm center/40 nm width. CD spectra were acquired using proteins that typically exhibit a red/green photocycle, which is the an Applied Photophysics Chirascan with a 2-nm bandwidth, as described (45). opposite of that seen in green/red CBCRs (17, 24). Such CBCRs All photochemical difference spectra are reported as (15Z − 15E).

also might change the bilin protonation state to induce changes in 15Z pH Titration Assays. Purified protein in the Pg state was diluted to give their absorption spectra. Indeed, L249H RcaE undergoes photo- 15Z a peak absorbance of ∼0.3 on the green band in a volume of 10 mL and then conversion between protonated Pr and an orange-absorbing 15E dialyzed for 10 h in 1 L of 1 M NaCl. Dialyzed samples were passed through species, mimicking the photocycle of the atypical red/green a 0.20-μm filter and then irradiated with green or red light to completely CBCR NpR1597g4 (24). In such a model, the conserved Asp photoisomerize the protein to the 15Z or 15E state. Subsequent manipu- residue of red/green CBCRs (Fig. S1, red box) might play a role lations were performed in the dark. Each protein sample (450 μL) was similar to that proposed for Glu217 in RcaE. transferred into an optical cuvette, and 50 μL of buffer was added. Buffers In this study, we demonstrated that the light-regulated kinase were 1 M sodium citrate·HCl for pH 3.0–4.5, 1 M MES·NaOH for pH 5.0–6.5, activity of RcaE in vitro is consistent with previous in vivo 1 M Hepes·NaOH for pH 7.0–8.5, and 1 M glycine·NaOH for pH 9.0–11.0. – Samples were mixed by gentle pipetting, and absorption spectra were analyses (37 39, 43, 48). Recent studies have shown that RcaE is μ involved not only in CCA but also in regulation of chlorophyll recorded immediately. After each measurement, 200 L of each titrated sample was mixed with 800 μL of 10 M urea (pH 2.0) to confirm that the pH synthesis and cellular morphology (49, 50). Our pH titration change did not alter the configuration of the C15-C16 double bond. For pH study shows that green absorption in CCA sensors is lost by the titration under denaturing conditions, 50 μL of one of the aforementioned 15Z state at low pH, whereas red absorption is lost by the 15E buffers and 400 μL of 7.5 M guanidinium chloride were added to 50 μLof state at high pH. Therefore, an efficient green/red photocycle dialyzed protein samples to give a final concentration of 6 M guanidinium can occur only in a narrow pH range from 6.5 to 7.5 (Fig. S3J), chloride. To analyze the pH titration data, absorbance values at selected perhaps allowing CCA sensors to integrate the incident light wavelengths were fit by nonlinear regression in KaleidaGraph (Synergy quality with the intracellular pH. Similarly, it has been suggested Software) to equations describing the titration of one or two groups, with fi that two-Cys CBCRs can sense the cellular redox potential via all species having nonzero extinction coef cients. Equations are described in SI Data Analysis. oxidation of the second Cys, which arrests the photocycle (21). There also are many examples of tandem CBCR sensors con- In Vitro Assembly Reactions. PCB was purified from powdered Spirulina cells taining multiple GAF domains that perceive light of various as described (45). 15Za-PCB was synthesized as described (47). For in vitro colors (1, 2, 14, 19, 23, 24). CBCRs thus are exceptionally so- reconstitution, apo-RcaE (3.3 μM) in disruption buffer was supplemented phisticated photosensors, able to integrate light with various with 1 mM Tris(2-carboxyethyl)phosphine. PCB or 15Za-PCB was added to

Hirose et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 16.5 μM, followed by a 2-h incubation in the dark and overnight dialysis to Katayama, Dr. Takami Ishizuka, and Dr. Takashi Shimada for helpful discussion. remove free bilin. Covalent binding of PCB and 15Za-PCB was confirmed by This work was supported by Grants-in-Aid for Scientific Research from the SDS/PAGE and zinc blotting (54). Japanese Society for the Promotion of Science 23370014 (to M.I.) and 09J09146 (to Y.H.); and by Grant DOE DE-FG02-09ER16117 from the Chemical Sciences, fi fi ACKNOWLEDGMENTS. We thank Dr. Takayuki Kohchi for the kind gift of the Geosciences, and Biosciences Division, Of ce of Basic Energy Sciences, Of ce of PCB-producing plasmid pKT271. We also thank Dr. David Kehoe, Dr. Mitsunori Science, US Department of Energy (to J.C.L.), which supported N.C.R. and J.C.L.

