Conformational differences between the Pfrand Pr states in Pseudomonasaeruginosa bacteriophytochromeXiaojing 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)

Phytochromes are red-light photoreceptors that regulate lightresponses in plants, fungi, and bacteria by means of reversiblephotoconversion between red (Pr) and far-red (Pfr) light-absorbingstates. Here, we report the crystal structure of the Q188L mutantof Pseudomonas aeruginosa bacteriophytochrome (PaBphP) pho-tosensory core module, which exhibits altered photoconversionbehavior and different crystal packing from wild type. We observetwo distinct chromophore conformations in the Q188L crystalstructure that we identify with the Pfr and Pr states. The Pr/Pfrcompositions, varying from crystal to crystal, seem to correlatewith light conditions under which the Q188L crystals are cryopro-tected. We also compare all known Pr and Pfr structures. Usingsite-directed mutagenesis, we identify residues that are involved instabilizing the 15Ea (Pfr) and 15Za (Pr) configurations of thebiliverdin chromophore. Specifically, Ser-261 appears to be essen-tial to form a stable Pr state in PaBphP, possibly by means of itsinteraction with the propionate group of ring C. We propose a‘‘flip-and-rotate’’ model that summarizes the major conforma-tional differences between the Pr and Pfr states of the chro-mophore and its binding pocket.

biliverdin � photoconversion � red-light photoreceptor

Phytochromes are red-light photoreceptors that undergo re-versible photoconversion between a red-light-absorbing

state (Pr) and a far-red-light-absorbing state (Pfr), and therebythey regulate a wide range of physiological responses in plants,fungi, and photosynthetic bacteria (1–5). Using linear tetrapyr-roles as chromophores to detect light in the long-wavelengthrange of the visible spectrum, the photosensory core module(PCM) of bacteriophytochromes contains three domains (PAS,GAF, and PHY). The PAS and GAF domains constitute thechromophore-binding module (CBM); and the PHY domain isessential for efficient photoconversion (5). Upon absorbing aphoton, 15Za/15Ea isomerization occurs about the C15AC16double bond between rings C and D of the bilin chromophore,followed by thermal relaxation events in the chromophore andthe protein matrix (6). Local conformational changes originatingin the photosensory domains propagate to the C-terminal his-tidine kinase (HK) domain, where they modulate the kinaseactivity and thus convert a light signal into a chemical signal (5).Fundamental questions about the molecular mechanisms ofphotoconversion and signal transduction remain unanswered.What are the local and long-range conformational changes?What molecular events are involved? In what sequence do theyoccur?

Extensive studies on a variety of phytochromes and bacteriophy-tochromes suggest that significant structural changes occur in boththe chromophore and protein moieties during Pr/Pfr photoconver-sion, but details of these changes are still lacking (7–11). Two crystalstructures of bacteriophytochromes with intact PCMs have beendetermined recently: that of Pseudomonas aeruginosa bacteriophy-tochrome (PaBphP) (12) and that of cyanobacterial phytochromeCph1 from Synechocystis sp. 6803 (13). Both structures include thePHY domain and represent their dark-adapted Pfr and Pr states,

respectively. Here, we report the crystal structure of a point mutantof the PaBphP-PCM (Q188L) that appears to exhibit mixed Pfr andPr states in the crystal. The Q188L crystals have distinct crystalpacking and grow under crystallization conditions different fromthat for WT. Based on the Q188L crystal structure, structuralcomparisons among the crystal structures in the Pr and Pfr states,and site-directed mutagenesis, we identify several residues andstructural elements that undergo conformational changes duringPr/Pfr photoconversion.

