Spectroscopy and a High-Resolution Crystal Structure of Tyr263 Mutants of Cyanobacterial Phytochrome Cph1

J. Mol. Biol. (2011) 413, 115–127

doi:10.1016/j.jmb.2011.08.023 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Spectroscopy and a High-Resolution Crystal Structure of Tyr263 Mutants of Cyanobacterial Phytochrome Cph1 Jo Mailliet 1 , Georgios Psakis 1 , Kathleen Feilke 1 , Vitaly Sineshchekov 2 , Lars-Oliver Essen 3 ⁎ and Jon Hughes 1 ⁎ 1

Institute for Plant Physiology, Justus Liebig University Giessen, Senckenbergstrasse 3, D35390 Giessen, Germany Biology Department, M. V. Lomonosov Moscow State University, Leninskiye Gory 1/12, RE-119992 Moscow, Russia 3 Structural Biochemistry, Philipps University Marburg, Hans-Meerwein-Strasse, D35032 Marburg, Germany 2

Received 29 June 2011; received in revised form 10 August 2011; accepted 11 August 2011 Available online 23 August 2011 Edited by R. Huber Keywords: photobiology; structural biology; photoreceptors; intramolecular signaling; spectroscopy

Phytochromes are biliprotein photoreceptors that can be photoswitched between red-light-absorbing state (Pr) and far-red-light-absorbing state (Pfr). Although three-dimensional structures of both states have been reported, the photoconversion and intramolecular signaling mechanisms are still unclear. Here, we report UV–Vis absorbance, fluorescence and CD spectroscopy along with various photochemical parameters of the wild type and Y263F, Y263H and Y263S mutants of the Cph1 photosensory module, as well as a 2.0-Å-resolution crystal structure of the Y263F mutant in its Pr ground state. Although Y263 is conserved, we show that the aromatic character but not the hydroxyl group of Y263 is important for Pfr formation. The crystal structure of the Y263F mutant (Protein Data Bank ID: 3ZQ5) reaffirms the ZZZssa chromophore configuration and provides a detailed picture of its binding pocket, particularly conformational heterogeneity around the chromophore. Comparison with other phytochrome structures reveals differences in the relative position of the PHY (phytochrome specific) domain and the interaction of the tongue with the extreme N-terminus. Our data support the notion that native phytochromes in their Pr state are structurally heterogeneous. © 2011 Elsevier Ltd. All rights reserved.

Introduction Plants use phytochromes to detect light and to measure the fluence rate difference between red and far-red, the resulting signal regulating many important developmental processes throughout the life cycle. 1 Members of the phytochrome superfamily are also found in eubacteria 2–4 and fungi. 5–7

*Corresponding authors. E-mail addresses: [email protected]; [email protected]. Abbreviations used: PCB, phycocyanobilin; SEC, size-exclusion chromatography; PDB, Protein Data Bank; MAS, magic angle spinning; Mes, 4-morpholineethanesulfonic acid.

In particular, Cph1 from the cyanobacterium Synechocystis 6803 has been the object of intense study. 8–11 Phytochromes use a covalently bound linear tetrapyrrole chromophore to absorb light, acting as bistable (flip-flop) photoswitches with a Pr ground state absorbing preferentially in the red region and a Pfr state absorbing preferentially in the far-red region. Crystal structures of the photosensory modules of prokaryotic phytochromes and bacteriophytochromes reveal a remarkable overall dumbbell structure. The N-terminal PAS (Per Arnt Sim)–GAF (cGMP-specific phosphodiesterase, adenylyl cyclase and FhlA) bidomain knotted around the chromophore 12 is attached to a PHY (phytochrome specific) domain that is held at a distance but nevertheless forms part of the chromophore

0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.

116 pocket with the help of an L-shaped, tongue-like protrusion. 13,14 To date, the protein database [Protein Data Bank (PDB)] entry 2VEA for Cph1 13 provides the only available crystal structure for a plant-type phytochrome photosensor (in which the chromophore is covalently attached to a Cys residue in the GAF domain) and for any photochemically competent superfamily member in the Pr state. 3C2W for PaBphP, 14 on the other hand, is the only available crystal structure for a photochemically competent bacteriophytochrome (in which the chromophore is attached near the N-terminus) and for any photochemically competent superfamily member in the Pfr state. How does Pr photoconvert into Pfr? In red light, the D-ring of the chromophore flips from the Z to the E configuration, leading to Pfr formation. 15,16 The D-ring flops back to its initial state upon absorption of far-red light to reform Pr. However, the photocycle is more complicated than this. The D-ring isomerization and the formation of the lumi-R intermediate is considered to be the first step of the photocycle. 17,18 This initial photoreaction and the subsequent intermediates have been investigated using time-resolved UV–Vis absorbance 18–21 and fluorescence 22,23 and infrared difference 24,25 and resonance Raman 19,26–33 spectroscopies. Upon photon absorption, the excited state relaxes to give a short-lived hot state, which can lead to an unstable pre-lumi-R via an activation energy barrier: this step occurs with a quantum efficiency close to unity. Pre-lumi-R has two possible fates: it can relax to form lumi-R with a quantum efficiency of about 0.15, corresponding to that of the overall Pr → Pfr phototransformation. Alternatively, it can revert to the initial Pr state with a probability of about 0.85. 17 Interestingly, the fast deprotonation that seems to quench lumi-R formation in bacteriophytochromes is not seen in plant phytochromes and Cph1. 21 Recent magic angle spinning (MAS) NMR studies 34 have revealed that the Pr ground state of Cph1 comprises two isoforms and that the facial disposition of the D-ring in Pfr in relation to the rest of the chromophore is different in Cph1 and plant phytochromes from that in bacteriophytochromes, as implied by CD spectroscopy. 35 What is the molecular mechanism underlying the light-driven Pr–Pfr flip-flop? It was expected that two tyrosine residues are essential for Pr → Pfr photoconversion. 36 One of these is Y176, 11,37 below ring D. The second is likely to be either Y203 or Y263, below and above ring D, respectively. Indeed, the 3C2W Pfr structure of the “bathy” bacteriophytochrome PaBphP shows significant shifts of both Y176 and Y203 side chains relative to Pr structures. 14 Y263 has been suggested to play a critical role in Pr → Pfr photoconversion, 12,13,38,39 whereby interactions with the hydroxyl group might be important. The bulky aromatic side chain