1. Rockwell NC, Lagarias JC (2010) A brief history of phytochromes. ChemPhysChem 28. Narikawa R, et al. (2013) Structures of cyanobacteriochromes from phototaxis regu- 11(6):1172–1180. lators AnPixJ and TePixJ reveal general and specific photoconversion mechanism. Proc 2. Auldridge ME, Forest KT (2011) Bacterial phytochromes: More than meets the light. Natl Acad Sci USA 110(3):918–923. Crit Rev Biochem Mol Biol 46(1):67–88. 29. Rockwell NC, Martin SS, Lagarias JC (2012) Mechanistic insight into the photosensory 3. Montgomery BL, Lagarias JC (2002) Phytochrome ancestry: Sensors of bilins and light. versatility of DXCF cyanobacteriochromes. Biochemistry 51(17):3576–3585. Trends Plant Sci 7(8):357–366. 30. Yoshihara S, Suzuki F, Fujita H, Geng XX, Ikeuchi M (2000) Novel putative photore- 4. Wagner JR, Brunzelle JS, Forest KT, Vierstra RD (2005) A light-sensing knot revealed ceptor and regulatory genes required for the positive phototactic movement of the by the structure of the chromophore-binding domain of phytochrome. Nature unicellular motile cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol – 438(7066):325–331. 41(12):1299 1304. 5. Rockwell NC, Su YS, Lagarias JC (2006) Phytochrome structure and signaling mecha- 31. Bhaya D, Takahashi A, Grossman AR (2001) Light regulation of type IV pilus-de- nisms. Annu Rev Plant Biol 57:837–858. pendent motility by chemosensor-like elements in Synechocystis PCC6803. Proc Natl – 6. Essen LO, Mailliet J, Hughes J (2008) The structure of a complete phytochrome sensory Acad Sci USA 98(13):7540 7545. module in the Pr ground state. Proc Natl Acad Sci USA 105(38):14709–14714. 32. Narikawa R, Suzuki F, Yoshihara S, Higashi S, Watanabe M, Ikeuchi M (2011) Novel 7. Song C, et al. (2011) Two ground state isoforms and a chromophore D-ring photoflip photosensory two-component system (PixA-NixB-NixC) involved in the regulation of triggering extensive intramolecular changes in a canonical phytochrome. Proc Natl positive and negative phototaxis of cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 52(12):2214–2224. Acad Sci USA 108(10):3842–3847. 33. Song JY, et al. (2011) Near-UV cyanobacteriochrome signaling system elicits negative 8. Yang X, Ren Z, Kuk J, Moffat K (2011) Temperature-scan cryocrystallography reveals phototaxis in the cyanobacterium Synechocystis sp. PCC 6803. Proc Natl Acad Sci USA reaction intermediates in bacteriophytochrome. Nature 479(7373):428–432. 108(26):10780–10785. 9. Yang X, Kuk J, Moffat K (2008) Crystal structure of Pseudomonas aeruginosa 34. Savakis P, et al. (2012) Light-induced alteration of c-di-GMP level controls motility of bacteriophytochrome: Photoconversion and signal transduction. Proc Natl Acad Sci Synechocystis sp. PCC 6803. Mol Microbiol 85(2):239–251. USA 105(38):14715–14720. 35. Kehoe DM, Gutu A (2006) Responding to color: The regulation of complementary 10. Strauss HM, Hughes J, Schmieder P (2005) Heteronuclear solution-state NMR studies chromatic adaptation. Annu Rev Plant Biol 57:127–150. of the chromophore in cyanobacterial phytochrome Cph1. Biochemistry 44(23): 36. Tandeau de Marsac N (1977) Occurrence and nature of chromatic adaptation in cy- 8244–8250. anobacteria. J Bacteriol 130(1):82–91. 