Results and DiscussionCrystal Structure of PaBphP-PCM Q188L. The Q188L mutant of thePaBphP PCM in solution adopts the Pfr state in the dark (figure2d of ref. 12). The mutant undergoes reversible Pr/Pfr photo-conversion, but its rate of reversion to the Pfr state in the darkis significantly slower than that of WT, with a half-time of about2 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�HCl, pH 7.7) werenot successful. Instead, crystals of the Q188L mutant with atypical size of 400�100�100 �m were obtained in hanging dropsvia 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-Q188Lat 2.9-Å resolution in space group P65 by the multiwavelengthanomalous dispersion (MAD) method using Se-Met-substitutedcrystals (table S1 in ref. 12). The overall arrangement of the PAS,GAF, and PHY domains in the Q188L structure is very similarto that in the WT structure, where all three sensory domainsconverge on the chromophore via a 41 knot between the PAS andGAF domains and an extended arm of the PHY domain. Thepairwise rms differences of the main-chain C� atoms betweenthe Q188L and WT structures are very small in the GAF domainbut are more significant in the PHY domain. The largestdifferences occur near both ends of the long helical bundle at thedimer interface (Fig. 1A). Although crystallized under condi-tions completely different from those for WT, the molecules ofthe Q188L mutant in the asymmetric unit are packed as aparallel, head-to-head dimer closely similar to the WT structure(12) but quite unlike the antiparallel dimer in the Cph1-PCMstructure (13). Two monomers in one asymmetric unit arerelated by near-perfect noncrystallographic twofold symmetrywhose 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 inthe Protein Data Bank, www.pdb.org (PDB ID codes 3G6O and 3IBR).

1To whom correspondence may be addressed. E-mail: xiaojingyang@uchicago.edu ormoffat@cars.uchicago.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0902178106/DCSupplemental.

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in contrast to the WT crystal structure, in which conformationalheterodimers are evidenced by two locations of the GAF-hAhelix (12). The Q188L dimer buries significantly less surface areaat the dimer interface compared with WT (2,690 Å2 versus 3,950Å2). Helices comprising the central helical bundle at the dimerinterface gradually separate when they extend into the PHYdomain of the Q188L structure (Fig. 1B).

Although the electron density of the PAS and GAF domains iswell defined, that of the PHY domain is less so, especially for theside chains of its core structural elements. The initial side-chainconformations of the Q188L model in the PHY domain weremostly adopted from the refined WT structure, in which theelectron 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 generalfeatures 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) struc-tures, the SA-omit maps are less well defined in Q188L, with visiblybroadened electron densities, especially in the regions of rings D/Cand the propionate groups of rings B and C (Fig. 2). Neither aZZEssa (Pfr) nor a ZZZssa (Pr) configuration alone can accountfor all observed densities. If a Q188L model is refined with thechromophore exclusively in the ZZEssa configuration, the residualFo � Fc difference maps clearly show positive densities (�3.0�)near the chromophore, indicating the existence of a secondconformation (data not shown). Alternatively, a single chro-mophore configuration can be modeled lying roughly betweenthose of the DrBphP-CBM (Pr) and PaBphP-PCM WT (Pfr)structures that approximately satisfies the electron density inQ188L, but such a model forms no reasonable hydrogen bondswith its surrounding protein. We further ruled out the possibilitythat X-ray radiation damage contributes to the broader densitydistribution in the chromophore. First, no significant differenceis observed between the SA-omit maps calculated with twodifferent Q188L datasets that were collected consecutively fromthe same Q188L crystal volume (data not shown). In addition,radiation damage has not been a determining factor for thechromophore conformations in other phytochrome structures,

although some evidence suggests that X-ray absorption rupturesthe covalent bond between the chromophore and its anchoringCys (14). We therefore propose that these broader map featuresresult from a mixture containing more than one chromophoreconformation.

Interestingly, but not surprisingly, these Q188L SA-omit mapscan be satisfactorily accounted for by superimposing the chro-mophores of the DrBphP-CBM and PaBphP-PCM WT structuresthat are individually transformed to a reference Q188L structurebased on the GAF core domains (equivalent residues: 157–280 inPaBphP and 170–293 in DrBphP; Fig. 2). We note that the Pr(DrBphP-CBM and RpBphP3-CBM) and the Pfr (PaBphP-PCM-WT) structures display distinct map features for the chromophorerelative to the GAF protein scaffold, in which main-chain atoms areclosely grouped among aligned BphP structures (Fig. 2 and TableS1). Furthermore, mixing two SA-omit maps in the Pr and Pfr statesleads to broader density around ring D and for the propionategroups of rings C and D, similar to those features observed in theSA-omit maps of Q188L (Fig. S1B). Therefore, we further proposethat the Q188L crystals contain a mixture of the Pfr and Pr states.To estimate the compositions of the Pr and Pfr states in the Q188LSA-omit maps, we developed a least-squares procedure. In thisprocedure, we used the SA-omit maps of the chromophores fromDrBphP-CBM and PaBphP-PCM-WT as basis maps to representthe ‘‘pure’’ Pr and Pfr states, respectively.