Tyr263 Mutants of Cyanobacterial Phytochrome Cph1

might also represent a significant obstacle in D-ring photoisomerization. To address the specific role of Y263 in the photoconversion mechanism, we created Y263F, Y263H and Y263S mutants and analyzed them spectroscopically, deriving various key photochemical parameters. Furthermore, we were able to crystallize and solve the structure of the Y263F mutant at 2.0 Å resolution. We discuss the implications of these data for phytochrome structure/ function relationships and the photoconversion process in particular.

Results Spectroscopic analyses The Cph1Δ2 wild-type sensor module and the three mutants overexpressed in vivo were highly soluble, fluorescence from Zn 2+-stained SDS gels confirming complete covalent chromophore attachment (Fig. S1). UV–Vis absorbance spectra, dark reversion and photoconversion kinetics are shown in Fig. 1a and Fig. S2, respectively; fluorescence excitation and emission spectra are shown in Fig. 1b, and CD spectra are shown in Fig. 1c. The principal photochemical parameters are given in Table 1 (see Supplementary Information for details). Data for the wild type generally correspond closely to those published earlier, 8,20,40 although slightly lower quantum yields for phototransformation were derived. The Y263F mutant showed photochromicity similar to that of the wild type, except that the lowest energy λmax values for Pr and Pfr were at 651 nm and 691 nm, respectively, representing hypsochromic shifts of 9 nm and 16 nm relative to the wild type. However, the oscillator ratio [relative absorbance strengths of the Pr λmax and near-UV (λSoret) peaks] and the extinction coefficient were almost unaffected, implying that the overall status of the chromophore is unchanged. Although the quantum yield for the Pr → Pfr forward reaction was much lower than that of the wild type, the value for the Pfr → Pr back reaction was unchanged, in harmony with the lower χPfr,max for Y263F relative to the wild type. Dark reversion of Y263F was negligible as in the wild type. The absorbance spectra of Y263H showed a broader red-orange shoulder with a much weakened Pr peak (reflected in the extinction coefficient of 47 mM − 1 cm − 1) shifted even further to the blue at 644 nm. Although irradiation at that wavelength generated a small far-red shoulder associated with a quantitatively similar bleaching of the Pr peak, this photochromicity was much weaker than that of the wild type and Y263F. Y263S showed a stronger Pr peak than Y263H, although a similar blue shift was

Tyr263 Mutants of Cyanobacterial Phytochrome Cph1

117

Fig. 1. Spectroscopy of the wild-type Cph1 sensory module and its Y263F, Y263H and Y263S mutants. (a) Absorbance spectra after far-red irradiation (brown) and red irradiation (orange). The difference, estimated Pfr and measured Pfr spectra are shown in green, dark blue and light blue, respectively, as appropriate. (b) Fluorescence excitation (red; λem = 720 nm) and emission (black; λex = 610 nm) spectra at 293 K. (c) CD spectra after far-red irradiation (brown) and red irradiation (orange). The estimated and measured Pfr spectra are shown in light blue and dark blue, respectively, as appropriate. The Pfr spectra were calculated from χPfr,max (see Table 1).

seen. Although bleaching of the Pr peak in red light was quantitatively similar to that seen for the wild type, the corresponding Pfr absorbance peak was much weaker. As a result of this, χPfr,max was returned to a value similar to that of the wild type. The quantum yield for the Pr → Pfr forward reaction (Φ P = 0.05) was lowered further, whereas the Pfr → Pr back reaction (ΦP = 0.09) lies between those of the wild type and Y263H. Dark reversion of Pfr was also accelerated significantly (t½ ∼ 1.5 h). Fluorescence studies (Fig. 1b and Table 1) yielded values of ΦP and χPfr,max similar to those derived from absorbance data. However, λmax values for absorbance and fluorescence excitation of the wild type were significantly different (660 nm and 648 nm, respectively), implying that a minor hypsochromically shifted species is responsible for fluorescence (see Discussion). However, this effect was not apparent in the three Y263 mutants.