11. Hahn J, Kühne R, Schmieder P (2007) Solution-state (15)N NMR spectroscopic study of 37. Kehoe DM, Grossman AR (1997) New classes of mutants in complementary chromatic alpha-C-phycocyanin: Implications for the structure of the chromophore-binding adaptation provide evidence for a novel four-step phosphorelay system. J Bacteriol – pocket of the cyanobacterial phytochrome Cph1. ChemBioChem 8(18):2249 2255. 179(12):3914–3921. 12. von Stetten D, et al. (2007) Highly conserved residues Asp-197 and His-250 in Agp1 38. Terauchi K, Montgomery BL, Grossman AR, Lagarias JC, Kehoe DM (2004) RcaE is fi phytochrome control the proton af nity of the chromophore and Pfr formation. a complementary chromatic adaptation photoreceptor required for green and red – J Biol Chem 282(3):2116 2123. light responsiveness. Mol Microbiol 51(2):567–577. 13. Yoshihara S, Katayama M, Geng X, Ikeuchi M (2004) Cyanobacterial phytochrome-like 39. Li L, Kehoe DM (2005) In vivo analysis of the roles of conserved aspartate and histidine PixJ1 holoprotein shows novel reversible photoconversion between blue- and green- residues within a complex response regulator. Mol Microbiol 55(5):1538–1552. absorbing forms. Plant Cell Physiol 45(12):1729–1737. 40. Hirose Y, Narikawa R, Katayama M, Ikeuchi M (2010) Cyanobacteriochrome CcaS 14. Ikeuchi M, Ishizuka T (2008) Cyanobacteriochromes: A new superfamily of tetra- regulates phycoerythrin accumulation in Nostoc punctiforme, a group II chromatic pyrrole-binding photoreceptors in cyanobacteria. Photochem Photobiol Sci 7(10): adapter. Proc Natl Acad Sci USA 107(19):8854–8859. 1159–1167. 41. Mukougawa K, Kanamoto H, Kobayashi T, Yokota A, Kohchi T (2006) Metabolic 15. Ishizuka T, et al. (2006) Characterization of cyanobacteriochrome TePixJ from a engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or thermophilic cyanobacterium Thermosynechococcus elongatus strain BP-1. Plant Cell phycoerythrobilin chromophore in Escherichia coli. FEBS Lett 580(5):1333–1338. Physiol 47(9):1251–1261. 42. Rockwell NC, Shang L, Martin SS, Lagarias JC (2009) Distinct classes of red/far-red 16. Hirose Y, Shimada T, Narikawa R, Katayama M, Ikeuchi M (2008) Cyanobacter- photochemistry within the phytochrome superfamily. Proc Natl Acad Sci USA 106(15): iochrome CcaS is the green light receptor that induces the expression of phycobili- 6123–6127. some linker protein. Proc Natl Acad Sci USA 105(28):9528–9533. 43. Kehoe DM, Grossman AR (1996) Similarity of a chromatic adaptation sensor to 17. Narikawa R, Fukushima Y, Ishizuka T, Itoh S, Ikeuchi M (2008) A novel photoactive phytochrome and ethylene receptors. Science 273(5280):1409–1412. GAF domain of cyanobacteriochrome AnPixJ that shows reversible green/red pho- 44. Sazuka T, Ohara O (1996) Sequence features surrounding the translation initiation toconversion. J Mol Biol 380(5):844–855. sites assigned on the genome sequence of Synechocystis sp. strain PCC6803 by amino- 18. Narikawa R, Kohchi T, Ikeuchi M (2008) Characterization of the photoactive GAF terminal protein sequencing. DNA Res 3(4):225–232. domain of the CikA homolog (SyCikA, Slr1969) of the cyanobacterium Synechocystis 45. Shang L, Rockwell NC, Martin SS, Lagarias JC (2010) Biliverdin amides reveal roles sp. PCC 6803. Photochem Photobiol Sci 7(10):1253–1259. for propionate side chains in bilin reductase recognition and in holophytochrome – 19. Rockwell NC, Martin SS, Feoktistova K, Lagarias JC (2011) Diverse two-cysteine pho- assembly and photoconversion. Biochemistry 49(29):6070 6082. tocycles in phytochromes and cyanobacteriochromes. Proc Natl Acad Sci USA 108(29): 46. Inomata K, et al. (2005) Sterically locked synthetic bilin derivatives and phytochrome 11854–11859. Agp1 from Agrobacterium tumefaciens form photoinsensitive Pr- and Pfr-like adducts. – 20. Chen Y, et al. (2012) Photophysical diversity of two novel cyanobacteriochromes with JBiolChem280(26):24491 24497. 