Báo cáo khoa học: Probing protein–chromophore interactions in Cph1 phytochrome by mutagenesis potx

Probing protein–chromophore interactions in Cph1 phytochrome by mutagenesis Janina Hahn 1 , Holger M. Strauss 1 , Frank T. Landgraf 2 , Hortensia Faus Gimene ` z 2 ,Gu ¨ nter Lochnit 3 , Peter Schmieder 1 and Jon Hughes 2 1 Forschungsinstitut fu ¨ r Molekulare Pharmakologie, Berlin, Germany 2 Pflanzenphysiologie, Fachbereich Biologie & Chemie, Justus-Liebig-Universita ¨ t, Giessen, Germany 3 Biochemisches Institut, Fachbereich Medizin, Justus-Liebig-Universita ¨ t, Giessen, Germany Phytochrome photoreceptors play a central role in the regulation of plant development. Phytochromes are red ⁄ far-red photochromic proteins with a covalently bound linear tetrapyrrole (bilin) prosthetic group. In the Pr ground state the chromophore preferentially absorbs red light, this leading to a Z fi E isomeriza- tion around the C 15 –C 16 double bond between rings C and D. Further conformational changes culminate in the formation of Pfr, the signalling state. This prefer- ably absorbs far-red light which converts the pigment back to Pr [1]. The active photoreceptor is formed by the apoprotein taking up and covalently attaching an Keywords biliprotein; photoreceptor; phytochrome; site directed mutagenesis; structure–function studies Correspondence J. Hahn, Forschungsinstitut fu ¨ r Molekulare Pharmakologie, Robert Ro ¨ ssle Str. 10, D-13125 Berlin, Germany Fax: +49 30 94793169 Tel: +49 30 94793316 E-mail: hahn@fmp-berlin.de (Received 28 October 2005, revised 27 January 2006, accepted 3 February 2006) doi:10.1111/j.1742-4658.2006.05164.x We have investigated mutants of phytochrome Cph1 from the cyanobacter- ium Synechocystis PCC6803 in order to study chromophore–protein inter- actions. Cph1D2, the 514-residue N-terminal sensor module produced as a recombinant His6-tagged apoprotein in Escherichia coli, autoassembles in vitro to form a holoprotein photochemically indistinguishable from the full-length product. We generated 12 site-directed mutants of Cph1D2, focusing on conserved residues which might be involved in chromophore– protein autoassembly and photoconversion. Folding, phycocyanobilin-bind- ing and Pr fi Pfr photoconversion were analysed using CD and UV–visible spectroscopy. MALDI-TOF-MS confirmed C259 as the chromophore attachment site. C259L is unable to attach the chromophore covalently but still autoassembles to form a red-shifted photochromic holoprotein. H260Q shows UV–visible properties similar to the wild-type at pH 7.0 but both Pr and Pfr (reversibly) bleach at pH 9.0, indicating that the imidazole side chain buffers chromophore protonation. Mutations at E189 disturbed fold- ing but the residue is not essential for chromophore–protein autoassembly. In D207A, whereas red irradiation of the ground state leads to bleaching of the red Pr band as in the wild-type, a Pfr-like peak does not arise, impli- cating D207 as a proton donor for a deprotonated intermediate prior to Pfr. UV-Vis spectra of both H260Q under alkaline conditions and D207A point to a particular significance of protonation in the Pfr state, possibly implying proton migration (release and re-uptake) during Pr fi Pfr photo- conversion. The findings are discussed in relation to the recently published 3D structure of a bacteriophytochrome fragment [Wagner JR, Brunzelle JS, Forest KT & Vierstra RD (2005) Nature 438, 325–331]. Abbreviations BV, biliverdin IXa; Cph1D2, the N-terminal 1–514 residue sensory module of Cph1 from Synechocystis PCC6803; e, extinction coefficient; FTRR, Fourier transform resonance Raman spectroscopy; FWHM, full width half maximum; IPTG, isopropyl thio-b- D-galactoside; LED, light- emitting diode; MeOH, methanol; Pr ⁄ Pfr, red ⁄ far-red absorbing form of phytochrome; PCB, phycocyanobilin; PFB, phytochromobilin; SAR, specific absorbance ratio; SEC, size-exclusion chromatography; k max , wavelength of the absorbance maximum. FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS 1415 appropriate bilin from the cytoplasm: this process is called autoassembly [2]. Phytochromes are exceedingly effective photoreceptors on account of their high extinction coefficients in the red⁄ far-red region, low fluorescence losses, high resistance to photobleaching and use of a thermodynamically stable signalling state to activate their response pathway. The molecular pro- cesses underlying autoassembly, hyper- and photochro- micity and signal transduction are thus of considerable interest. The unexpected discovery of a prokaryotic phyto- chrome, Cph1 [3,4], fundamentally changed our view of evolution and function of this class of photorecep- tors, relating them to histidine sensor kinases, a pro- tein family involved in a wide variety of perception systems in prokaryotes, fungi and plants [5]. Cph1 has numerous features in common with plant phyto- chromes. Furthermore, large amounts of pure, highly concentrated holoCph1 can easily be produced by apo- protein overexpression in Escherichia coli and in vitro autoassembly with an appropriate bilin [6]. HoloCph1 can also be produced in E. coli by coexpressing haem oxygenase and appropriate bilin reductase genes together with Cph1 [7,8]. Cph1 is thereby well suited to studies of autoassembly as well as of the photocon- version