Relative to the wild type, the quantum yield of Pr fluorescence (ΦF) was 3.5-fold higher in Y263F, consistent with less efficient photochemistry (ΦP). Interestingly, despite the dramatic difference seen in the absorbance spectra, the excitation spectrum of Y263H was almost identical with that of the wild type. The emission spectrum was significantly broader than that of the wild type and Y263F. Y263S showed excitation and emission spectra similar to that of Y263H, whereas ΦF was 5-fold higher than the wild type. This increase was mirrored by ΦP. Far-UV CD spectra give information about the overall protein secondary structure; thus, the data (Fig. S3) implied similar folding of the wild type and mutants. Positive and negative signals in the red region, on the other hand, reflect the tilts of the chromophore rings 35 (Fig. 1c). All three mutants showed much weaker negative signals in the red

Tyr263 Mutants of Cyanobacterial Phytochrome Cph1

118

Table 1. UV–Vis absorbance, fluorescence, photoconversion and SEC characteristics of the Cph1 photosensor wild type and Y263 mutants Absorbance

Photochemistry

Fluorescence excitation

Fluorescence emission

SEC

Protein

Wild type

Y263F

Y263H

Y263S

λmax (Pr) (nm) λmax (Pfr) (nm) λSoret (Pr) (nm) A (λmax)/A (λSoret) (Pr) FWHM (Pr) (nm) Δλmax (nm) Δλibp (nm) Δλmin (nm) ΔAλmax/ΔAλmin ΔAλmax/Aλmax ɛ (Pr λmax) (mM− 1 cm− 1) χPfr,max ΦP (Pr → Pfr) ΦP (Pfr → Pr) Dark reversion: t (h-1) Dark reversion: t½ (h) λmax (nm) λSoret (nm) I(λmax)/I(λSoret) FWHM (nm) λmax (nm) FWHM (nm) λmax (A) − λmax (Ex) (nm) λmax (Em) − λmax (Ex) (nm) ΦF χPfr,max ΦP (Pr → Pfr) Pr dimerization Pfr dimerization

660 707 360 2.9 42 656 678 704 0.84 0.36 85 0.70 0.13 0.11 0.0019 N360 648 401 9.7 62 676 50 16 28 0.024 0.70 0.13 Yes Yes

651 691 357 2.8 50 642 669 698 0.76 0.24 85 0.50 0.05 0.10 0.0026 N264 654 400 15 52 678 44 27 24 0.084 0.50 0.06 No Yes

644 — 356 1.5 52 643 666 688 0.96 0.16 47 0.46 0.07 0.11 0.049 14 644 399 8.9 62 669 60 25 25 0.067 0.40 0.08 No No

643 — 355 2.2 50 643 673 690 3.40 0.36 80 0.60 0.05 0.09 0.45 1.5 640 402 11 60 663 66 20 23 0.1 0.50 0.08 No No

FWHM (full width at half maximum) at λmax for the absorbance and excitation spectra were determined by Gaussian approximation of the main red band, derived from the λmax to the 700-nm data. FWHM at λmax for the emission spectra was determined by Gaussian approximation of the main red band, derived from the 650-nm data to the λmax data. The Stokes shift is the λmax (A) − λmax (Ex) parameter. χPfr,max and ΦP were determined by both absorbance and fluorescence methods (see Supplementary Information)

region than the wild type, effects poorly correlated with ɛ values. A positive signal around 670 nm for both wild type and Y263F after red irradiation appeared associated with the D-ring photoflip. 35 In the Y263S mutant, an additional second positive peak was seen around 600 nm. In harmony with earlier work, 41 size-exclusion chromatography (SEC) showed that the wild-type photosensory module dimerized preferentially in the Pfr state. Pr and Pfr of Y263F migrated as a monomer and a dimer, respectively, Pr eluting as a monomer even at concentrations at 30 mg/ml (Fig. S4). In the case of Y263H and Y263S, both Pr and Pfr states eluted as monomers, although a small dimeric fraction was seen, possibly attributable to Pfr (Fig. S4). Structure of the Y263F mutant Crystals of Y263F in its Pr state were only generated under conditions different from those effective for the wild type. The new, monoclinic crystal form found for Y263F has one molecule per asymmetric unit and diffracted isotropically to 2.0 Å resolution. In contrast, the previously reported

crystals of the wild type were tetragonal and showed strongly anisotropic diffraction along the z-axis. The latter effect was also observed for the equivalent photosensory module of the bathyphytochrome PaBphP, 13,14 although the latter crystallized as parallel dimers in contrast to the antiparallel configuration in both Cph1Δ2 crystal forms. The improved diffraction characteristics of Y263F might derive from either packing differences resulting from slight changes in the quaternary structure or improved homogeneity of the material. 13 The refined structure of Y263F (PDB ID: 3ZQ5) has an overall temperature factor (28 Å 2 ) significantly lowered than that of the wild type (66 Å 2 ). Consequently, the improved electron density for the chromophore and its local interactions revealed additional details regarding the chromophore conformation, water molecules and alternative sidechain conformations not traceable in the wild-type structure. Likewise, the unresolved gaps in the 2VEA wild-type structure, Q73–E80 and G100– D101 in the PAS domain, R148–Q150 in the GAF domain and A460–K466 at the tip of the tongue are now defined by electron density. The only other high-resolution phytochrome structure available is

Tyr263 Mutants of Cyanobacterial Phytochrome Cph1

the photochemically impotent PAS–GAF bidomain of the DrBphP Y307S mutant. 42 The overall structure of the photosensory module in Y263F is similar to that of the wild type (Fig. 2), showing an N-terminal PAS–GAF lobe separated