Table S2 shows that the Pr and Pfr states coexist in all Q188Lcrystals we have examined, but to a different extent from crystal tocrystal. The composition of the Pr state, the ‘‘light’’ state for Q188L,seems to correlate with the light conditions under which the Q188Lcrystals were cryoprotected. The Q188L crystals exposed only todouble-filtered (green and blue) microscope light contain less Prstate than crystals exposed to more illumination. From more than30 crystals exposed to singly green-filtered microscope light, onlyone crystal (Pa125) diffracts beyond 3.5-Å resolution. This crystalexhibits the most extensive map features characteristic of the Prstate (Fig. 2). We subjected two Q188L datasets (Pa125 and Pa62)to further map analysis and structure refinement. The SeMet Pa125dataset contained the most Pr state (�45% Pr and �55% Pfr),whereas the native Pa62 crystal, with brief exposure to double-filtered microscope light, retained more Pfr state (�33% Pr and

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 DataBank (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 structuresare aligned based on the left monomer. Same view as in A.

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�67% Pfr) and yielded a dataset with the best resolution for anyQ188L crystal (Table S2).

Because of limited diffraction resolution, the electron-densitymap alone does not definitively distinguish the E versus Z config-uration for ring D in the mixed Pr/Pfr states. However, based onconserved side chains that interact with ring D in the Pr and Pfrstructures, such as Asp-194 in the Pfr state and His-277 in the Prstate, we assigned 15Ea to the configuration in cyan and 15Za to thesecond configuration in gray, which corresponded to the Pfr and Prstates, respectively (Figs. 2 and 3A). To explore conformationalchanges beyond the chromophore, we also calculated the SA-omitmaps in which the side-chain atoms for 11 residues (Cys-12,

Tyr-163, Tyr-190, Asp-194, Tyr-203, Arg-209, Arg-241, His-247,His-277, Tyr-250, and Ser-261) that immediately surround thechromophore were omitted. Tyr-190, Tyr-163 and Arg-209 exhibitbranched side-chain densities that are most evident in the SA-omitmap of Pa125 (Fig. 3C). The centroids of the side chains densitiesin Cys-12, Tyr-203, and Tyr-250 are slightly shifted between thealigned Pa62 and Pa125 maps, whereas Asp-194, Arg-241, His-247,Ser-261, and His-277 display no significant differences betweenthese maps. We thus model dual conformations for Tyr-163,Tyr-190 Arg-209, Cys-12, Tyr-203, and Tyr-250 based on theSA-omit maps as well as the known Pr/Pfr structures. The covalentbonds between the SG atom of Cys-12 and the C32 atom of ring Ain the Pr and Pfr states are modeled based on the best fitting of theSA-omit maps of the region; any effect of X-ray radiation on thecovalent bond was not modeled. The occupancies of the Pr and Pfrstates are estimated by the least-squares fitting of the SA-omit mapsand the map contours of related Pr/Pfr features. We also validatedthe Pr/Pfr occupancies in Pa125 by using an absorption spectrummeasured from the Pa125 crystal at 100 K after X-ray datacollection (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 freeR 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 factorand free R factor of 23% and 29.4%, respectively. The final modelcontains two PaBphP-PCM-Q188L monomers, each with onechromophore and one water. Segments spanning residues 1–5,368–370, 396–405, 417–421, 434–447, 495–497, and eight C-terminal tag residues are not modeled in either monomer becauseof disorder in these regions (Table S3).