mechanism. Numerous related photoreceptors have subsequently been identified in prokaryotes, nota- bly bacteriophytochrome from Deinococcus radiodu- rans, DrBphP, the 3D structure of whose N-terminal domain was recently published [9]. Phytochrome sequences show highly conserved regions probably representing functionally essential subdomains [6,10]. The UV-Vis absorbance and vibra- tional spectroscopic characteristics of phytochromes assembled with the same chromophore are remarkably similar, whereas significant and characteristic changes are associated with subtle changes in the bilin pros- thetic group. It was thus expected that the pocket in which the chromophore is held is constructed from various functionally conserved subdomains reflected at the sequence level in all phytochromes. The new X-ray structure [9] indeed bears this out although it must be born in mind that DrBphP differs functionally from plant-like phytochromes in many respects and that the fragment crystallized is photochemically impotent. In oat phytochrome A the phytochromobilin (PFB) chromophore is attached by a thioether link to C322 #380 [11–13], a residue conserved in plant-type phytochromes including Cph1 but not in bacteriophytochromes (the residue number is that of the named phytochrome, # indicating its position in the alignment at www. uni-giessen.de/gf1251/Phytochrome/align2x.htm). Phyco- cyanobilin (PCB) is probably the native Cph1 chromophore [14], but no direct evidence for its expec- ted attachment at C259 #380 has been published [5]. A substitution at this putative ligation site should abolish covalent attachment, but not necessarily other protein– bilin interactions, as studies with blocking reagents and of autoassembly kinetics have implied [15–17]. In free PCB at neutral pH 7 the two central ring nitrogens share a single proton, but a second is added under acid conditions. Protonation occurs during phytochrome autoassembly too, but the donor is unknown. Con- versely, homology studies implied that a basic residue homologous to R254 #375 close to the presumed chromo- phore attachment site interacts with the propionate side chain of chromophore ring B [18,19]. Additionally, the strength and position of the dominant red and far-red absorbance bands of Pr and Pfr, respectively, are pH-dependent, an H residue near the chromophore being implicated [20]. H260 #381 adjacent to the putative ligation site is perfectly conserved and hence a prime candidate for this function. Such conserved interactions probably central to phytochrome action can be probed by modifying the protein moiety via site-directed mutagenesis of the cog- nate gene [21–25], with the important proviso that, except in the case of null phenotypes, all conclusions based purely on site-directed mutagenesis are confoun- ded by unknown possible side-effects on folding. Ide- ally, the mutations are guided by 3D structural data. Such information for phytochrome [9] were not avail- able at the time of this study. The N-terminal 514 residue sensory module of recombinant Cph1 ) that is, Cph1D2 ) is photochemi- cally autonomous. We generated 12 amino acid replacement mutants in Cph1D2 and analysed their expression, autoassembly, UV-Vis absorbance, photo- chromicity and thermal reversion properties. We also used CD spectroscopy to detect gross changes in sec- ondary structure: only correctly folded products were considered to offer interpretable information. We pro- vide the first direct evidence that the PCB chromo- phore is ligated to C259 #380 , analogously to oat phyA, and also describe the effects of mutations at this resi- due. The H260 #381 Q mutant showed massive, reversible pH effects on the absorbance spectra, obliterating the characteristic Pfr peak, implying that the imidazole side chain buffers chromophore protonation, partic- ularly in the case of Pfr. A perhaps related effect was seen for the conserved acidic residue D207 #328 : when this was replaced by A, although Pr (reversibly) photo- bleached, no Pfr-like peak was formed in its place. Mutations of R254 #375 had similar small effects on UV-Vis properties: that the R residue is nevertheless perfectly conserved implies a role in signal transduc- Protein–chromophore interactions in Cph1 J. Hahn et al. 1416 FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS tion. We also show that E189 #130 is not required for covalent autoassembly, as had been proposed. We dis- cuss these findings in relation to phytochrome function and the bacteriophytochrome X-ray structure [9] which has subsequently become available. Results Characterization of Cph1 D1 and D2 At the start of this work two deletion clones with C-terminal His6-tags were created, Cph1D1 and Cph1D2, in which the N-terminal 492 and 514 resi- dues, respectively, of Cph1 were overproduced as apo- proteins in E. coli , autoassembled with and purified by nickel affinity chromatography. Although most Cph1D1 was expressed as insoluble inclusion bodies so that the final yield of soluble apoprotein was only  500 lgÆL )1 culture, addition of PCB resulted in covalent autoassembly (as apparent from Zn 2+ - induced bilin fluorescence in SDS ⁄ PAGE) and red ⁄ far-red photochromicity. The Pfr-like band was weak and significantly blue-shifted, however, in comparison to full-length