119 from the C-terminal PHY domain by the long linker helix α9 (P298–T344). Despite a crystal packing quite different from that of the wild type and unlike all other known phytochrome crystal structures, Y263F and the wild type crystallize as similar antiparallel

Fig. 2. Superimposition of the overall structure (a) and the chromophore pocket (b) of the Y263F mutant (colored) and the Cph1 wild type (gray) in their Pr states. In the Y263F structure, the PHY domain is shifted by 17° in relation to the PAS–GAF bidomain. Due to the Y263F substitution, the D-ring of the chromophore is inclined further, which in turn requires a rearrangement of conserved residues (M267 and F475) in its vicinity.

120 dimers (Fig. S5a). The structures of the individual lobes are very similar, yielding low r.m.s.d. values following superimpositions of 0.36 Å and 0.32 Å for the PAS–GAF (231 C α positions) and PHY (133 C α positions) lobes, respectively. Neither the PAS–GAF interface nor the light-sensing knot is affected by the Y263F substitution. Indeed, most side-chain conformations are similar in the two structures, exceptions concentrating at the surface of the monomer. The Y263F dimer interface along the linker helix α9 and the accompanying helix α4 from the PAS domain overall resembles that of the wild type (differences are detailed in Fig. S5b and Table S2) with interface areas of 1205 Å 2 and 1239 Å 2 for the wild type and Y263F, respectively. The 47-residue α9 helix includes two hinges, I316–S317 and V329–Q330, located close to the domain surfaces of the GAF and PHY domains, respectively (Fig. S6). Although the hydrogen-bonding pattern within the α9 helix remains unbroken, slight differences at the I316– S317 hinge cause its straightening in the Y263F structure, resulting in a 17° offset for the PHY domain (and necessitating that molecular replacement for the PAS–GAF and PHY regions be carried out independently). Other differences are localized in the packing of helix α4 to the C-terminal portion of α9. In two different crystal forms (3C2W and 2G6O) of the photosensory module of bacteriophytochrome PaBphP, 14,43 a similar plasticity of the α9 helix is seen at the hinge proximal to the GAF domain. This suggests that the α9 linker helix is flexible enough to adopt various distinct conformational states in solution. Interestingly, despite this relative shift of the PAS–GAF and PHY domains, the tongue protruding from the PHY domain faithfully contacts the chromophore within the GAF domain. Accordingly, changes in the relative domain orientations are compensated by the degree of kinking of the L-shaped tongue. Two hinges, G451–G452 and W478–K479, mediate these changes by adopting different main-chain conformations. The Ψ/Φ values of the G451–G452 peptide bond change from 74°/108° (wild type) to − 13°/158° (Y263F) and those of W478–K479 change from 101°/− 76° to 135°/− 110°, whereas the main-chain conformation between the PHY domain and these hinges remains mostly unaffected. The hinges are associated with highly conserved WGG and WxE motifs, implying that they are functionally significant. The loss of the hydroxyl group in Y263F induces only small changes in the chromophore configuration. Overall, the phycocyanobilin (PCB) chromophore is slightly more twisted than in the wild type. In particular, the D-ring adopts a more inclined position (37° versus 26° relative to ring C) but shows polar water-mediated contacts with H290 the same as those of the wild type (Fig. 3). Slightly larger angles are apparent between the

Tyr263 Mutants of Cyanobacterial Phytochrome Cph1

B-ring and the C-ring (5.8° versus 1.5°). The C-ring propionate is rotationally shifted, losing its polar contacts with H260 but still interacting via a hydrogen bond with T274, while the B-ring propionate retains its interaction with R254. The A-ring adopts a steeper inclination (16° versus 10° relative to ring B) than in the wild-type structure and interacts with R472 via a water molecule that is only present in the 3ZQ5 structure of Y263F. As the cysteine side chain of C259 adopts alternative conformations in the Y263F structure, its thioether linkage to the C3 1 atom of the PCB chromophore is stereochemically heterogeneous with a 0.55:0.45 distribution between the R configuration and the S configuration. 13,44,45 However, radiation damage, as reported before, in the crystal structures of a biliverdin-binding bacteriophytochrome 46 to cause breakage of the thioether bond could not be observed at the C3 1 atom. Likewise, the stereocenter at the C2 atom is found to occur exclusively in the R configuration. Furthermore, two methionines, M174 and M267, located above and below the D-ring of the chromophore show two alternative rotamers (Fig. S7). Although the salt bridge between R472 of the conserved PRxSF motif of the tongue and D207 of the GAF domain is retained, S474 and F475 undergo a major rotational shift, now pointing out of the chromophore pocket (Fig. 2b). The new structure shows significant shifts in the β20 strand such that W478 in the conserved WxE motif of the tongue adopts a more outward position. Further notable differences are associated with the tongue and the α1 helix. In the 2VEA wild-type structure, the helix extends to the extreme N-terminus of the model, whereas in 3ZQ5, A2–D9 is unwound, forming several backbone hydrogen bonds to the tongue. The UV–Vis absorbance spectra of the Y263F Pr crystal at − 160 °C showed sharper, slightly redshifted peaks relative to the spectra in solution at room temperature. In situ photoconversion to the Pfr state was possible for small crystals (Fig. S8) but resulted in a loss of diffraction as observed for the wild type. 13

