Conformational differences between the Pfr and Pr states in Pseudomonas aeruginosa bacteriophytochrome Xiaojing Yanga,1, Jane Kuka, and Keith Moffata,b,1 aDepartment
of Biochemistry and Molecular Biology and bInstitute for Biophysical Dynamics, University of Chicago, 929 East 57th Street, Chicago, IL 60637
Edited by J. Clark Lagarias, University of California, Davis, CA, and approved July 29, 2009 (received for review February 26, 2009)
biliverdin 兩 photoconversion 兩 red-light photoreceptor
P
hytochromes are red-light photoreceptors that undergo reversible photoconversion between a red-light-absorbing state (Pr) and a far-red-light-absorbing state (Pfr), and thereby they regulate a wide range of physiological responses in plants, fungi, and photosynthetic bacteria (1–5). Using linear tetrapyrroles as chromophores to detect light in the long-wavelength range of the visible spectrum, the photosensory core module (PCM) of bacteriophytochromes contains three domains (PAS, GAF, and PHY). The PAS and GAF domains constitute the chromophore-binding module (CBM); and the PHY domain is essential for efficient photoconversion (5). Upon absorbing a photon, 15Za/15Ea isomerization occurs about the C15AC16 double bond between rings C and D of the bilin chromophore, followed by thermal relaxation events in the chromophore and the protein matrix (6). Local conformational changes originating in the photosensory domains propagate to the C-terminal histidine kinase (HK) domain, where they modulate the kinase activity and thus convert a light signal into a chemical signal (5). Fundamental questions about the molecular mechanisms of photoconversion and signal transduction remain unanswered. What are the local and long-range conformational changes? What molecular events are involved? In what sequence do they occur? Extensive studies on a variety of phytochromes and bacteriophytochromes suggest that significant structural changes occur in both the chromophore and protein moieties during Pr/Pfr photoconversion, but details of these changes are still lacking (7–11). Two crystal structures of bacteriophytochromes with intact PCMs have been determined recently: that of Pseudomonas aeruginosa bacteriophytochrome (PaBphP) (12) and that of cyanobacterial phytochrome Cph1 from Synechocystis sp. 6803 (13). Both structures include the PHY domain and represent their dark-adapted Pfr and Pr states, www.pnas.org兾cgi兾doi兾10.1073兾pnas.0902178106
respectively. Here, we report the crystal structure of a point mutant of the PaBphP-PCM (Q188L) that appears to exhibit mixed Pfr and Pr states in the crystal. The Q188L crystals have distinct crystal packing and grow under crystallization conditions different from that for WT. Based on the Q188L crystal structure, structural comparisons among the crystal structures in the Pr and Pfr states, and site-directed mutagenesis, we identify several residues and structural elements that undergo conformational changes during Pr/Pfr photoconversion. Results and Discussion Crystal Structure of PaBphP-PCM Q188L. The Q188L mutant of the
PaBphP PCM in solution adopts the Pfr state in the dark (figure 2d of ref. 12). The mutant undergoes reversible Pr/Pfr photoconversion, but its rate of reversion to the Pfr state in the dark is significantly slower than that of WT, with a half-time of about 2 h; full reversion requires overnight incubation in the dark. Attempts to crystallize the Q188L mutant under WT conditions (0.45 M ammonium phosphate and 0.1 M Tris䡠HCl, pH 7.7) were not successful. Instead, crystals of the Q188L mutant with a typical size of 400⫻100⫻100 m were obtained in hanging drops via vapor diffusion with the mother liquor 0.5% PEG4000 (wt/vol) and 0.01 M sodium acetate, pH 4.6. We determined the crystal structure of PaBphP-PCM-Q188L at 2.9-Å resolution in space group P65 by the multiwavelength anomalous dispersion (MAD) method using Se-Met-substituted crystals (table S1 in ref. 12). The overall arrangement of the PAS, GAF, and PHY domains in the Q188L structure is very similar to that in the WT structure, where all three sensory domains converge on the chromophore via a 41 knot between the PAS and GAF domains and an extended arm of the PHY domain. The pairwise rms differences of the main-chain C␣ atoms between the Q188L and WT structures are very small in the GAF domain but are more significant in the PHY domain. The largest differences occur near both ends of the long helical bundle at the dimer interface (Fig. 1A). Although crystallized under conditions completely different from those for WT, the molecules of the Q188L mutant in the asymmetric unit are packed as a parallel, head-to-head dimer closely similar to the WT structure (12) but quite unlike the antiparallel dimer in the Cph1-PCM structure (13). Two monomers in one asymmetric unit are related by near-perfect noncrystallographic twofold symmetry whose axis is perpendicular to the crystallographic 65 screw axis, Author contributions: X.Y. designed research; X.Y. and J.K. performed research; X.Y. analyzed data; and X.Y. and K.M. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The coordinates and structure factor amplitudes have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3G6O and 3IBR). 1To
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This article contains supporting information online at www.pnas.org/cgi/content/full/ 0902178106/DCSupplemental.