Although we grew our WT and Q188L crystals in the dark, it isnot possible in practice to totally avoid light when setting upcrystallization and handling photoactive crystals during cryofreez-ing. The significantly reduced dark-reversion rate of Q188L makesit much more difficult to retain the Pfr state at 100% occupancythan in WT crystals. When cryoprotected under the same lightconditions, the Q188L crystals always exhibit absorption spectra ofthe mixed Pr and Pfr states at 100 K (Fig. S2) and revert in the darkto the Pfr state very slowly, even at room temperature, whereas theWT crystals exhibit spectra typical of the Pfr state at 100 K (datanot shown).

The mixed Pr/Pfr states likely contribute to the disorderedelectron densities in the Q188L crystals, especially in the PHYdomain. The PHY domain and its extended arm region are packedalong the principal 65 screw axis and form a cylindrical solventchannel with a diameter of 35 Å (Fig. S3). The Q188L crystals, withweaker packing constraints on the PHY domain, evidently exhibithigher tolerance of the coexistence of the Pr and Pfr states in thecrystal lattice than the tightly packed WT crystals in space groupC2221.

Conformational Differences in the Chromophore-Binding Pocket. Thetwo chromophore configurations (ZZEssa and ZZZssa) in theQ188L crystal structure are related by 15Ea/15Za isomerizationabout the C15AC16 double bond of the methine linkage betweenrings C and D, and by rotation roughly around ring A (Fig. 2). Thisis consistent with structural differences between the chromophoresin 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 struc-ture) show remarkable uniformity in the interactions between theZZZssa chromophore and the surrounding protein environment,regardless of the presence or absence of the PHY domain or thechemical nature of the chromophore (Fig. S1D) (13). The PaBphP-PCM structure in the Pfr state, on the other hand, exhibits a distinctchromophore configuration (ZZEssa) and differs in stabilizinginteractions from the Pr structures (Fig. 3A and Fig. S1D) (12).However, the overall structures of the CBMs in the Pr and Pfr statesare remarkably similar, with small pairwise rmsd values between

Fig. 2. Stereoviews of the SA-omit maps of the chromophore in the Pr, Pfr, andmixed 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 andPaBphP-PCM, but a mixture of the two models is required for both Q188L crystals.

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aligned C� atoms of 0.4–1.3 Å (Table S1). We argue that given therelatively uniform protein backbones, localized structural differ-ences near the chromophore reflect ‘‘true’’ conformational differ-ences between the Pr and Pfr states. They are less likely to arisefrom the fact that we are comparing different bacteriophyto-chromes crystallized under different conditions.

The major conformational difference in the chromophore be-tween the Pr and Pfr states can be summarized by ‘‘flip-and-rotate’’motions. Upon absorbing a photon, ring D flips about theC15AC16 double bond. After several intermediate events, theentire chromophore eventually rotates relative to the proteinmoiety, with an overall rotation centered around ring A and alonga rotation axis approximately perpendicular to the plane of rings Band C (Fig. 3A). Consequently, interactions with ring D and thepropionate groups of rings B and C are rearranged. In the Pfr stateof PaBphP-PCM, the side chain of Arg-209 interacts directly withthe propionate groups of ring B, and the side chains of Ser-275,His-277, and Tyr-163 are within hydrogen-bonding distance fromthe propionate group of ring C. In the Pr state, in contrast, residuesArg-241 and Ser-261 interact with the propionate groups (Fig. 3).All of these residues except Ser-275 are highly conserved among allphytochromes.

We examined the role of residues that may serve as proteinanchors for the propionate groups in the Pfr state with single-alanine substitutions in PaBphP-PCM (Fig. 4). The single mutantsS275A and Y163A retain the Pfr dark state, whereas R209Adisplays the mixed Pr/Pfr state, even after dark incubation; theyform the Pr state after illumination at 750 nm. H277A adopts thePr dark state and photoconverts to the Pfr state with limitedefficiency, a photoconversion phenotype similar to that found incanonical bacteriophytochromes, such as RpBphP2 and Agp1 (15,16). The rate of dark reversion to the Pfr state is reduced in theR209A and S275A mutants compared with WT but is increased inthe Y163A mutant. These data suggest that Arg-209, Ser-275, andHis-277 contribute to stabilize the Pfr state in PaBphP, likely viahydrogen bonds with the propionate groups, and His-277 seems toplay an important role.