Discussion The combination of blue-shifted absorbance peaks but otherwise seemingly normal photochromicity of Y263F has been reported for several other Cph1 mutations close to the chromophore. 10 However, neither the oscillator ratio nor the Pr extinction coefficient itself is significantly affected, implying that the overall geometry and protonation of the chromophore are unchanged in Y263F. The 9-nm hypsochromic shift of the red peak might thus be explained by the stronger tilt of the D-ring (26° and 37° in 2VEA and 3ZQ5, respectively), as ring tilts

Tyr263 Mutants of Cyanobacterial Phytochrome Cph1

121

Fig. 3. (a) High-resolution three-dimensional structure of the PCB chromophore (cyan) and its interacting residues. The substituted residue Y263F is shown in magenta. (b and c) Conformational differences between the Y263F structure (cyan) and the wild-type structure (gray transparent) lead to a 1-Å constriction of the chromophore.

N 40° rapidly lead to π-orbital uncoupling. D-ring tilts exceeding 40° are not unusual in tetrapyrrolebinding proteins: 47 the high-resolution structures of several bacteriophytochrome PAS–GAF bidomains (2O9C, 2O9B and 2OOL) have tilts of 42–55°. With these minor differences in mind and the fact that both the wild type and Y263F perform Pr → Pfr photoconversion both in solution and in the crystalline state, a further sign of local structural plasticity of the Cph1 Pr ground state is given by the heterogeneous attachment of the bilin chromophore

to C259 and the variable packing of the D-ring. The latter is made by the alternative side chains of M174 and M269, as well as the different conformations adopted by Y263/F475 and F263/F475 in the wildtype 2VEA and Y263F 3ZQ5 structures, respectively. These subtle structural differences around the chromophore nevertheless suffice to reduce the Pr → Pfr photoconversion quantum efficiency (ΦP) of Y263F to only a third of the wild type, whereas that of the Pfr → Pr back reaction is unchanged, thereby explaining the lower χPfr,max of the mutant.

122 The lower ΦP is mirrored as an increase in the fluorescence quantum yield (ΦF), indicating that the changes associated with the mutation affect the system prior to lumi-R formation. On a more global scale, structural plasticity is reflected by the different domain arrangements found between the chromophore-bearing PAS– GAF bidomain and the C-terminal PHY domain. The 17° offset of the PHY domain in Y263F relative to the wild type is caused by the I316–S317 hinge in the α9 linker helix (Fig. S6). The other discontinuity from a straight helix at V329–Q330 is unaffected. Similar hinge effects are apparent in the structures of the PaBphP sensory module even though these crystallize as parallel rather than antiparallel dimers as in the case of Cph1. It would thus seem that the helix α9 hinges are functionally significant. Given the major shift in the position of the PHY domain associated with the Y263F mutation, it is interesting that the tongue still faithfully contacts with the chromophore lobe, indicating that its function is remarkably tolerant of PHY domain movements. Although several intimate interactions between the tip of the tongue and the extreme N-terminus are apparent in 3ZQ5 but not 2VEA, in 3ZQ5, the tongue retains the conserved salt bridge between R472 of the PRxSF motif 13 and D207 of the DIP motif of the GAF domain. Two hinge regions allow the tongue to kink differently so that the R472–D207 interaction is maintained despite the relative shift of the GAF and PHY domains. Both hinges are part of two highly conserved sequence motifs of Cph1/plant phytochromes, WGG and WxE. 13 As the D207–R472 salt bridge is disturbed during Pr → Pfr photoconversion and as R472 34 packs closely to the glycines of the WGG hinge, we propose that changes associated with photoisomerization are transmitted directly to the tongue at that point and thereby bring about the quaternary structural change in the complete Cph1 homodimer associated with signaling. Although the observed conformational changes in the residues S474, F475 and W478 may be direct results of the Y263F mutation, they could instead derive from the different crystal packings in the 2VEA and 3ZQ5 structures. Indeed, as several differences are directly attributable to contacts between symmetry mates (notably, interactions between the tip of the tongue and the extreme N-terminus) and as most of the side-chain rotamer differences are associated with superficial residues, we expect that many particular features of 3ZQ5 reflect those of the native structure. Local structural plasticity of the protein environment around the PCB chromophore correlates with the presence of various Pr isoforms in solution. Advanced MAS NMR methods recently provided detailed descriptions of two Pr isoforms in Cph1. 34 Earlier MAS NMR 48, solution-state NMR 49 and