PNAS 兩 September 15, 2009 兩 vol. 106 兩 no. 37 兩 15639 –15644
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Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria by means of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here, we report the crystal structure of the Q188L mutant of Pseudomonas aeruginosa bacteriophytochrome (PaBphP) photosensory core module, which exhibits altered photoconversion behavior and different crystal packing from wild type. We observe two distinct chromophore conformations in the Q188L crystal structure that we identify with the Pfr and Pr states. The Pr/Pfr compositions, varying from crystal to crystal, seem to correlate with light conditions under which the Q188L crystals are cryoprotected. We also compare all known Pr and Pfr structures. Using site-directed mutagenesis, we identify residues that are involved in stabilizing the 15Ea (Pfr) and 15Za (Pr) configurations of the biliverdin chromophore. Specifically, Ser-261 appears to be essential to form a stable Pr state in PaBphP, possibly by means of its interaction with the propionate group of ring C. We propose a ‘‘flip-and-rotate’’ model that summarizes the major conformational differences between the Pr and Pfr states of the chromophore and its binding pocket.
Fig. 1. Crystal structure of the Q188L mutant of PaBphP-PCM. (A) Ribbon diagram colored by the main-chain rmsd values between the Q188L [Protein Data Bank (PDB) ID code 3G6O] and WT (PDB ID code 3C2W) structures. (B) Superposition of the Q188L (gray) and WT (green) structures as a dimer. The structures are aligned based on the left monomer. Same view as in A.
in contrast to the WT crystal structure, in which conformational heterodimers are evidenced by two locations of the GAF-hA helix (12). The Q188L dimer buries significantly less surface area at the dimer interface compared with WT (2,690 Å2 versus 3,950 Å2). Helices comprising the central helical bundle at the dimer interface gradually separate when they extend into the PHY domain of the Q188L structure (Fig. 1B). Although the electron density of the PAS and GAF domains is well defined, that of the PHY domain is less so, especially for the side chains of its core structural elements. The initial side-chain conformations of the Q188L model in the PHY domain were mostly adopted from the refined WT structure, in which the electron density of the PHY domain is much more ordered (12). Mixed Pr/Pfr States in the Q188L Crystals. The simulated annealing
(SA)-omit maps for the chromophore of Q188L exhibit general features typical of a linear tetrapyrrole in a 5s10s15a configuration (Fig. 2). Compared with those from the DrBphP-CBM (2O9C), RpBphP3-CBM (2OOL), and PaBphP-PCM WT (3C2W) structures, the SA-omit maps are less well defined in Q188L, with visibly broadened electron densities, especially in the regions of rings D/C and the propionate groups of rings B and C (Fig. 2). Neither a ZZEssa (Pfr) nor a ZZZssa (Pr) configuration alone can account for all observed densities. If a Q188L model is refined with the chromophore exclusively in the ZZEssa configuration, the residual Fo ⫺ Fc difference maps clearly show positive densities (⬎3.0) near the chromophore, indicating the existence of a second conformation (data not shown). Alternatively, a single chromophore configuration can be modeled lying roughly between those of the DrBphP-CBM (Pr) and PaBphP-PCM WT (Pfr) structures that approximately satisfies the electron density in Q188L, but such a model forms no reasonable hydrogen bonds with its surrounding protein. We further ruled out the possibility that X-ray radiation damage contributes to the broader density distribution in the chromophore. First, no significant difference is observed between the SA-omit maps calculated with two different Q188L datasets that were collected consecutively from the same Q188L crystal volume (data not shown). In addition, radiation damage has not been a determining factor for the chromophore conformations in other phytochrome structures, 15640 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0902178106