Cph1 (Fig. 1); an effect was also seen in a similar deletion mutant [26]. In contrast, Cph1D2 yielded up to 80 mg apoproteinÆL )1 culture and showed a difference spectrum almost identical to that of full-length Cph1 (Fig. 1), confirming the results of Yeh et al. [4]. Coomassie-stained SDS ⁄ PAGE indicated a purity of  80% for Cph1D2at this stage. Further purification via Superose 200 (Amersham Pharmacia ⁄ GE) size-exclusion chromato- graphy (SEC) yielded essentially pure holoprotein. Cph1D2, unlike full-length Cph1, shows no tendency to aggregate in vitro (data not shown). The extinction coefficient of Cph1D2 Pr was 82 mm )1 Æcm )1 at 654 nm (kmax), a value of 85 mm )1 Æcm )1 for full-length Cph1 confirming that earlier reported [16]. Further UV-Vis data are summar- ized in Table 1. Extinction coefficients for free PCB were 16, 20, 29, 30 and 46 mm )1 Æcm )1 at each kmax in Tris ⁄ HCl pH 7.8, MES pH 5.5, sodium acetate pH 3.0, 0.5 m HCl pH 0.3 and CH 3 Cl ⁄ HCl (1 : 19), respectively. The relevant UV-Vis spectra are shown in Fig. 2. The maximal 654 nm ⁄ 280 nm specific absorb- ance ratio (SAR) of Cph1D2 Pr obtained was 1.3, sig- nificantly higher than that for full-length Cph1 at equivalent purity (1.0 [16]). e 280 nm was calculated to be 59 and 83 mm )1 Æcm )1 for Cph1D2 and full-length Cph1 apoproteins, respectively (Vector NTI, Infor- max). The contribution of PCB attached to the holo- protein is about 5 mm -1 Æcm -1 [16], yielding 64 and 88 mm )1 Æcm )1 at 280 nm for the holoproteins. Taking the Pr e kmax in the red region from Table 1, the max- imal SAR would be 1.28 and 0.98, respectively, in close agreement with that of our purest samples. Quantum efficiencies of photoconversion were not measured directly, but kinetics under red and far-red irradiation were similar for Cph1D2 and full-length Cph1 holoproteins. As the e-values are similar, we thus expect quantum efficiencies to be similar too, that is  0.16 in each direction [6,20]. A maximal 0.70 mole fraction of Pfr at photoequilibrium in red light is seen in Cph1D2 as in full-length Cph1: the calculated UV-Vis spectrum for 100% Pfr derived from this is identical to that of purified Cph1D2 Pfr (unlike full-length Cph1, Cph1D2 as Pr is monomeric except at very high (> 10 mm) concentrations; the Pfr form, however, homodimerises, readily allowing it to be purified by SEC [27]). Dark reversion is insignificant: none was detected after 2 weeks at 20 °C (data not shown). λ [nm] ∆A 500 600 700 800 -1.0 -0.5 0.0 0.5 1.0 Fig. 1. UV-Vis difference spectra of full-length Cph1 (n) and deletion mutants Cph1D1(d) and Cph1D2(m). Table 1. Summary of UV-Vis absorbance data for full-length Cph1 and the deletion mutant Cph1D2. ND, not determined; ibp, isosbe- stic point. Parameter Cph1 (full-length) Cph1D2 (N514D) Pr Pfr Pr Pfr k max, red 656 nm 704 nm 654 nm 702 nm k max, UV ⁄ A 359 nm ND 358 nm ND e at k max, red 86 mM )1 Æcm )1 ND 82 mM )1 Æcm )1 ND k DA,max 655 nm 707 nm 655 nm 707 nm k DA,0 , ibp 677 nm 677 nm SAR (A 655 ⁄ A 280 ) 1.0 1.3 Molecular mass (Apoprotein + His tag) 85.3 kDa 58.7 kDa J. Hahn et al. Protein–chromophore interactions in Cph1 FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS 1417 Direct determination of the chromophore attachment site Tryptic fragments of PCB-Cph1D2 holoprotein were separated by HPLC, the chromopeptide eluting as a single peak with a spectrum closely fitting that expec- ted for protonated PCB covalently linked via ring A to the peptide (Fig. 3A). The chromopeptide showed weak 214 nm absorbance, implying a poor release efficiency. MALDI-TOF MS of this fraction showed major peaks ([M + H] + ) with a typical isotopic profile correspond- ing to predicted tryptic fragments 98–112 (m ⁄ z 1753.849 and in the methionine oxidized form at m ⁄ z 1769.851), 64–80 (m ⁄ z 1951.986) and 399–420 (m ⁄ z 2534.271) of Cph1 (Fig. 3B). The expected PCB-coupled chromopep- tide SAYHC*HLTYLK (residues 255–279) is predicted to have a molecular mass of 1920.923 Da. A small but distinct double peak corresponding to the expected [M + H] + at m ⁄ z 1921.9384 and to [M–H] + at m ⁄ z 1919.929 was seen, the latter probably presenting an oxidized derivative (a similar effect was seen in a study of Agp1 where the Biliverdin IXa (BV) chromopeptide ion detected was also 2 Da lighter than expected [28]). MS 2 analysis of the double peak showed fragment ions at m ⁄ z 585.991 ⁄ 587.939 (reflecting the expected and A B Fig. 2. pH dependence of PCB UV-Vis absorption spectra. (A) Absorption spectra of free PCB in different buffers at different pH-values. Spectra are plotted for PCB in 100 m M Tris ⁄ HCl pH 7.7 (n), 10 m M MES pH 5.5 (d), 5 mM sodium acetate pH 3.0 (m), 0.5 M HCl pH 0.3 (.), HCl ⁄ MeOH (1 : 19) (e) and CH 3 Cl ⁄ HCl (1 : 19) (n). For comparison the absorption spectrum of Cph1D2in the Pr state is shown (dotted line). (B) pH difference spectra for free PCB. Absorbance changes are plotted for pH values 5.5, 3.0, 0.3 (solid lines) and HCl ⁄ MeOH (dashed line) after subtraction of the pH 7.7 spectrum. A B C Fig. 3. Tryptic profiles and MALDI spectra of Cph1D2. (A) HPLC elution profiles at 214 nm (peptide absorbance, upper panel) and 370 nm (bilin UV ⁄ A absorbance, lower panel); inset: UV-Vis-spec- trum of chromopeptide peak. (B) MALDI-TOF spectrum of chromo- peptide