Tyr263 Mutants of Cyanobacterial Phytochrome Cph1

fluorescence spectroscopy 9,23 also implied heterogeneity in the Cph1 ground state, as did Raman, 28 ultrafast kinetics 21 and even crystallographic 46 studies of bacteriophytochrome RpBphP3 as Pr. Kinetic 50 and fluorescence 51–54 studies of plant phyA reached the same conclusion. That Pr crystal structures generally show only one isoform is not inconsistent with there being more than one isoform in solution. In the case of the wild-type Cph1 sensory module, isoform II corresponds to the 2VEA crystal structure, whereas isoform I shows important differences in the position and charge of the H260 side chain and the hydrogen bonding of chromophore ring D, both likely to be critically important in the photochemistry of the molecule. 34 An explanation of the low quantum efficiency of photoconversion by phytochromes is that only one isoform is photochemically competent. It might be that the high-resolution crystal structure of Y263F presented here does not predominate in solution. Indeed, the weak CD signal in the red region would imply a flat chromophore in Y263F rather than the stronger D-ring tilt seen in the structure. However, as in the wild type, the absorbance spectra of the Y263F crystals at 100 K (λmax values of 668 nm and 663 nm, respectively; see Ref. 13 and Fig. S8) are consistent with those for equivalent Pr solutions at room temperature. 55 There is thus little reason to expect that the 3ZQ5 structure is affected to a large extent by crystal packing and crystallization conditions. Also, the blue-shifted Pr absorbance peak of Y263F both in solution and in the crystal is consistent with the stronger tilt seen in the new structure. The weak CD signal of Y263F also does not result from reduced absorbance, as ɛ is unchanged. It might alternatively be explained by the local differences in the chemical environment of the D-ring or the presence of a mixture of isoforms with different tilt angles in solution. MAS NMR experiments with this mutant should clarify the situation. We hypothesize that Y263F Pr in solution comprises at least two chromophore conformations and that the predominant conformation/s are photochemically impotent. Given that the extinction coefficients are similar, the lower quantum yield of Y263F implies that the proportion of photochemically competent molecules is about 2.5-fold lower than that of the wild type. The D-ring tilt is likely to be an important factor in phytochrome photochemistry, explaining why the UV–Vis absorbance and fluorescence excitation spectra are closely similar in the case of Y263F, whereas in the wild type, the fluorescence spectrum is blue shifted relative to the absorbance spectrum. Rockwell et al. suggested that the twisted C15 = C16 double bond might provide the energy to overcome the C13′ methyl/NH barrier. 35 Thus, the D-ring tilt cocks the trigger for Pr photochemistry.

Tyr263 Mutants of Cyanobacterial Phytochrome Cph1

Including the other two mutants described here helps further in understanding phytochrome molecular function. Whereas the wild type, Y263F and Y263S all show ɛ values of ca 85 mM − 1 cm − 1 for Pr at λmax, the value for Y263H is substantially lower (as seen in the homologous mutant of DrBph 38). A closer examination of the Y263H absorbance spectrum reveals that, whereas the red peak is much weaker, the broad orange shoulder is scarcely affected, implying that the Pr chromophore is incompletely protonated. 10 Interestingly, the Pr peak in Y263S is similar to that of the wild type, perhaps indicating that either side chain or an intruded water molecule can act in proton exchange. In Y263H, the quantum efficiency of the forward photoreaction is slightly higher than that in Y263F (reflected in reduced fluorescence yield), whereas that of the back reaction is unchanged. Together, these parameters explain why the difference spectrum for Y263H with its smaller and still aromatic side chain is very weak but symmetrical. In remarkable contrast to the aberrant absorbance spectrum of Y263H, however, the fluorescence emission spectrum of this mutant resembles that of the wild type. The fluorescence emission spectrum of this mutant is broadened, implying that the chromophore might be loosened by adopting various energetically favorable conformations in the Pr state, many of which might be photochemically impotent. This possibility is supported by CD data, the red region showing a very weak Pr signal and almost no state-related change. The Pr signal is likely to derive from predominantly planar or symmetrically tilted chromophores, as well as from the weak absorbance. The Y263S mutant is characterized by a highly asymmetrical difference spectrum: red irradiation bleaches the Pr peak, while the Pfr counterpart is scarcely apparent. This phenotype is seen in many mutations affecting the tongue of the PHY domain. A possible explanation is that the effect results from either imperfect sealing of the chromophore pocket or inclusion of additional water molecules and might be associated with the meta-R state that fails to reprotonate. CD data (Fig. 1c) for Y263S show a second positive peak around 600 nm. This could result from the increased mobility of the chromophore in the pocket, the loss of the aromatic residue at position 263 perhaps allowing the chromophore to adopt a different configuration altogether. A similar effect was observed with studies on PCB chromopeptides. 56 Considering the mutants at position 263 together, the weak red absorbance peak of the Pr state in Y263H correlates with a potential role for the side chain in abstracting a proton from the chromophore. On the other hand, the weak Pfr absorbance peak seen in both Y263H and Y263S but not Y263F implies that, at least, a phenyl ring is important for stabilizing the Pfr state. MAS NMR data 34 showed