although some evidence suggests that X-ray absorption ruptures the covalent bond between the chromophore and its anchoring Cys (14). We therefore propose that these broader map features result from a mixture containing more than one chromophore conformation. Interestingly, but not surprisingly, these Q188L SA-omit maps can be satisfactorily accounted for by superimposing the chromophores of the DrBphP-CBM and PaBphP-PCM WT structures that are individually transformed to a reference Q188L structure based on the GAF core domains (equivalent residues: 157–280 in PaBphP and 170–293 in DrBphP; Fig. 2). We note that the Pr (DrBphP-CBM and RpBphP3-CBM) and the Pfr (PaBphP-PCMWT) structures display distinct map features for the chromophore relative to the GAF protein scaffold, in which main-chain atoms are closely grouped among aligned BphP structures (Fig. 2 and Table S1). Furthermore, mixing two SA-omit maps in the Pr and Pfr states leads to broader density around ring D and for the propionate groups of rings C and D, similar to those features observed in the SA-omit maps of Q188L (Fig. S1B). Therefore, we further propose that the Q188L crystals contain a mixture of the Pfr and Pr states. To estimate the compositions of the Pr and Pfr states in the Q188L SA-omit maps, we developed a least-squares procedure. In this procedure, we used the SA-omit maps of the chromophores from DrBphP-CBM and PaBphP-PCM-WT as basis maps to represent the ‘‘pure’’ Pr and Pfr states, respectively. Table S2 shows that the Pr and Pfr states coexist in all Q188L crystals we have examined, but to a different extent from crystal to crystal. The composition of the Pr state, the ‘‘light’’ state for Q188L, seems to correlate with the light conditions under which the Q188L crystals were cryoprotected. The Q188L crystals exposed only to double-filtered (green and blue) microscope light contain less Pr state than crystals exposed to more illumination. From more than 30 crystals exposed to singly green-filtered microscope light, only one crystal (Pa125) diffracts beyond 3.5-Å resolution. This crystal exhibits the most extensive map features characteristic of the Pr state (Fig. 2). We subjected two Q188L datasets (Pa125 and Pa62) to further map analysis and structure refinement. The SeMet Pa125 dataset contained the most Pr state (⬇45% Pr and ⬇55% Pfr), whereas the native Pa62 crystal, with brief exposure to doublefiltered microscope light, retained more Pfr state (⬇33% Pr and Yang et al.
⬇67% Pfr) and yielded a dataset with the best resolution for any Q188L crystal (Table S2). Because of limited diffraction resolution, the electron-density map alone does not definitively distinguish the E versus Z configuration for ring D in the mixed Pr/Pfr states. However, based on conserved side chains that interact with ring D in the Pr and Pfr structures, such as Asp-194 in the Pfr state and His-277 in the Pr state, we assigned 15Ea to the configuration in cyan and 15Za to the second configuration in gray, which corresponded to the Pfr and Pr states, respectively (Figs. 2 and 3A). To explore conformational changes beyond the chromophore, we also calculated the SA-omit maps in which the side-chain atoms for 11 residues (Cys-12, Yang et al.
Conformational Differences in the Chromophore-Binding Pocket. The
two chromophore configurations (ZZEssa and ZZZssa) in the Q188L crystal structure are related by 15Ea/15Za isomerization about the C15AC16 double bond of the methine linkage between rings C and D, and by rotation roughly around ring A (Fig. 2). This is consistent with structural differences between the chromophores in the Pr structures and PaBphP-PCM-WT Pfr structures (Fig. S1D). All known Pr structures (DrBphP-CBM, RpBphP3-CBM, and Cph1-PCM crystal structures and the SyB-Cph1 NMR structure) show remarkable uniformity in the interactions between the ZZZssa chromophore and the surrounding protein environment, regardless of the presence or absence of the PHY domain or the chemical nature of the chromophore (Fig. S1D) (13). The PaBphPPCM structure in the Pfr state, on the other hand, exhibits a distinct chromophore configuration (ZZEssa) and differs in stabilizing interactions from the Pr structures (Fig. 3A and Fig. S1D) (12). However, the overall structures of the CBMs in the Pr and Pfr states are remarkably similar, with small pairwise rmsd values between PNAS 兩 September 15, 2009 兩 vol. 106 兩 no. 37 兩 15641
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Fig. 2. Stereoviews of the SA-omit maps of the chromophore in the Pr, Pfr, and mixed states. The chromophore models of the Pfr (ZZEssa; cyan) and Pr (ZZZssa, gray) states are from the DrBphP-CBM (2O9C) and PaBphP-PCM WT (3C2W) structures, respectively. The SA-omit maps from DrBphP-CBM, PaBphP-PCM-WT, Q188L-Pa62, and Q188L-Pa125 are aligned based on their GAF core domains. Note that a single model accounts for the SA-omit density in DrBphP-CBM and PaBphP-PCM, but a mixture of the two models is required for both Q188L crystals.