Alanine substitutions of the side chains interacting with thepropionate groups in the Pr state also affect photoconversion inPaBphP. The S261A mutant adopts the Pfr dark state but shows nodetectable formation of the Pr state upon illumination at 750 nm(Fig. 4). This may result either from completely blocked photocon-version to the Pr state or from formation of a Pr state that is tooshort-lived to be detected in our experiments. In the R241A mutant,

the absorbance ratio of the Q band and Soret band (A750/A400) isreduced. Although R241A photoconverts to the Pr state, its darkreversion is faster than that of WT. These data suggest that Ser-261is essential and Arg-241 is also involved in forming a stable Pr statein PaBphP through interactions with the propionate group ofring B.

The roles of residues corresponding to Arg-241 and Arg-209in stabilizing the Pr and Pfr states have been explored in otherbacteriophytochromes, such as DrBphP (17), Cph1 (18), and aphytochrome Synechococcus OS-B� Cph1 (SyB-Cph1) that lacksa PAS domain (19). Substitution of Arg-254 in DrBphP (corre-sponding to Arg-241 of PaBphP) with alanine resulted in a lowerratio of the Q-band absorbance (700 nm) relative to the Soretband (400 nm). The R254A mutant of DrBphP did not affect theformation of the Pfr state upon illumination but displayed nodetectable dark reversion to the Pr state (17). The recent NMRstructure of the GAF domain of SyB-Cph1 also revealed highmobility around the residues Arg-133 and Arg-101, correspond-ing to Arg-241 and Arg-209 in PaBphP, which might arise fromthe propionate groups switching between the Pr and Pfr states(19). These results are consistent with the structural basis of theflip-and-rotate model.

Comparisons between the Pr and Pfr structures show thatTyr-163 in the Pr state and Tyr-190 in the Pfr state occupy the samelocation flanking the cavity for ring D (figure 3a in ref. 12). Wesuggested previously that Tyr-190 and Tyr-163 switch their side-chain rotamers in concert with motions in the chromophore duringphotoconversion. This proposal is supported by the SA-omit mapsof Q188L Pa125 and Pa62, in which 11 surrounding residues,including Tyr-163 and Tyr-190, are omitted in addition to thechromophore. Electron density for the side chain of Tyr-163 is nolonger consistent with a single conformation in the Pfr structure inboth maps. Tyr-163 exhibits significant additional density overlap-ping with Tyr-190 in the Pfr state, which is most evident in Pa125.The disordered side chain of Tyr-190 also displays density indicatingdual conformations (Fig. 3C).

Although both the Y163A and Y190A mutants retain the Pfrground state, the Y163A mutant exhibits greatly reduced efficiencyof photoconversion to the Pr state and quickly reverts to the Pfrstate (Fig. 4). The Y190A mutant, on the other hand, exhibits asignificantly weakened and broadened absorption band in the red,in which �max is blue-shifted by 17 nm to 733 nm. The differentphotoconversion behaviors in the Y163A and Y190A mutants mayarise partly from increased mobility of ring D upon removal of the

Fig. 3. Conformational differences in the chromophore-binding pocket between the Pfr (cyan) and Pr (gray) states. (A) Interactions between the propionategroups 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 theside chains of Tyr-163 and Tyr-190 flanking ring D in Q188L-Pa125 and Q188L-Pa62.

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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 isevident from the properties of the Y163H mutant. This mutant canbe converted from the Pfr to the Pr state by illumination at 750 nm,but illumination at 690 nm cannot drive the reverse process, andspontaneous reversion is significantly slower than in WT (half-time�90 min versus 5 min; Fig. S4A). The Y163H mutant is alsomodestly fluorescent upon excitation at 400 nm, with two emissionpeaks near 615 and 722 nm (Fig. S4B). The equivalent mutantsY176H in Cph1 (20) and Y276H in PhyB (21) do not undergodetectable photoconversion from their ground Pr state to the Pfr