123 not only that Pfr forms a hydrogen bond between the chromophore D-ring carbonyl and the hydroxyl of Y263 but also that several packing interactions are affected. Our data for Y263H indicate a dramatic loss of photochemical efficiency likely to derive from predominantly disordered and/or deprotonated chromophores. Although an attractive explanation of this would be that the protein itself is grossly misfolded, far-UV CD data (Fig. S3) provide no support for this. Contrary to studies of the noncanonical phytochrome SyB-Cph1, which assigned a key role to the hydroxyl group of Y263, 39 we show that this hydroxyl group is not essential for Pfr formation in Cph1. The Y263F mutant showed a much more subtle phenotype in which the quantum energy requirements for excitation to S1 were increased slightly (hypsochromic shifts of λmax for Pr and Pfr), while the quantum efficiency of Pr → Pfr photoconversion was substantially reduced. This implies that the hydroxyl group promotes efficient C14 = C15 isomerization around ring D during photoconversion from Pr to Pfr. Liquid NMR studies of the isolated GAF domain of SyB-Cph1 were initially interpreted to show that Pr → Pfr photoconversion involved isomerization of the A-ring. 39 The highresolution Y263F structure presented here clearly shows the chromophore in a periplanar ZZZssa conformation as seen in all the Pr crystal structures to date, 12,13,42,46,57 whereas that of PaBphP in its Pfr ground state was ZZEssa and is likely to be ZZZssa for its Pr state in the Q188L mutant, 14,43 with only minor changes associated with the A-ring. Recent MAS NMR studies of Cph1 34 have reaffirmed ZZZssa and ZZEssa as conformations in Pr and Pfr, respectively. 14,16 We also note that, whereas several crystal structures of Cph1 and PaBphP and the MAS NMR data for Cph1 show very different rotamers for several side chains near ring D in Pr and Pfr, the published SyB-Cph1(GAF) ensembles do not. One explanation would be that SyB-Cph1 functions quite differently from canonical phytochromes. We note, however, that the published model ensembles 39 have now been “revised” (PDB entries 2LB9 and 2LB5 replacing 2KOI and 2KLI). Whereas full-length phytochromes form stable dimers via a likely helix–loop–helix structure of their C-terminal dimerization and phosphoacceptor domain, the N-terminal photosensory module of Cph1 shows a state-dependent dimerization affinity, KA for Pfr being at least 20-fold higher than that for Pr. 41 Whereas the Y263F mutant behaved similarly to the wild type in the Pfr state, Pr showed no propensity to dimerize (Fig. S4). Similar effects on dimerization affinity have been described for mutations affecting residues near the chromophore B-ring. 13 Interestingly, the equivalent of one of these residues (R254), R352K in plant phyB, was shown to be defective in signaling. 58,59 We consider

124 therefore that state-dependent dimerization might be relevant to the signaling process. It should be noted, however, that the detached photosensor studied here and in Essen et al. has full freedom of movement, allowing, for example, the formation of antiparallel dimers as seen in the crystal structures. Although these might represent the dimers seen in solution, this is not necessarily the case—as implied by the fact that ground state Y263F and wild type show very different dimerization propensities in solution, while the crystal dimers are very similar. The distinction is biologically important, as the fulllength molecule forms stable parallel dimers, constraining the movement of the photosensory module. In addition to its significance in relation to the phytochrome family, Cph1 represents a particularly useful paradigm for investigating the likely common switching mechanism used in “twocomponent” sensory histidine protein kinases. State-dependent interactions between the photosensory modules might provide the basis for regulating autokinase/phosphotransferase activity of the transmitter, analogously to the rotary switch model. 60 Preliminary studies of the Cph1 signaling mechanism using both in vitro methods and on the basis of engineered chimeras in vivo have been successful. 34 Spectroscopic and structural studies of the wildtype Cph1 photosensor and Y263 mutants have allowed us to make tentative conclusions regarding the relationship between photochemistry, the photocycle and structure. The energetics of the photoreaction has been studied by low-temperature fluorescence spectroscopy (unpublished results). Numerous further mutants have been created and are being characterized, expanding the experimental horizon of our work on phytochrome structure/ function relationships. We hope that quantum mechanics/molecular mechanics methods based on wild-type and mutant structures will be able provide thoroughgoing explanations of the photochemistry and dynamics of the molecule, although this is likely to be complicated by the apparent heterogeneity of the Pr ground state.

Experimental Procedures Production of Y263F, Y263H and Y263S Site-directed mutants of the histidine-tagged Cph1 photosensory module (Cph1Δ2; comprising the first 514 amino acids) were generated by the back-to-back PCR method as previously described. 10 The protein was expressed as described previously. 13,61 For dimerization studies, SEC was performed with a red-irradiated 3 mg/ml sample using a 16/60 Superdex 200 column (GE Healthcare; Vt = 120.6 ml, V0 = 39.8 ml and flow rate = 1 ml/min) in 50 mM Tris–HCl (pH 7.8), 1 mM