Tyr-163, Tyr-190, Asp-194, Tyr-203, Arg-209, Arg-241, His-247, His-277, Tyr-250, and Ser-261) that immediately surround the chromophore were omitted. Tyr-190, Tyr-163 and Arg-209 exhibit branched side-chain densities that are most evident in the SA-omit map of Pa125 (Fig. 3C). The centroids of the side chains densities in Cys-12, Tyr-203, and Tyr-250 are slightly shifted between the aligned Pa62 and Pa125 maps, whereas Asp-194, Arg-241, His-247, Ser-261, and His-277 display no significant differences between these maps. We thus model dual conformations for Tyr-163, Tyr-190 Arg-209, Cys-12, Tyr-203, and Tyr-250 based on the SA-omit maps as well as the known Pr/Pfr structures. The covalent bonds between the SG atom of Cys-12 and the C32 atom of ring A in the Pr and Pfr states are modeled based on the best fitting of the SA-omit maps of the region; any effect of X-ray radiation on the covalent bond was not modeled. The occupancies of the Pr and Pfr states are estimated by the least-squares fitting of the SA-omit maps and the map contours of related Pr/Pfr features. We also validated the Pr/Pfr occupancies in Pa125 by using an absorption spectrum measured from the Pa125 crystal at 100 K after X-ray data collection (Fig. S2). The Pa125 data, consisting of 55% Pfr and 45% Pr, were refined at 2.97-Å resolution with a final R factor and free R factor of 23.3% and 30.7%, respectively. The Pa62 data with 67% Pfr and 33% Pr were refined at 2.85-Å resolution to a final R factor and free R factor of 23% and 29.4%, respectively. The final model contains two PaBphP-PCM-Q188L monomers, each with one chromophore and one water. Segments spanning residues 1–5, 368–370, 396–405, 417–421, 434–447, 495–497, and eight Cterminal tag residues are not modeled in either monomer because of disorder in these regions (Table S3). Although we grew our WT and Q188L crystals in the dark, it is not possible in practice to totally avoid light when setting up crystallization and handling photoactive crystals during cryofreezing. The significantly reduced dark-reversion rate of Q188L makes it much more difficult to retain the Pfr state at 100% occupancy than in WT crystals. When cryoprotected under the same light conditions, the Q188L crystals always exhibit absorption spectra of the mixed Pr and Pfr states at 100 K (Fig. S2) and revert in the dark to the Pfr state very slowly, even at room temperature, whereas the WT crystals exhibit spectra typical of the Pfr state at 100 K (data not shown). The mixed Pr/Pfr states likely contribute to the disordered electron densities in the Q188L crystals, especially in the PHY domain. The PHY domain and its extended arm region are packed along the principal 65 screw axis and form a cylindrical solvent channel with a diameter of 35 Å (Fig. S3). The Q188L crystals, with weaker packing constraints on the PHY domain, evidently exhibit higher tolerance of the coexistence of the Pr and Pfr states in the crystal lattice than the tightly packed WT crystals in space group C2221.
Fig. 3. Conformational differences in the chromophore-binding pocket between the Pfr (cyan) and Pr (gray) states. (A) Interactions between the propionate groups of rings B/C and the protein moiety in the Pfr (red dashed line) and Pr (green dashed line) states (PDB ID code 3G6O). (B and C) The SA-omit maps of the side chains of Tyr-163 and Tyr-190 flanking ring D in Q188L-Pa125 and Q188L-Pa62.