fraction; inset: enlarged. (C) MALDI-TOF ⁄ TOF spectrum; inset: enlarged. Protein–chromophore interactions in Cph1 J. Hahn et al. 1418 FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS oxidized forms of the cleaved chromophore) and 1336.915 (reflecting the peptide backbone) (unlike full- length Cph1, Cph1D2 as Pr is monomeric except at very high (> 10 mm) concentrations; the Pfr form, however, homodimerizes readily allowing it to be purified by SEC [28]). Edman microsequence data (not shown) from the same fraction were consistent with the sequences of the fragments identified in MALDI: the fifth residue of the chromopeptide ) C259 ) was absent, as would be expected for a cysteinyl–PCB complex. Taken together these data show that the PCB chromophore is ligated to C259 via a thioether bond. Characterization of Cph1 D2 site-directed mutants To determine the role of specific conserved residues in the Synechocystis phytochrome Cph1, 12 site-directed mutations were introduced into the N-terminal sensory module Cph1D2. The mutants were heterologously expressed as C-terminally His6-tagged apoproteins in E. coli, purified and tested for PCB-binding, apopro- tein folding, Pr–Pfr photochromicity and thermal reversion using SDS ⁄ PAGE ⁄ zinc fluorescence and CD and UV-Vis spectroscopy. The appropriate data is summarized in Table 2 and in Figs 4 and 5. Y257 #378 , h258 #379 , l261 #382 These residues lie close to the chromophore binding site but are not conserved in other phytochromes and are thus, in contrast to conserved residues, probably not functionally important. Indeed, the Y257H, H258F and L261A holoproteins showed no significant differences in chromophore autoassembly, UV-Vis or CD properties relative to the wild-type (Table 2). C259 #380 As MALDI studies showed, this is the residue in Cph1 to which PCB becomes attached via a thioether bond. Thus mutations at C259 should abolish covalent attach- ment and have dramatic effects on photochemistry. Both C259M and C259L mutants autoassembled with PCB to give red ⁄ far-red photochromic holoproteins although, as expected, covalent attachment did not occur (Figs 4 and 6). The autoassembly reaction was much slower than in the wild-type especially under nonreducing conditions, taking many hours for chromophore binding and photochromicity to become saturated even with a large PCB molar excess (as seen in [29]). After brief incubation of apoprotein with a small molar excess of PCB under nonreducing condi- tions, holoC259L showed an almost symmetrical differ- ence spectrum following red irradiation, with lowest energy bands at 674 and 735 nm, representing a  25 nm bathochromic shift relative to the wild-type. Subsequent irradiation with FR did not repopulate the Pr-like species, however (Fig. 6A,B). HoloC259L allowed to assemble to completion under reducing con- ditions showed similarly shifted lowest energy bands. Table 2. Characterization of Cph1D1, Cph1D2 and Cph1D2-mutants. ND, not determined; ibp, isosbestic point. Mutation Soluble expression relative to Cph1D2 a CD like wild-type Cph1D2 Covalent PCB attachment Difference spectrum Absorption spectrum k max (Pr) [nm] k DA,0 ibp [nm] k max (Pfr) [nm] k max (Pr) [nm] k max (Pfr) [nm] Cph1D1 + ND Yes 649 676 695 ND ND Cph1D2 + + + Yes Yes 655 677 706 654 702 E189A – – – – – – – – E189Q + No Yes – – – 665 – D207A + + Yes Yes 653 – – 653 – D207N + ND Yes 653 – – ND ND (+ E196G) R254A + Yes Yes 645 667 702 645 705 R254K + + Yes Yes 644 668 704 647 702 Y257H + Yes Yes 654 677 707 664 702 H258F + ND yes 651 676 706 ND ND C259L + Yes no 674 704 736 683 734 C259M + ND no 664 702 731 ND ND H260F – – – – – – – – H260Q + + Yes Yes 643 673 698 639 700 L261A + ND Yes 644 674 705 ND ND a Expression yield of Cph1D2: 80 mgÆL )1 culture: + + +, 100 – 50%; + +, 40–10%; +, < 10%; –, insoluble expression. J. Hahn et al. Protein–chromophore interactions in Cph1 FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS 1419 The Pr peak was much weaker than that of Pfr, but this photochromicity was now stable (Fig. 6C,D). Attempts to remove unbound bilins by chromatography lead to chromophore escape as all reversibility was lost. H260 #381 This residue is perfectly conserved in all phytochromes, even those in which the canonical C #380 attachment site itself is missing. The H260Q mutant of Cph1 D2 bound PCB covalently (Fig. 4A) to give an only slightly blue-shifted Pr absorbance maximum at 639 nm (Fig. 4B). As an H residue imidazole side chain was expected to be involved in (de)protonation of the chromophore [20], we measured absorption spectra of Cph1D2 wild-type and H260Q holoproteins at different pH values following far-red and red irradi- ation, uncovering a remarkable phenotype (Fig. 7). At pH 7 the spectra of the mutant and wild-type Pfr forms were similar (kmax 700 nm and 703 nm for the lowest energy bands, respectively) while mutant Pr was  14 nm downshifted (kmax 641 nm and 656 nm, respectively). At pH 9, however, the mutant behaved differently from the wild-type: Pr-typical red absorb- ance band weakened almost 10-fold while that of Pfr disappeared completely. Weaker bands at 549 nm and 577 nm, respectively, appeared in their place. The effect was fully reversed by returning the pigment to pH 7. Not surprisingly, far-red irradiation of the