Crystal structure of the chromophore binding domain of an unusual bacteriophytochrome, RpBphP3, reveals residues that modulate photoconversion

Abstract

Bacteriophytochromes RpBphP2 and RpBphP3 from the photosynthetic bacterium Rhodopseudomonas palustris work in tandem to modulate synthesis of the light-harvesting complex LH4 in response to light. Although RpBphP2 and RpBphP3 share the same domain structure with 52% sequence identity, they demonstrate distinct photoconversion behaviors. RpBphP2 exhibits the “classical” phytochrome behavior of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states, whereas RpBphP3 exhibits novel photoconversion between Pr and a near-red (Pnr) light-absorbing states. We have determined the crystal structure at 2.2-Å resolution of the chromophore binding domains of RpBphP3, covalently bound with chromophore biliverdin IXα. By combining structural and sequence analyses with site-directed mutagenesis, we identify key residues that directly modulate the photochemical properties of RpBphP3 and RpBphP2. Remarkably, we identify a region spanning residues 207–212 in RpBphP3, in which a single mutation, L207Y, causes this unusual bacteriophytochrome to revert to the classical phenotype that undergoes reversible photoconversion between the Pr and Pfr states. The reverse mutation, Y193L, in the corresponding region in RpBphP2 significantly diminishes the formation of the Pfr state. We propose that residues 207–212 and the spatially adjacent conserved residues, Asp-216 and Tyr-272, interact with the chromophore and form part of the interface between the chromophore binding domains and the PHY domain that modulates photoconversion.

Phytochromes are photoreceptors found in plants, cyanobacteria, fungi, and nonphotosynthetic bacteria that regulate a range of physiological responses such as chlorophyll synthesis, seed germination, floral induction, and phototaxis by using light in the red/far-red region of the spectrum (1). Upon absorption of a photon in the appropriate wavelength range, their linear tetrapyrrole chromophores (bilins) switch between two stable, spectrally distinct, red- and far-red-light absorbing forms, denoted Pr and Pfr, respectively. In most phytochromes Pr is the dark-adapted, ground state; in others, it is Pfr. The primary photochemical event for the Pr/Pfr photoconversion in plant phytochromes (Phys) and bacteriophytochromes (Bphs) is believed to involve rapid 15Z anti to 15E anti (15Za/15Ea) isomerization of the C15C16 double bond between rings C and D of the bilin chromophore. Isomerization is followed by slower transitions via several spectrally distinct intermediates (2–4) that are presumably accompanied by structural changes in the chromophore and the surrounding protein.

A pair of Bphs from the photosynthetic bacterium Rhodopseudomonas palustris, denoted RpBphP2 and RpBphP3, was characterized that in tandem modulates synthesis of the LH4 light-harvesting complex (5). RpBphP2 and RpBphP3 share the same biliverdin IXα (BV) chromophore and the same domain structure, in which three N-terminal photosensory domains, PAS (Per-ARNT-Sim), GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA), and PHY (phytochrome), are linked to the C-terminal histidine kinase domain (Fig. 1a). Despite 52% sequence identity and similar spectral characteristics in their dark-adapted Pr states, RpBphP2 and RpBphP3 demonstrate quite different photoconversion behaviors. Upon illumination in the red, RpBphP2 exhibits the classical phytochrome behavior of reversible Pr/Pfr photoconversion. In contrast, RpBphP3 exhibits an unusual, reversible transition from the Pr state to a novel state characterized by an absorption band in the near-red centered at 650 nm; this state is denoted Pnr (5).

Fig. 1.

Crystal structure of RpBphP3-CBD. (a) Domain structure of the full-length Bph RpBphP3. (b) Ribbon diagram of the two RpBphP3-CBD molecules in the asymmetric unit. The PAS domain is in yellow, and the GAF domain is in green. (c) Superposition of the crystal structures of RpBphP3-CBD (PDB ID code 2OOL, in yellow and green) and DrBphP-CBD (PDB ID code 2O9C, in light blue). (d) The F_o − F_c omit map in the region of chromophore. The chromophore is shown as 2(S),3(E)-PΦB covalently linked to Cys-28.