aligned C␣ atoms of 0.4–1.3 Å (Table S1). We argue that given the relatively uniform protein backbones, localized structural differences near the chromophore reflect ‘‘true’’ conformational differences between the Pr and Pfr states. They are less likely to arise from the fact that we are comparing different bacteriophytochromes crystallized under different conditions. The major conformational difference in the chromophore between the Pr and Pfr states can be summarized by ‘‘flip-and-rotate’’ motions. Upon absorbing a photon, ring D flips about the C15AC16 double bond. After several intermediate events, the entire chromophore eventually rotates relative to the protein moiety, with an overall rotation centered around ring A and along a rotation axis approximately perpendicular to the plane of rings B and C (Fig. 3A). Consequently, interactions with ring D and the propionate groups of rings B and C are rearranged. In the Pfr state of PaBphP-PCM, the side chain of Arg-209 interacts directly with the propionate groups of ring B, and the side chains of Ser-275, His-277, and Tyr-163 are within hydrogen-bonding distance from the propionate group of ring C. In the Pr state, in contrast, residues Arg-241 and Ser-261 interact with the propionate groups (Fig. 3). All of these residues except Ser-275 are highly conserved among all phytochromes. We examined the role of residues that may serve as protein anchors for the propionate groups in the Pfr state with singlealanine substitutions in PaBphP-PCM (Fig. 4). The single mutants S275A and Y163A retain the Pfr dark state, whereas R209A displays the mixed Pr/Pfr state, even after dark incubation; they form the Pr state after illumination at 750 nm. H277A adopts the Pr dark state and photoconverts to the Pfr state with limited efficiency, a photoconversion phenotype similar to that found in canonical bacteriophytochromes, such as RpBphP2 and Agp1 (15, 16). The rate of dark reversion to the Pfr state is reduced in the R209A and S275A mutants compared with WT but is increased in the Y163A mutant. These data suggest that Arg-209, Ser-275, and His-277 contribute to stabilize the Pfr state in PaBphP, likely via hydrogen bonds with the propionate groups, and His-277 seems to play an important role. Alanine substitutions of the side chains interacting with the propionate groups in the Pr state also affect photoconversion in PaBphP. The S261A mutant adopts the Pfr dark state but shows no detectable formation of the Pr state upon illumination at 750 nm (Fig. 4). This may result either from completely blocked photoconversion to the Pr state or from formation of a Pr state that is too short-lived to be detected in our experiments. In the R241A mutant, 15642 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0902178106
the absorbance ratio of the Q band and Soret band (A750/A400) is reduced. Although R241A photoconverts to the Pr state, its dark reversion is faster than that of WT. These data suggest that Ser-261 is essential and Arg-241 is also involved in forming a stable Pr state in PaBphP through interactions with the propionate group of ring B. The roles of residues corresponding to Arg-241 and Arg-209 in stabilizing the Pr and Pfr states have been explored in other bacteriophytochromes, such as DrBphP (17), Cph1 (18), and a phytochrome Synechococcus OS-B⬘ Cph1 (SyB-Cph1) that lacks a PAS domain (19). Substitution of Arg-254 in DrBphP (corresponding to Arg-241 of PaBphP) with alanine resulted in a lower ratio of the Q-band absorbance (700 nm) relative to the Soret band (400 nm). The R254A mutant of DrBphP did not affect the formation of the Pfr state upon illumination but displayed no detectable dark reversion to the Pr state (17). The recent NMR structure of the GAF domain of SyB-Cph1 also revealed high mobility around the residues Arg-133 and Arg-101, corresponding to Arg-241 and Arg-209 in PaBphP, which might arise from the propionate groups switching between the Pr and Pfr states (19). These results are consistent with the structural basis of the flip-and-rotate model. Comparisons between the Pr and Pfr structures show that Tyr-163 in the Pr state and Tyr-190 in the Pfr state occupy the same location flanking the cavity for ring D (figure 3a in ref. 12). We suggested previously that Tyr-190 and Tyr-163 switch their sidechain rotamers in concert with motions in the chromophore during photoconversion. This proposal is supported by the SA-omit maps of Q188L Pa125 and Pa62, in which 11 surrounding residues, including Tyr-163 and Tyr-190, are omitted in addition to the chromophore. Electron density for the side chain of Tyr-163 is no longer consistent with a single conformation in the Pfr structure in both maps. Tyr-163 exhibits significant additional density overlapping with Tyr-190 in the Pfr state, which is most evident in Pa125. The disordered side chain of Tyr-190 also displays density indicating dual conformations (Fig. 3C). Although both the Y163A and Y190A mutants retain the Pfr ground state, the Y163A mutant exhibits greatly reduced efficiency of photoconversion to the Pr state and quickly reverts to the Pfr state (Fig. 4). The Y190A mutant, on the other hand, exhibits a significantly weakened and broadened absorption band in the red, in which max is blue-shifted by 17 nm to 733 nm. The different photoconversion behaviors in the Y163A and Y190A mutants may arise partly from increased mobility of ring D upon removal of the Yang et al.