bleached form at pH 9 induced no photochemistry, whereas 550 nm irradiation of the bleached ground state did lead to photoconversion as the product revealed itself as Pfr once the pH 7 was restored (data not shown). D207 #328 This acidic amino acid is conserved in all phyto- chromes and might be involved in chromophore proto- nation. CD spectroscopy showed that the D207A replacement was similarly folded to the wild-type, binding PCB covalently to form an apparently normal Pr state (k max at 653 nm, Figs 4 and 5). Upon red irra- diation the Pr band bleached as in the wild-type, but no Pfr-like peak appeared. The Pr-like form reap- peared in darkness, however, reversion being complete within an hour (Fig. 8). The extinction coefficient of D207A was estimated to be 62.9 mm )1 Æcm )1 .A D207N ⁄ E196G double mutant behaved similarly (Table 2). R254 #375 As this residue is perfectly conserved in plant as well as prokaryotic phytochromes, it is likely to be func- tionally important. Therefore R254 was mutated to K and to A. Whereas the CD spectrum of the conserva- tive K mutant was almost identical to that of the wild- Coomassie wt D207A R254K H260Q C259L E189Q Zn-fluorescence A B Fig. 4. Cph1D2 and site-directed mutants. (A) Coomassie stain and Zn 2+ fluorescence of Cph1D2 and of selected Cph1D2 site-directed mutants after SDS ⁄ PAGE. (B) UV-Vis difference spectra of Cph1D2 and of selected Cph1D2 site-directed mutants. Absorbance differ- ence maxima and isosbestic points are given. The dotted vertical lines are drawn through the absorption maxima of the Pr and Pfr state of Cph1D2 to highlight shifts in the mutants. Protein–chromophore interactions in Cph1 J. Hahn et al. 1420 FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS type, the CD spectrum of R254A implied slight folding differences (Fig. 5). Nevertheless, both bound PCB covalently to form a red⁄ far-red photochromic holo- protein with the red kmax of Pr  10 nm downshifted but Pfr spectra indistinguishable from that of the wild- type. E189 #310 This acidic residue was the focus of earlier mutagenesis studies which inferred a central role in bilin ligation [25]. Our E189A mutant was expressed as insoluble protein bodies, attempts at refolding by solubilization in urea followed by slow dialysis proving unsuccessful. The E189Q mutation was better tolerated although the expression yield of soluble protein was much lower than for the Cph1D2 wild-type. CD spectroscopy implied, furthermore, that folding was significantly dif- ferent from that of the wild-type. However, when this mutant apoprotein was presented with PCB, a low level of covalent attachment accompanied by a weak Pr-like band at 665 nm was seen (Figs 4A and 9), implying normal protonation of a thioether-linked bilin. No photochromicity signal associated with red ⁄ far-red irradiation was measurable, however. Discussion In this study we focused on Cph1D2, the N-terminal 514 residue sensory module of Cph1. The smaller dele- tion product, Cph1D1 (N1–492) was functionally compromised (Table 1 and Fig. 1), showing very poor solubility and a weak Pfr-like absorbance typical of phytochromes in which the PHY subdomain (see http://www.sanger.ac.uk//Software/Pfam) is incom- plete. On the other hand the UV-Vis absorbance prop- erties of holoCph1D2 ) whose C-terminus corresponds exactly to that of the PHY subdomain ) closely resem- ble those of full-length holoCph1 (see Table 1 and Fig. 1). Thus the sensory module is photochemically autonomous, as implied in an earlier study [25]. Cph1D2 can therefore be used as a convenient model for investigating phytochrome functions, as ) unlike full-length Cph1 ) it does not aggregate under normal in vitro conditions. Indeed, as pure Pfr can be obtained by SEC [27], it might also be possible to obtain struc- tural data for that form too. UV-Vis absorbance properties of bilins and other tetrapyrroles are determined both by their protonation state and by the extent and linearity of the conjugated p orbital system. Coiled bilins (like free PCB) show a Fig. 5. Circular dichroism spectra of selec- ted mutants. For comparison Cph1D2-wt spectra are shown (dotted lines) and molar ellipticities were calculated. J. Hahn et al. Protein–chromophore interactions in Cph1 FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS 1421 strong UVA band but weak absorbance at longer wavelengths, while in linear bilins the situation is reversed, the dipole moment perpendicular to the long molecular axis giving strong red absorbance at the expense of the UV ⁄ A band (the UV ⁄ A and lowest energy red ⁄ far-red bands are sometimes called Soret Fig. 6. UV-Vis absorbance properties of Cph1D2-C259L in the presence of excess PCB. Absorbance and difference spectra under nonreducing (A,B) and reducing (C,D) conditions. (A,C) (n) Pr[1], after autoassem- bly with PCB in the dark; (d) Pfr (7 ⁄ 3Pfr⁄ Pr mixture) after R irradiation; (h) Pr[2], after FR irradiation. (B, D) (n) Pr[1]–Pfr; (d) Pfr– Pr[2]. A B Fig. 7. pH-dependence of UV-Vis absorbance properties of Cph1D2 and Cph1D 2-H260Q after far-red (100% Pr) and after red (7 ⁄ 3 Pfr ⁄ Pr photoequilibrium) irradiation. (A) Cph1D2 wild-type at pH 7 (after far-red n, after red d) and pH 9 (after far-red h, after red s). (B) Cph1D2-H260Q at pH 7-start (after far-red n, after