The crystal structure of the chromophore binding domain (CBD) of the Bph from Deinococcus radiodurans, DrBphP, recently provided the first structural insight into the photosensory domains of phytochromes (6). However, the molecular and mechanistic details of Pr/Pfr photoconversion remain largely unknown. To explore the structural basis of reversible Pnr/Pr/Pfr photoconversion and the factors that confer unusual photoconversion behavior on RpBphP3, we have determined the crystal structure of the CBD of RpBphP3 (RpBphP3-CBD) in the Pr state. RpBphP3-CBD contains the PAS and GAF domains, but as in the DrBphP-CBD structure it lacks the PHY domain and therefore displays limited photoconversion (7). We identify key residues that directly modulate the photoconversion by combining structural and sequence analyses with site-directed mutagenesis carried out on longer constructs of RpBphP3 and RpBphP2 comprising the PAS, GAF, and PHY domains, which display photoconversion efficiency comparable to that of their full-length proteins. We specifically examine how residues surrounding ring D in the 15Za or 15Ea configuration affect the photoconversion behaviors of RpBphP3 and RpBphP2. We also explore the roles of the conserved residues (Asp-216 and Tyr-272 in RpBphP3 and their equivalents Asp-202 and Tyr-258 in RpBphP2) in photoconversion.

Results and Discussion

Crystal Structure of RpBphP3-CBD.

The crystal structure of RpBphP3-CBD was determined in its Pr state, containing residues 1–337 and a covalently bound BV chromophore (Fig. 1a). The structure was solved at 2.2-Å resolution by molecular replacement using the crystal structure of DrBphP-CBD [Protein Data Bank (PDB) ID code 1ZTU] as search model (6) and refined to an R-factor of 18.8% and a free R-factor of 23.1% [supporting information (SI) Table 1]. There are two molecules in the asymmetric unit related by noncrystallographic twofold symmetry. Electron density for the N-terminal tag, the first 26 residues, a short loop (residues 97–100), and five residues at the C terminus is not visible in either molecule.

The crystal structure of RpBphP3-CBD includes two photosensory domains, the PAS and GAF domains, which are spatially connected by a knot identical to that in DrBphP-CBD (6). In the GAF domain, a central, twisted, six-stranded, antiparallel β-sheet accommodates the chromophore binding pocket on one side and a three-helix bundle on the other. In the PAS domain, a five-stranded, antiparallel β-sheet and four short helices tightly pack around a hydrophobic core. The N-terminal extension of the PAS domain threads through a 30-residue loop spanning residues 237–267 that protrudes from the GAF domain to form a knot, stabilized by hydrophobic interactions between the extension and loop residues and by hydrogen bonds between the main chains of the extension and loop where they cross. The root mean square displacement between main-chain atoms is 1.49 Å for the core structural elements with 273 aligned residues between RpBphP3-CBD and the recently published high resolution DrBphP-CBD structure (PDB ID code 2O9C) (8), thus illustrating the close similarity of their tertiary structures. Major structural differences reside in loops and short helices located at the distal side of core β-sheet in the PAS domain (Fig. 1c). Each GAF domain contributes three helices to a tightly packed six-helix bundle forming the noncrystallographic dimer interface that contains both hydrophobic and polar interactions and buries ≈2,030 Ų (Fig. 2b). In the DrBphP-CBD structures (6, 8), the similarly arranged monomers are related by strict crystallographic twofold symmetry. The presence of the same intermolecular six-helix bundle in different Bphs under different crystallization conditions suggests an important role for the GAF domain in assembling biologically functional, dimeric phytochromes. This is consistent with observations on Cph1 and Agp1 photosensory domains (9, 10) and contrasts with the proposal that the C-terminal histidine kinase domain of phytochromes is responsible for their dimerization (1, 11, 12).

Fig. 2.

The chromophore binding pocket. (a) Residues in the chromophore (cyan) pocket examined by site-directed mutagenesis. Lys-183, Ser-297, and His-299 form the 15Za pocket; Leu-207, Phe-210, and Phe-212 form part of the 15Ea pocket; and Tyr-272 and Asp-216 are colored in pink. Potential hydrogen bonds are shown in red dashed lines, and the corresponding distances are given in the text. (b) The chromophore cavity surrounding ring D. (c) Sequence alignment of representative Bphs and Phy-like photoreceptors in the regions of the 15Za and 15Ea pockets in the GAF domain (residues colored as in a). The consensus sequence motif, PASDIP, is highlighted in green. The sequences are as follows: RpBphP3 (R. palustris CGA009 PhyB2), RpBphP2 (R. palustris CGA009 PhyB1), DrBphP (D. radiodurans R1 BphP), AtBphP1/Agp1 (Agrobacterium tumefaciens BphP1), PsBphP (Pseudomonas syringae DC3000 BphP), XaBphP (Xanthomonas axonopodis BphP), XcBphP (Xanthomonas campestris ATCC 33913 BphP), RcPPH (R. centenum PPH), TtPPD (Thermochromatium tepidum Ppd), AtBphP2/Agp2 (A. tumefaciens BphP2), RpBphP5 (R. palustris CGA009 PhyB5), PaBphP (Pseudomonas aeruginosa PA01 BphP), Cph1 (Synechocystis PCC6803 Cph1), and Cph2 (Synechocystis PCC6803 Cph2).