state, and the Y176H mutant in Cph1 is intensely fluorescent, with an emission maximum at 650–670 nm. Saturation mutagenesis at Tyr-176 of Cph1 implied that its hydroxyl group is important for efficient Pr-to-Pfr photoconversion (22). In the PaBphP Pfr structure, the side chain of Tyr-163 makes a hydrogen bond with the propionate group of ring C, an interaction that is absent in the Pr structures (Fig. 3B). We speculate that the conserved Tyr-163 residue is important for mediating the Pr-to-Pfr reaction but is not essential for the reverse pathway from the Pfr to the Pr state. This is not unexpected; time-resolved spectroscopic studies on plant phytochromes and Agp1 reveal quite distinct intermediates for the forward and reverse photoconversion reactions (23, 24). However, unlike Cph1 and Phys, Y163H of PaBphP exhibits no fluorescence enhancement compared with WT, suggesting that Tyr-163 plays a distinct role in the Pr-to-Pfr reaction in PaBphP from its equivalents in Cph1 and Phys (22). Taken together, our structural and mutational analyses on PaBphP-PCM suggest that the hydrogen-bonding network between the chromophore and its protein environment undergoes extensive rearrangement between the Pr and Pfr states to accommodate the flip-and-rotate motion of the biliverdin IX␣ (BV) chromophore. The mixed Pr and Pfr states in our Q188L crystal structures provide evidence that these conformational changes occur in the photoactive crystals. This flip-and-rotate model is consistent with recent 13C and 15N magic-angle spinning NMR studies that detected changes in the electronic structure of the chromophore and interactions with its environment, based on chemical shift differences between the Pr and Pfr states of the PCMs in Cph1 and plant phyA (10). More significant chemical shifts are associated with rings C/D than with rings A/B (rotation). Rohmer et al. (10) inferred that hydrogenbonding interaction with the ring D carbonyl increases in the Pfr state (flip), and a significant change in the protein environment occurs around the propionate carboxylate group of ring C (rotate). It is plausible that the reverse Pr-to-Pfr reaction undergoes similar flip-and-rotate motions, probably with structural intermediates that differ in detail from those in the Pfr-to-Pr reaction. Although crystal structures probe the static Pr and Pfr states, molecular events lying between these end states hold the key to molecular mechanism of reversible Pr/Pfr photoconversion, and they remain to be experimentally resolved in both space and time.
Fig. 4. Absorption properties of the PaBphP-PCM WT and selected mutant proteins in solution. The spectra in solid lines represent the dark-adapted state; spectra in dotted lines are taken immediately after 5-min illumination at a wavelength of 750 or 690 nm; and spectra in dashed lines are recorded after 5-min dark reversion. Estimated half-times of dark reversion are indicated in parentheses.
bulky side chains of Tyr-163 or Tyr-190 flanking its cavity. However, Tyr-163 seems to play a role beyond simple space filling, as is evident from the properties of the Y163H mutant. This mutant can be converted from the Pfr to the Pr state by illumination at 750 nm, but illumination at 690 nm cannot drive the reverse process, and spontaneous reversion is significantly slower than in WT (half-time ⬎90 min versus 5 min; Fig. S4A). The Y163H mutant is also modestly fluorescent upon excitation at 400 nm, with two emission peaks near 615 and 722 nm (Fig. S4B). The equivalent mutants Y176H in Cph1 (20) and Y276H in PhyB (21) do not undergo detectable photoconversion from their ground Pr state to the Pfr Yang et al.