red, m), pH 9 (after far-red h, after red n ) and at pH 7-end (after far-red r, after red .). A B Fig. 8. UV-Vis absorbance properties of Cph1D2-D207A. (A) Spectra recorded after autoassembly with PCB in the dark (n), after 90 s red irradiation (d) and after 90 s far red irradiation (m). (B) Thermal reversion of Cph1D2-D207A. After PCB assembly in the dark (n) and saturating red irradiation (d), the sample was kept in the dark and absorption spectra were recorded after 10 (m), 20 (.), 30 (r) and 45 (b) min. Protein–chromophore interactions in Cph1 J. Hahn et al. 1422 FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS and Qy in analogy to closed-ring tetrapyrroles; this possibly misleading terminology has been avoided here) [30,31]. While the ratio of the two ‘oscillator strengths’ changes with uncoiling, the total absorptivity remains constant. FTRR (e.g. [32] for oat phyA) gives more specific information about the conformation of the chromophore, as of course can more direct meth- ods like NMR (e.g. [33,34] and Rohmer T. and Matysik J., University of Leiden, the Netherlands, unpublished data). and X-ray crystallography (e.g. [9]). On the other hand, protonation has only subtle effects in the UV ⁄ A region, while the red peak strengthens approximately threefold at low pH [34,35]. In fact, in PCB a new band at 688 nm appears and strengthens with protonation, but its k max does not shift, while the broad, weaker shoulder centred at  615 nm (also seen in phytochrome spectra) remains unchanged (Fig. 2). Recently, Go ¨ ller et al. (2005) [36] have successfully modelled the role of protonation in strengthening e red by enhancing electronic coupling between PCB rings, while NMR studies [33] have proved that all four rings are fully protonated in both Pr and Pfr states of Cph1. The current model for the autoassembly reaction [15,16,37], important to the present study, envisages three-steps: (1) an initial chromophore recognition pro- cess (< 1 ms) of the unprotonated, coiled bilin with weak absorbance in the orange region; (2) entrance into a pocket within the protein (100–200 ms) during which uncoiling and protonation occur, leading to a fourfold hyperchromicity of the lowest energy absor- bance band in red ⁄ far-red and the appearance of photochromicity in that region; (3) a final covalent ligation (1–10 S) to a C residue through the formation of a thioether bond. While pioneering studies showed that PUB is attached to oat phyA at C322 #380 [13] at least some bacteriophytochromes attach a BV chromo- phore at a C residue close to the N-terminus [9,28,38], contradicting earlier data [39]. A further important dif- ference is that bacteriophytochromes covalently ligate to the ring A vinyl side chain of BV, forming a two- carbon linker, while in oat phyA the ring A ethylidene side chain of PFB yields a single-carbon linker. Here we present direct evidence that in Cph1 PCB is simi- larly ligated to C259 #380 (Fig. 3). As shown by our C259L mutant, if step 3 of autoassembly is prevented by mutating this residue, many holophytochrome-like features appear, but k max values are shifted  25 nm bathochromically (Fig. 4) as would be expected if the ethylidene group double bond was left intact to contri- bute to the PCB delocalized p-electron system. This is seen also in the wild-type if C residues are blocked nonspecifically by iodacetamide [16]. No effect is seen with blocked Agp1 and BV, however, in accordance with the vinyl group double bond in that case not being connected to the p-system [40]. Redox conditions seem to be important in autoassembly steps 1 and ⁄ or 2. PCB binding was weak under nonreducing condi- tions, requiring at least 15 lm PCB, and even then only a single round of R ⁄ FR photoconversion was possible – as though the chromophore was lost as a consequence of photoconversion (Fig. 6A,B). Under reducing conditions (Fig. 6C,D) the relative strengths of the UV ⁄ A and red bands were approximately equal, implying a more chiral chromophore conformation than in the wild-type, a conclusion consistent with experiments using methoxy-PCB [41]. Thus conforma- tional changes leading to uncoiling are associated with both step 2 and step 3 of autoassembly. The long wavelength band in the Pfr-like state of the C259L mutant was even weaker than that of the ground state. R #375 near the ligation site is perfectly conserved amongst all known phytochromes, bacteriophyto- chromes and even several other biliproteins. It thus might be expected to be important in chromophore binding and conformation. Indeed, X-ray structural data shows that it forms a salt bridge with the pro- pionate side chain of ring B, apparently pulling on the chromophore from deep within the protein [9,17–19]. However, R #375 I and T mutants of pea phyA showed only  5 nm hypsochromic shifts [22]. Here we mutated R254 #375 to K (likewise a basic residue) and to A (a smaller, moderately hydrophobic residue). Both mutants fold similarly to the wild-type and bind PCB effectively (Figs 4A and 5). Whereas their Pfr absorbance characteristics match those of the wild-type almost exactly, the Pr peak shows a 10-nm hypsochro- mic shift in both cases (Table 2, Fig. 4B). This might Fig. 9. UV-Vis absorbance properties of Cph1D2-E189Q. Spectra recorded after autoassembly with