The chromophore interacts primarily with residues in the GAF domain. The vinyl group of ring A is covalently linked via its C3² atom to Cys-28, located in the N-terminal extension of the knot. The chromophore is positioned between helices in the GAF domain with an opening at the outer surface of the dimer. The methine linkers between rings A, B, and C are in the syn conformation in which rings B and C are nearly coplanar, whereas rings A and D lie out of the B-C plane by 16° and 40°, respectively, defined by angles between normals to the planes of rings. The linkage between rings C and D clearly adopts the 15Za configuration (Fig. 2a). The overall 5Zs/10Zs/15Za configuration of chromophore in RpBphP3-CBD is consistent with the DrBphP-CBD structure in the Pr state but differs in the angle between the B-C plane and ring D (51° in DrBphP-CBD versus 40° in RpBphP3-CBD).

The recently published DrBphP-CBD crystal structure at 1.45-Å resolution proposed a different conformation for ring A in the covalently attached form, which generates a chiral center at C2 (8). During refinement of the RpBphP3-CBD structure, we identified significant positive and negative difference densities in the region of ring A in F_o − F_c maps. We explored whether these residual difference densities arose from an inaccurate model for the covalently attached chromophore or from x-ray radiation damage. We divided 480 diffraction images obtained from a single RpBphP3-CBD crystal into four data sets of 120 images each, distinguished by the order in which they were collected. After refinement, the resulting four F_o − F_c maps clearly show that negative difference density on the bond between the C3² atom of ring A and the sulfur atom of Cys-28 increased as data collection proceeded, indicating rupture of the covalent bond between the chromophore and Cys-28 due to radiation damage (SI Fig. 5). Bond rupture is also correlated with decay of positive difference density flanking the C2 atom of ring A. This confirms that these positive difference densities are indeed associated with ring A of a covalently attached chromophore, in agreement with the DrBphP-CBD structure (PDB ID codes 2O9B and 2O9C). However, the proposed chemical structure of the chromophore, 2(R),3(E)-phytochromobilin (PΦB), does not fully account for all positive densities. Our F_o − F_c maps of RpBphP-CBD refined with BV suggest that one molecule in the asymmetric unit exhibits only the negative hand in ring A at the chiral center C2, arising from 2(S),3(E)-PΦB; the other molecule exhibits a mixed population of both positive and negative hands at C2 (SI Fig. 5). We further refine the RpBphP3-CBD structure with both 2(R),3(E)-PΦB and 2(S),3(E)-PΦB (PDB ID code 2OOL).

Sequence alignment of all known phytochromes reveals a consensus sequence motif that signifies phytochrome families, PASDIP, which spans residues 213–218 in RpBphP3 (Fig. 2c). This sequence forms a one-turn 3₁₀-helix, which positions the main-chain carbonyl group of Asp-216 within hydrogen bonding distance of the pyrrole nitrogens in rings A, B, and C (3.42 Å, 3.15 Å, and 3.02 Å, respectively). The side chain of this conserved aspartate is also close to the carbonyl group of ring A (3.45 Å) (Fig. 2a). On the other side of rings A, B, and C, a highly conserved residue His-269 interacts with their pyrrole nitrogens via a water molecule. In addition, the chromophore makes extensive interactions with conserved residues in the GAF domain via the propionate substituents of rings B (to Arg-263 and Tyr-225) and C (to Arg-231, Ser-283, and His-269). The carbonyl group of ring D is surrounded by three polar residues, Lys-183, Ser-297, and His-299 (distances are 3.37 Å, 2.57 Å, and 2.97 Å, respectively). The conserved aromatic residues Tyr-185, Tyr-272, and Phe-212 approach ring D from the side opposite to the carbonyl group. Their bulky side chains entirely bury ring D and form a cavity in the GAF domain (Fig. 2b). This cavity is evidently sufficiently large to permit ring D to rotate around the C15C16 bond when undergoing fast 15Za/15Ea isomerization upon absorbing a photon.