binding pocket, the most striking structural differences between the PaBphP-PCM and Cph1-PCM structures are located at the Nterminal extension of the PAS domain and the arm of the PHY domain that interact with the GAF domain (Fig. S5A). The N terminus of the PAS domain threads through a 41 knot and shields ring A of the chromophore. In the Cph1-PCM structure, the N-terminal extension of the PAS domain adopts a three-turn helical conformation, but in both PaBphP-PCM WT and Q188L structures, it forms an unstructured coil that contains the covalent Cys-12 anchor for the BV chromophore. Although the arm regions of the PHY domains are comparable in length, the Cph1-PCM and PaBphP-PCM structures adopt very distinct secondary structure elements. In Cph1-PCM, this region consists of several -strands connected by extended coils. In contrast, the arm of the PHY domain in the PaBphP structures contains a three-turn helix preceded by a structural segment largely consisting of random coil. The surface patches of the GAF domains buried by the PAS and PHY domains do not overlap exactly, with 2,713 Å2 of buried surface area in the Cph1-PCM structure and 3,234 Å2 in the PaBphP-PCM-WT structure. Such secondary structural differences in the arm regions (␣-helix versus -strand) and the N termini of the PAS domains (coil versus ␣-helix) are probably too extensive for them to represent the end points of a single photoconversion trajectory. There is no evidence for such drastic structural differences in the photoactive Q188L PNAS 兩 September 15, 2009 兩 vol. 106 兩 no. 37 兩 15643
BIOCHEMISTRY
Global Structural Differences. More distant from the chromophore-
crystal structure containing the mixed Pr/Pfr states. In addition, the DrBphP-CBM Pr structure adopts a very similar conformation to the PaBphP Pfr structure in the N-terminal extension, despite the absence of a PHY domain. These large differences may originate in different chromophores (BV in PaBphP versus PCB in Cph1) covalently linked to different Cys anchors (Cys-12 in the PAS domain of PaBphP versus Cys-259 in the GAF domain of Cph1). The relative orientation of the PHY and GAF domains differs markedly between the PaBphP-PCM-WT and Cph1-PCM structures (Fig. S5B). The long helices connecting the GAF and PHY domains bend in opposite directions in the Pfr and Pr structures, thus demonstrating flexibility of helices at the dimer interface (Fig. S5C). These global structural differences might simply derive from distinct molecular packing under different crystallization conditions or from sequence differences, but other evidence is relevant. First, illumination of photoactive PaBphP-PCM-WT and PaBphPPCM-Q188L crystals at room temperature results in severe loss of crystal order. A similar observation was reported for the Cph1PCM crystals (13). Light-induced lattice disorder implies that substantial tertiary and/or quaternary structural changes occur in the photoactive crystals, as is also evident in weaker densities for the entire core PHY domain in the Q188L mutant structure. Second, attempts to cross-crystallize PaBphP-PCM-WT and PaBphPPCM-Q188L in the dark were not successful. Because most side chains in the central helices of the Q188L structure are wellordered, we argue against the otherwise plausible suggestion that the mixed Pr/Pfr states lead to the quaternary structural differences at the dimer interface. It is also possible that general structural perturbations from the single-residue substitution (Q188L) affect dimerization and crystal packing. But disorder in the PHY domain, on the other hand, might arise directly from the mixed Pr/Pfr states in the Q188L crystals. Despite their varied origins, these global structural differences certainly reflect intrinsic plasticity required by signaling proteins to transmit signals over significant distances to the effector domains. 1. Montgomery BL, Lagarias JC (2002) Phytochrome ancestry: Sensors of bilins and light. Trends Plant Sci 7:357–366. 2. Hughes J, Lamparter T (1999) Prokaryotes and phytochrome. The connection to chromophores and signaling. Plant Physiol 121:1059 –1068. 3. Davis SJ, Vener AV, Vierstra RD (1999) Bacteriophytochromes: Phytochrome-like photoreceptors from nonphotosynthetic eubacteria. Science 286:2517–2520. 4. Bhoo SH, Davis SJ, Walker J, Karniol B, Vierstra RD (2001) Bacteriophytochromes are photochromic histidine kinases using a biliverdin chromophore. Nature 414:776 –779. 5. Rockwell NC, Su YS, Lagarias JC (2006) Phytochrome structure and signaling mechanisms. Annu Rev Plant Biol 57:837– 858. 