Tyr263 Mutants of Cyanobacterial Phytochrome Cph1 ethylenediaminetetraacetic acid, 300 mM NaCl and 1 mM β-mercaptoethanol as buffer. For work in darkness, appropriate infrared (940 nm ± 45 nm) visualization equipment was used. 61 For crystallization trials, a sample of Y263F at a concentration of 10 mg/ml was irradiated with far-red light to populate the Pr state and further purified using a preparative Superdex 200 column (Vt = 318 ml and V0 = 105 ml; GE Healthcare). UV–Vis, fluorescence and CD spectroscopy Absorbance spectra in solution were recorded at room temperature using a modified Agilent 8453 UV–Vis diode array detector spectrophotometer. 10,61 Samples were irradiated with light-emitting diodes and appropriate interference filters (651 nm for Y263F and 640 nm for Y263H and Y263S). Extinction coefficients were determined by measuring equal concentrations of the holoproteins in β-mercaptoethanol buffer (native conditions) and in 8 M urea at pH 2.0 (acidic denaturing conditions) based on the extinction coefficient of phycocyanin. 62 Because Pfr absorbs significantly in the red region, irradiation never leads to complete occupancy of the Pfr state. The mole fraction of Pfr present at photoequilibrium at λmax,Pr (χPfr,max) was estimated by proportionally subtracting the spectrum of Pr (following far-red irradiation) from that following irradiation at Pr λmax to fit the spectrum of 100% Pfr purified by SEC. 41 The quantum yield of Pr → Pfr photoconversion [ΦP(Pr → Pfr)] was determined from quantitative measurement of the photoconversion rate in red light, 20,63,64 as described in Supplementary Information. Samples were pre-irradiated to photoequilibrium using appropriate interference filters (see above). Dark reversion was monitored at appropriate intervals at 20 °C at the respective λmax of the Pr and Pfr states and at λibp to estimate state-independent absorbance changes. The measuring light was occluded, or the samples were removed between measurements. Fluorescence spectra and kinetics were recorded at room temperature using a Fluoromax4 spectrofluorometer (Horiba/Jobin Yvon) as described previously. 23,65 To minimize spectral artifacts deriving from self-absorption and scattering, we used a protein concentration of approximately 0.1 mg/ml. Emission spectra were measured using excitation at 610 nm, slit bandwidth of 0.3 nm and a red interference filter (λ = 610 nm, Tmax = 40%), that is, with low intensity excitation light to minimize phytochrome photoconversion during the scan. To exclude interference from scattered excitation light, we placed a 600-nm-cutoff filter in the emission optical path. Emission spectra were recorded from 630 nm to 750 nm with a slit bandwidth of 10 nm and an integration time of 0.1 s. Determination of quantum yields of the photochemical processes and χPfr,max are described in Supplementary Information. CD spectra were recorded at room temperature with a Jasco J-810 spectropolarimeter. Experiments were conducted as previously described, 66 except that 1-mm-pathlength cells were used. Near-UV (260–800 nm) CD spectra of Pr and the Pr/Pfr photoequilibrium mixture were recorded following saturating irradiation at 730 nm and at the appropriate λmax, respectively. The spectra for Pfr were obtained by subtraction of the Pr contribution from

Tyr263 Mutants of Cyanobacterial Phytochrome Cph1 the Pr/Pfr photoequilibrium and, where possible, verified by comparison with that of 100% Pfr as obtained by SEC (Fig. S4). All purified samples (concentration of 10 mg/ml) were handled in complete darkness using infrared visualization equipment (940 ± 45 nm). 61 To minimize photoconversion by the measuring light of the instrument, we irradiated samples with the appropriate actinic light source prior to each scan. A total of 10 scans were collected and averaged. Confirmation of the structural integrity of the constructed mutants was obtained by the acquisition of far-UV (190–260 nm) CD spectra, following saturating red irradiation and their comparison to those of the wild-type sensory module following the same irradiation. Far-UV spectra of the tested proteins (1 μM) are presented as the smoothed average of five accumulations (see Fig. S3). Secondary structure prediction analysis was performed with the supplied J-810 software for Windows, using the Yang statistical algorithm. 67 All recorded CD spectra were baseline corrected by subtraction of the “buffer-only” spectrum. Crystallization, data collection and structure solution Initial crystallization screens were carried out in 96-well MRC sitting-drop plates (Jena Bioscience) at 18 °C in darkness as described previously. 13 Crystals appeared under a condition containing 1 μl of 10 mg/ml Y263F Pr and 1 μl of reservoir solution [2.5 M sodium acetate and 0.1 M 4-morpholineethanesulfonic acid (Mes) (pH 6.5)]. Further optimization utilized the hanging-drop vapor diffusion technique. Optimally diffracting crystals were obtained under a condition containing 1.9 M sodium acetate and 0.4 M Mes (pH 7.2). We added 30% glycerol as a cryoprotectant, and crystals were frozen in liquid nitrogen using 1.75 M sodium acetate, 0.2 M Mes (pH 7.2) and 30% glycerol. Data sets were collected at beamline ID14-2, European Synchrotron Radiation Facility, Grenoble. The completeness of the 2.05-Å data set was 99.8% and revealed the monoclinic space group C2 (a = 107.57 Å, b = 95.15 Å, c = 73.58 Å and β = 99.67°) with one molecule per asymmetric symmetry unit. Molecular replacement by PHASER 68 was performed with the photosensory module of wild-type Cph1 (PDB ID: 2VEA) for the PAS–GAF bidomain and the PHY domain independently, followed by alternating cycles of automated and manual refinement using REFMAC5 69 and Coot. 70 The final model of Cph1Y263F photosensory module (PDB ID: 3ZQ5) converged at R-factor/Rfree of 17.0/21.4% (Table S1) and is structurally defined from A2 to H518. Accession numbers Coordinates and structure factors have been deposited in the PDB with accession number 3ZQ5.

Acknowledgements This work was supported by Deutsche Forschungsgemeinschaft grants HU702/6-1, HU702/7-1 and

125 ES152/6-1 and by the Russian Foundation for Fundamental Investigation grant 11-04-01732. The authors are grateful for support from Tobias Klar and David von Stetten at European Synchrotron Radiation Facility, Grenoble (beamline ID14-2 and cryobench), and for the technical assistance provided by Tina Lang and Petra Gnau.

Supplementary Data Supplementary data to this article can be found online at doi:10.1016/j.jmb.2011.08.023

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