state, and the Y176H mutant in Cph1 is intensely fluorescent, withan emission maximum at 650–670 nm. Saturation mutagenesis atTyr-176 of Cph1 implied that its hydroxyl group is important forefficient Pr-to-Pfr photoconversion (22). In the PaBphP Pfr struc-ture, the side chain of Tyr-163 makes a hydrogen bond with thepropionate group of ring C, an interaction that is absent in the Prstructures (Fig. 3B). We speculate that the conserved Tyr-163residue is important for mediating the Pr-to-Pfr reaction but is notessential for the reverse pathway from the Pfr to the Pr state. Thisis not unexpected; time-resolved spectroscopic studies on plantphytochromes and Agp1 reveal quite distinct intermediates for theforward and reverse photoconversion reactions (23, 24). However,unlike Cph1 and Phys, Y163H of PaBphP exhibits no fluorescenceenhancement compared with WT, suggesting that Tyr-163 plays adistinct role in the Pr-to-Pfr reaction in PaBphP from its equivalentsin Cph1 and Phys (22).

Taken together, our structural and mutational analyses onPaBphP-PCM suggest that the hydrogen-bonding network be-tween the chromophore and its protein environment undergoesextensive rearrangement between the Pr and Pfr states toaccommodate the flip-and-rotate motion of the biliverdin IX�(BV) chromophore. The mixed Pr and Pfr states in our Q188Lcrystal structures provide evidence that these conformationalchanges occur in the photoactive crystals.

This flip-and-rotate model is consistent with recent 13C and 15Nmagic-angle spinning NMR studies that detected changes in theelectronic structure of the chromophore and interactions with itsenvironment, based on chemical shift differences between the Prand Pfr states of the PCMs in Cph1 and plant phyA (10). Moresignificant chemical shifts are associated with rings C/D than withrings A/B (rotation). Rohmer et al. (10) inferred that hydrogen-bonding interaction with the ring D carbonyl increases in the Pfrstate (flip), and a significant change in the protein environmentoccurs around the propionate carboxylate group of ring C (rotate).It is plausible that the reverse Pr-to-Pfr reaction undergoes similarflip-and-rotate motions, probably with structural intermediates thatdiffer in detail from those in the Pfr-to-Pr reaction. Althoughcrystal structures probe the static Pr and Pfr states, molecularevents lying between these end states hold the key to molecularmechanism of reversible Pr/Pfr photoconversion, and they remainto be experimentally resolved in both space and time.

Global Structural Differences. More distant from the chromophore-binding pocket, the most striking structural differences between thePaBphP-PCM and Cph1-PCM structures are located at the N-terminal extension of the PAS domain and the arm of the PHYdomain that interact with the GAF domain (Fig. S5A). The Nterminus of the PAS domain threads through a 41 knot and shieldsring A of the chromophore. In the Cph1-PCM structure, theN-terminal extension of the PAS domain adopts a three-turn helicalconformation, but in both PaBphP-PCM WT and Q188L struc-tures, it forms an unstructured coil that contains the covalentCys-12 anchor for the BV chromophore. Although the arm regionsof the PHY domains are comparable in length, the Cph1-PCM andPaBphP-PCM structures adopt very distinct secondary structureelements. In Cph1-PCM, this region consists of several �-strandsconnected by extended coils. In contrast, the arm of the PHYdomain in the PaBphP structures contains a three-turn helixpreceded by a structural segment largely consisting of random coil.The surface patches of the GAF domains buried by the PAS andPHY domains do not overlap exactly, with 2,713 Å2 of buriedsurface area in the Cph1-PCM structure and 3,234 Å2 in thePaBphP-PCM-WT structure.

Such secondary structural differences in the arm regions (�-helixversus �-strand) and the N termini of the PAS domains (coil versus�-helix) are probably too extensive for them to represent the endpoints of a single photoconversion trajectory. There is no evidencefor such drastic structural differences in the photoactive Q188L

Fig. 4. Absorption properties of the PaBphP-PCM WT and selected mutantproteins in solution. The spectra in solid lines represent the dark-adaptedstate; spectra in dotted lines are taken immediately after 5-min illuminationat a wavelength of 750 or 690 nm; and spectra in dashed lines are recordedafter 5-min dark reversion. Estimated half-times of dark reversion are indi-cated in parentheses.