PCB in the dark (n) and subse- quent red irradiation (d). J. Hahn et al. Protein–chromophore interactions in Cph1 FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS 1423 arise from a rotation around the C5–C6 bond specific to the Pr form (H. Scheer, LMU Munich, personal communication), although such a shift is also predicted for (de)protonation of the propionate [36]. Either way, the UV-Vis shift is much too subtle to explain the degree of conservation seen, thus it is very likely that R254 #375 instead plays a central role in signal trans- duction: indeed, the 1ZTU structure implies that even a slight movement of the chromophore would break the salt bridge, fitting with the UV-Vis phenotype of our R254K mutant. Overlooked to date, the X-ray structure shows an intriguing  2.5-A ˚ diameter channel leading from the salt bridge to the other side of the protein, wide enough for water molecules or hydroxo- nium ions. It would in any case be worthwhile investi- gating R254 #375 mutants at the physiological level. Go ¨ ller et al. [36] also calculated that ring B ⁄ C pro- tonation of chiral PCB leads to dramatically increased electron coupling associated with a bathochromic shift of e max of 130 nm: we observe a shift of 160 nm on acidification of free PCB compared with 150 nm for holoCph1 prior to ligation (i.e. in C259L), a reason- able fit considering likely conformation differences. Protonation requires a donor with a pKa of < 4.6, and is thus just possible for E or D carboxyl side chains [35]. None of the 12 highly conserved E or D residues are near C259 #380 in the primary sequence, furthermore, the recent 1ZTU structure shows that only two of these E189 #310 and D207 #328 are posi- tioned anywhere near the bilin. Coincidentally, these are the two residues we had focused on in the present study. As we show, E189Q mutations are better toler- ated than others [25,42], UV-Vis properties being con- sistent with a ground state resembling protonated Pr (Figs 4 and 9, Table 2). Thus the proposed central role for E189 #310 in ligation [25] is unlikely, neither is it likely to be the proton donor in autoassembly step 2. Unfortunately, D207 #328 too is most unlikely to fulfil this role. The mutant apoprotein bound PCB covalent- ly (Fig. 4A) to yield a ground state with a similar e max and k red compared to wild-type, implying a partially coiled, protonated chromophore. Furthermore, 1ZTU shows that the carboxyl group of D207 #328 is directed away from the chromophore, forming a hydrophilic acid patch exposed to the solvent, at least in this BphP deletion mutant. The main chain carbonyl oxygen of D207 #328 interacts with the nitrogens of rings A, B and C, so that they might share their protons – but this is a proton acceptor, not a donor, and of course any mutation at this site could fulfil this role. Our mutants imply that D207 #328 is important in Pfr formation. Red irradiation leads to bleaching (as in the wild-type), but no Pfr-like band appeared, rather a broad peak centred at 590 nm remained. Not surprisingly, FR irra- diation had no effect, but the bleached form reverts thermally to the Pr-like state. D207 #328 N behaved simi- larly (Table 2). As proton exchange is probably associ- ated with photoconversion (see below), D207 #328 might be involved in reprotonation prior to Pfr formation. Such a role would not be apparent from 1ZTU because this cannot form bona fide Pfr. It seems clear, however, that neither E189 #310 nor D207 #328 can be the proton donor in step 2 of autoassembly. Thus the donor for bilin protonation, even in the light of the 1ZTU structure, remains unknown. Although it is now certain that all four chromophore nitrogens are protonated in neutral buffers in both Pr and Pfr states [33], transient deprotonation of the chro- mophore seems to be a feature of Pr fi Pfr photocon- version [20,43,44]. Significant deprotonation of both Pr and Pfr can be induced by shifting the pH, however, one pKa component being close to neutral, thus possibly representing an imidazole (H side chain) and ⁄ or a direct effect on PCB [20]. In the present study we show that H260 #381 plays a crucial role in this process. Mutagen- esis of H260 #381 to L, R, F, G and Q has already been reported for recombinant phytochrome A from pea and oat. In the first four cases covalent ligation and photo- chromicity were obliterated probably because of misfolding, whereas H260 #381 Q retained covalent attachment and photochromicity [21,22,24]. While Q resembles H regarding its steric demands and its hydro- gen bonding ability [45], its buffering capacity is very weak. While our H260Q mutant is wild-type like in its folding, photochromicity and covalent autoassembly under normal conditions (Table 2; Figs 4 and 5), it shows dramatically increased sensitivity to buffer pH (Fig. 7), the long wavelength absorbance peak of Pr weakening drastically and that of Pfr disappearing com- pletely at pH 9.0, much weaker, broader bands centred at 549 nm and 577 nm, respectively, appearing in their place. The UV ⁄ A bands show smaller changes for both states. These effects are fully pH-reversible. We con- clude that H260 #381 plays a crucial role in buffering both Pr and Pfr protonation. As it is easy to titrate chromo- phore protonation in the mutant, this offers