47. Nishiyama K, et al. (2010) Total syntheses of sterically locked phycocyanobilin de- phycocyanobilin chromophores: Photochemistry and dark reversion kinetics. FEBS J rivatives bearing a 15Z-anti or a 15E-anti CD-ring component. Bull Chem Soc Jpn 279(1):40–54. 83(11):1309–1322. 21. Enomoto G, Hirose Y, Narikawa R, Ikeuchi M (2012) Thiol-based photocycle of the 48. Li L, Kehoe DM (2008) Abundance changes of the response regulator RcaC require blue and teal light-sensing cyanobacteriochrome Tlr1999. Biochemistry 51(14): specific aspartate and histidine residues and are necessary for normal light color 3050–3058. responsiveness. J Bacteriol 190(21):7241–7250. 22. Ma Q, et al. (2012) A rising tide of blue-absorbing biliprotein photoreceptors: Char- 49. Bordowitz JR, Montgomery BL (2008) Photoregulation of cellular morphology during acterization of seven such bilin-binding GAF domains in Nostoc sp. PCC7120. FEBS J complementary chromatic adaptation requires sensor-kinase-class protein RcaE in 279(21):4095–4108. Fremyella diplosiphon. J Bacteriol 190(11):4069–4074. 23. Rockwell NC, Martin SS, Gulevich AG, Lagarias JC (2012) Phycoviolobilin formation 50. Shui J, et al. (2009) Light-dependent and light-independent protochlorophyllide ox- and spectral tuning in the DXCF cyanobacteriochrome subfamily. Biochemistry 51(7): idoreductases in the chromatically adapting cyanobacterium Fremyella diplosiphon – 1449 1463. UTEX 481. Plant Cell Physiol 50(8):1507–1521. 24. Rockwell NC, Martin SS, Lagarias JC (2012) Red/green cyanobacteriochromes: Sensors 51. Gutu A, Alvey RM, Bashour S, Zingg D, Kehoe DM (2011) Sulfate-driven elemental – of color and power. Biochemistry 51(48):9667 9677. sparing is regulated at the transcriptional and posttranscriptional levels in a fila- 25. Rockwell NC, et al. (2008) A second conserved GAF domain cysteine is required for the mentous cyanobacterium. J Bacteriol 193(6):1449–1460. blue/green photoreversibility of cyanobacteriochrome Tlr0924 from Thermosy- 52. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: Mutant nechococcus elongatus. Biochemistry 47(27):7304–7316. hosts that allow synthesis of some membrane proteins and globular proteins at high 26. Ishizuka T, et al. (2011) The cyanobacteriochrome, TePixJ, isomerizes its own levels. J Mol Biol 260(3):289–298. chromophore by converting phycocyanobilin to phycoviolobilin. Biochemistry 50(6): 53. Kim PW, et al. (2012) Femtosecond photodynamics of the red/green cyanobacter- 953–961. iochrome NpR6012g4 from Nostoc punctiforme. 1. Forward dynamics. Biochemistry 27. Burgie ES, Walker JM, Phillips GN, Jr., Vierstra RD (2013) A photo-labile thioether 51(2):608–618. linkage to phycoviolobilin provides the foundation for the blue/green photocycles in 54. Berkelman TR, Lagarias JC (1986) Visualization of bilin-linked peptides and proteins in DXCF-cyanobacteriochromes. Structure 21(1):88–97. polyacrylamide gels. Anal Biochem 156(1):194–201.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1302909110 Hirose et al. Downloaded by guest on September 27, 2021