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crystal structure containing the mixed Pr/Pfr states. In addition, theDrBphP-CBM Pr structure adopts a very similar conformation tothe PaBphP Pfr structure in the N-terminal extension, despite theabsence of a PHY domain. These large differences may originate indifferent chromophores (BV in PaBphP versus PCB in Cph1)covalently linked to different Cys anchors (Cys-12 in the PASdomain of PaBphP versus Cys-259 in the GAF domain of Cph1).

The relative orientation of the PHY and GAF domains differsmarkedly between the PaBphP-PCM-WT and Cph1-PCM struc-tures (Fig. S5B). The long helices connecting the GAF and PHYdomains 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 fromdistinct molecular packing under different crystallization condi-tions or from sequence differences, but other evidence is relevant.First, illumination of photoactive PaBphP-PCM-WT and PaBphP-PCM-Q188L crystals at room temperature results in severe loss ofcrystal order. A similar observation was reported for the Cph1-PCM crystals (13). Light-induced lattice disorder implies thatsubstantial tertiary and/or quaternary structural changes occur inthe photoactive crystals, as is also evident in weaker densities for theentire core PHY domain in the Q188L mutant structure. Second,attempts to cross-crystallize PaBphP-PCM-WT and PaBphP-PCM-Q188L in the dark were not successful. Because most sidechains in the central helices of the Q188L structure are well-ordered, we argue against the otherwise plausible suggestion thatthe mixed Pr/Pfr states lead to the quaternary structural differencesat the dimer interface. It is also possible that general structuralperturbations from the single-residue substitution (Q188L) affectdimerization and crystal packing. But disorder in the PHY domain,on the other hand, might arise directly from the mixed Pr/Pfr statesin the Q188L crystals. Despite their varied origins, these globalstructural differences certainly reflect intrinsic plasticity requiredby signaling proteins to transmit signals over significant distances tothe effector domains.

Materials and MethodsCloning, Mutagenesis, and Purification. Site-directed mutagenesis and proteinpurification were carried out as described previously (12, 14).

UV-Visible Spectroscopy. UV-visible spectra of purified WT and mutant PaBphP-PCMproteins in solutionwererecordedat roomtemperature from230to900nmwith a Shimadzu UV-1650 PC spectrophotometer. Spectra were recorded eitherin 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). Visiblespectra of crystals were recorded at ambient and cryogenic temperatures with amicrospectrophotometer (Xspectra) at BioCARS, Advanced Photon Source (APS;Argonne National Laboratory, Argonne, IL).

Crystallization and Data Collection. The Q188L mutant was crystallized from0.5% (wt/vol) PEG4000 (Fluka) and 0.01 M sodium acetate, pH 4.6, with a finalprotein concentration of 10 mg/mL at 20 °C in the dark. Crystals were handledand frozen as described previously for WT (12). Microspectrophotometryshows that the Q188L crystals undergo Pr/Pfr photoconversion at ambienttemperature (Fig. S2). All diffraction data from the Se-Met and native crystalswere collected at 100 K at the Structural Biology Center 19-ID and BioCARS14-ID beam stations at the APS. All images were indexed, integrated, andscaled by using HKL2000 or HKL3000 (HKL Research).

Structure Determination and Refinement. The crystal structure of the Q188Lmutant was determined by MAD method using Solve (25) and Sharp (26) at2.9-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/Pfrcompositions by using PHENIX (29). The buried surface areas were calculatedwith CNS. Coot (30) was used for all model building and map fitting. Aleast-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 summa-rized in Table S3.

ACKNOWLEDGMENTS. We thank Ying Pigli and Yuen-Ling Chan for help andadvice in cloning and mutagenesis; Vukica Srajer of BioCARS for assistance inmicrospectroscopic experiments on crystals; Zhong Ren of BioCARS for mapanalysis algorithms; and the reviewers for helpful comments. We also thankthe staff of the Structural Biology Center and BioCARS at the APS, ArgonneNational Laboratory for beam line access. This work was supported by Na-tional Institutes of Health Grant GM036452 (to K.M.).

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