6. Rockwell NC, Lagarias JC (2006) The structure of phytochrome: A picture is worth a thousand spectra. Plant Cell 18:4 –14. 7. Noack S, Michael N, Rosen R, Lamparter T (2007) Protein conformational changes of Agrobacterium phytochrome Agp1 during chromophore assembly and photoconversion. Biochemistry 46:4164 – 4176. 8. Mroginski MA, Murgida DH, Hildebrandt P (2007) The chromophore structural changes during the photocycle of phytochrome: A combined resonance Raman and quantum chemical approach. Acc Chem Res 40:258 –266. 9. Hahn J, Strauss HM, Schmieder P (2008) Heteronuclear NMR investigation on the structure and dynamics of the chromophore binding pocket of the cyanobacterial phytochrome Cph1. J Am Chem Soc 130:11170 –11178. 10. Rohmer T, et al. (2008) Light-induced chromophore activity and signal transduction in phytochromes observed by 13C and 15N magic-angle spinning NMR. Proc Natl Acad Sci USA 105:15229 –15234. 11. Esteban B, Carrascal M, Abian J, Lamparter T (2005) Light-induced conformational changes of cyanobacterial phytochrome Cph1 probed by limited proteolysis and autophosphorylation. Biochemistry 44:450 – 461. 12. Yang X, Kuk J, Moffat K (2008) Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: Photoconversion and signal transduction. Proc Natl Acad Sci USA 105:14715–14720. 13. Essen LO, Mailliet J, Hughes J (2008) The structure of a complete phytochrome sensory module in the Pr ground state. Proc Natl Acad Sci USA 105:14709 –14714. 14. Yang X, Stojkovic EA, Kuk J, Moffat K (2007) Crystal structure of the chromophore binding domain of an unusual bacteriophytochrome, RpBphP3, reveals residues that modulate photoconversion. Proc Natl Acad Sci USA 104:12571–12576. 15. Giraud E, et al. (2005) A new type of bacteriophytochrome acts in tandem with a classical bacteriophytochrome to control the antennae synthesis in Rhodopseudomonas palustris. J Biol Chem 280:32389 –32397.
15644 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0902178106
Materials and Methods Cloning, Mutagenesis, and Purification. Site-directed mutagenesis and protein purification were carried out as described previously (12, 14). UV-Visible Spectroscopy. UV-visible spectra of purified WT and mutant PaBphPPCM proteins in solution were recorded at room temperature from 230 to 900 nm with a Shimadzu UV-1650 PC spectrophotometer. Spectra were recorded either in the dark-adapted state or after illumination at 750 nm (far red), 690 nm (red) provided by interference filters with a 10-nm bandwidth (Andover). Visible spectra of crystals were recorded at ambient and cryogenic temperatures with a microspectrophotometer (Xspectra) at BioCARS, Advanced Photon Source (APS; Argonne National Laboratory, Argonne, IL). Crystallization and Data Collection. The Q188L mutant was crystallized from 0.5% (wt/vol) PEG4000 (Fluka) and 0.01 M sodium acetate, pH 4.6, with a final protein concentration of 10 mg/mL at 20 °C in the dark. Crystals were handled and frozen as described previously for WT (12). Microspectrophotometry shows that the Q188L crystals undergo Pr/Pfr photoconversion at ambient temperature (Fig. S2). All diffraction data from the Se-Met and native crystals were collected at 100 K at the Structural Biology Center 19-ID and BioCARS 14-ID beam stations at the APS. All images were indexed, integrated, and scaled by using HKL2000 or HKL3000 (HKL Research). Structure Determination and Refinement. The crystal structure of the Q188L mutant was determined by MAD method using Solve (25) and Sharp (26) at 2.9-Å resolution, and it was initially refined with CNS (25) and Refmac5 (27, 28). The two Q188L models (Pa62 and Pa125) were refined with different Pr/Pfr compositions by using PHENIX (29). The buried surface areas were calculated with CNS. Coot (30) was used for all model building and map fitting. A least-square procedure (lsqkab in CCP4) was used for structural alignment. Structures and electron-density maps were illustrated by using PyMOL (http:// pymol.org). Data collection, phasing, and refinement statistics are summarized in Table S3. ACKNOWLEDGMENTS. We thank Ying Pigli and Yuen-Ling Chan for help and advice in cloning and mutagenesis; Vukica Sˇrajer of BioCARS for assistance in microspectroscopic experiments on crystals; Zhong Ren of BioCARS for map analysis algorithms; and the reviewers for helpful comments. We also thank the staff of the Structural Biology Center and BioCARS at the APS, Argonne National Laboratory for beam line access. This work was supported by National Institutes of Health Grant GM036452 (to K.M.).
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