a poten- tially useful degree of freedom for more sophisticated analytical methods. H260 #381 is likely to be important according to the 1ZTU X-ray structure [9]. The chromophore nitrogens of rings A, B and C are hydrogen bonded to the D207 #328 backbone nitrogen, perhaps sharing their protons. On the other side of the pocket, the d1 nitro- gen of H260 #381 and chromophore ring C nitrogen are separated by 3.3 A ˚ , just outside van der Waals’ con- tact, but a hydrogen bonding bridge is provided by Protein–chromophore interactions in Cph1 J. Hahn et al. 1424 FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... deprotonation of Protein–chromophore interactions in Cph1 47 48 49 50 51 52 the chromophore in the bleached intermediate Biochemistry 33, 153–158 Lamparter T, Michael N, Caspani O, Miyata T, Shirai K & Inomata K (2003) Biliverdin binds covalently to Agrobacterium phytochrome AgP1 via its ring A vinyl side-chain J Biol Chem 278, 33786–33792 Fischer AJ & Lagarias JC (2004) Harnessing phytochrome s glowing potential... point induces a major change in the chromophore pocket relative to that seen in 1ZTU Of the five residues interacting with the D ring in 1ZTU, two are not conserved in planttype phytochromes Moreover, while mutation of one of these (Y176#297H) in Cph1 gives rise to strong fluorescence [48], this is not seen in bacteriophytochromes [42] The 1ZTU structure is also notable in that, while the A, B and C rings... chromophore was examined by SDS ⁄ PAGE followed by Zn2+-induced fluorescence [6,52] The C259L mutant was handled somewhat differently Incubation of the purified apoprotein with PCB for 10 min resulted in low levels of holoprotein in which Pr fi Pfr photoconversion was compromised Following prolonged incubation in the presence of b-mercaptoethanol, however, fully reversible holoprotein was obtained As subsequent... were then recorded and the corresponding ekmax determined relative to the known value in HCl ⁄ MeOH Holophytochrome Holoproteins were prepared in darkness by mixing the apoprotein at 5–100 lm with a 10-fold molar excess of PCB in TES Subsequent operations were carried out with minimal exposure to 520 nm LED safelight After 10 min incubation free PCB was removed by gel filtration over PD-10 columns (Amersham... N-terminal chromophore binding PAS ⁄ GAF domains), the missing PHY domain precluding the formation of bona fide Pfr There may also be other problems in extrapolating from 1ZTU to the phytochrome family For example, the BV chromophore in 1ZTU is modelled as ZZZssa (but with a 44° C–D ring rotation), more chiral than the ZZZasa expected from recent resonance Raman data for oat phyA [32] Interestingly:... overexpression clones in pQE12 (Qiagen, Hilden, Germany) were generated by PCR using error-checking DNA polymerase (TakaraEx, Otsu, Japan) pF10.His (full-length Cph1 with a C-terminal His-tag) has been described else- FEBS Journal 273 (2006) 1415–1429 ª 2006 The Authors Journal compilation ª 2006 FEBS 1425 Protein–chromophore interactions in Cph1 J Hahn et al where [6] p920.B3 and p926.5 (Cph1D1 and Cph1D2, respectively)... PCC6083 (kindly provided by A Wilde, Humboldt University, Berlin, Germany) following 25 amplification cycles using appropriate header-primers, restriction and ligation into EcoRI and BamHI of pQE12, thereby generating clones to overexpress the N-terminal sensory module of Cph1 with a His6-tag followed by a stop codon immediately downstream of V492 and E514, respectively Site-directed mutagenesis of Cph1D2... (Pbl) appears in place of Pfr Pbl probably represents a deprotonated intermediate (sometimes called Ibl or metaRc) which has been detected in some kinetic and freeze-trapping studies [35,43,46] Both H260#381Q at elevated pH and D207#328A produce a Pbl-like form after red irradiation (Figs 7 and 8), either because they indirectly compromise PHY Protein–chromophore interactions in Cph1 domain function... of mutations in the chromophore pocket of recombinant phytochrome on chromoprotein assembly and Pr-to-Pfr photoconversion Eur J Biochem 266, 201–208 Wu S-H & Lagarias JC (2000) Defining the bilin lyase domain: lessons from the extended phytochrome superfamily Biochemistry 39, 13487–13495 Park CM, Shim JY, Yang SS, Kang JG, Kim JI, Luka Z & Song PS (2000) Chromophore–apoprotein interactions in Synechocystis... Fluorescence investigations of the recombinant cyanobacterial phytochrome (Cph1) and its C-terminally truncated monomeric species (Cph1D2): implication for holoprotein assembly, chromophore– apoprotein interaction and photochemistry J Photochem Photobiol B 67, 39–50 Tasler R, Moises T & Frankenberg-Dinkel N (2005) Biochemical and spectroscopic characterization of the bacterial phytochrome of Pseudomonas aeruginosa . Probing protein–chromophore interactions in Cph1 phytochrome by mutagenesis Janina Hahn 1 , Holger M. Strauss 1 , Frank T. Landgraf 2 ,. impotent. In oat phytochrome A the phytochromobilin (PFB) chromophore is attached by a thioether link to C322 #380 [11–13], a residue conserved in plant-type phytochromes including Cph1 but not in bacteriophytochromes. holoCph1 can easily be produced by apo- protein overexpression in Escherichia coli and in vitro autoassembly with an appropriate bilin [6]. HoloCph1 can also be produced in E. coli by coexpressing