The 15Za and 15Ea Pockets.

We compared the local protein environments of the chromophores in the RpBphP3-CBD and DrBphP-CBD structures to understand the quite different photoconversion behaviors of their full-length proteins. Although their crystal structures are very similar overall, we focus on small but significant differences between them. In our RpBphP3-CBD structure, three polar side chains, Lys-183, Ser-297, and His-299, interact with the carbonyl group of ring D and stabilize the 15Za configuration in the Pr state; but in the DrBphP-CBD structure only the corresponding His-290 forms such a hydrogen bond. The additional interactions provided by Lys-183 and Ser-297 in the RpBphP3-CBD structure constrain ring D to an offset of 17° (defined by the torsion angle around the C14C15 single bond) versus 23° in DrBphP-CBD. Sequence alignment of phytochromes around these three residues reveals that, although the histidine is widely conserved, RpBphP3 is unique among phytochromes in containing two additional polar residues, Lys-183 and Ser-297, in the vicinity of the carbonyl group of ring D in the 15Za configuration. The corresponding residues are Met and Ala in most Bphs (Fig. 2c) and Met and Val in plant phytochromes. We designate the structural region containing Lys-183-Ser-297-His-299 as the “15Za pocket.”

It is believed that the primary photochemical event in Bphs is Z/E isomerization about the C15C16 double bond (1). Recent studies on adducts of the related Bph Agp1 reconstituted with sterically locked BV analogs (13) support the assignment of the 15Za configuration to the Pr state and 15Ea to the Pfr state. To explore potential chromophore–protein interactions in the Pfr state, we modeled the 15Ea configuration by mimicking torsion around the C15C16 double bond in both RpBphP3-CBD and DrBphP-CBD. Even if we assume a rigid protein framework, this motion is permitted by the spacious cavity in which ring D is located. In the modeled 15Ea configuration in RpBphP3-CBD, a stretch of residues (207–212) immediately N-terminal to the signature PASDIP sequence motif shields ring D from the surface. Residues Leu-207, Phe-210, and Phe-212 are directed toward the carbonyl group of ring D whereas Leu-208, Asp-209, and His-211 point away from ring D. Asp-209 in RpBphP3 is distinctive in the sequence alignment of this region: a glycine is present in all other Bphs (Fig. 2c).

We note that in most known phytochromes that exhibit Pr/Pfr photoconversion one or more polar residues are found at positions corresponding to Leu-207, Phe-210, and Phe-212 (Fig. 2c). For example, Tyr replaces Leu-207 in RpBphP2 and His replaces Phe-210 in DrBphP. In RpBphP3 and Pph from Rhodospirillum centenum, no polar residues are present at any of these three positions and no Pr/Pfr photoconversion occurs (5). Furthermore, Pfr (rather than Pr) is the dark-adapted state in a subfamily of phytochromes such as AtBphP2, RpBphP1, RpBphP5, and PaBphP (14–16). In all members of this subfamily, either Gln or Asn is present at the position corresponding to Phe-210. We therefore designate the region spanning residues 207–212 as the “15Ea pocket.”

We sought to test these correlations among sequence, structure, and photoconversion and to confirm the roles of residues identified in the 15Za and 15Ea pockets via site-directed mutagenesis on longer constructs, RpBphP3–521 (residues 1–521) and RpBphP2–505 (residues 1–505). Both constructs include the PHY domain C-terminal to the PAS and GAF domains and demonstrate the full photoconversion behavior of full-length proteins (data not shown). We tested three specific hypotheses by interchanging residues between RpBphP2 and RpBphP3 in the 15Ea and 15Za pockets: (i) the two additional polar residues (Lys-183 and Ser-297) in the 15Za pocket stabilize the Pr state and hinder formation of the Pfr state in RpBphP3; (ii) replacing Leu-207 in the 15Ea pocket of RpBphP3 with the corresponding residue Tyr in RpBphP2 enables formation of the Pfr state in RpBphP3; and (iii) the conserved Gly in the 15Ea pockets facilitates the Pr/Pfr photoconversion.

In RpBphP3–521, substitution of Lys-183 and Ser-297 in the 15Za pocket with their equivalents in RpBphP2 (K183M, S297A, or both) did not enable formation of the Pfr state upon illumination at 690 nm; and conversely, introduction of these polar residues into RpBphP2–505 by reverse mutations (M169K, A283S, or both) did not significantly affect formation of the Pfr state (Fig. 3). These results suggest that neither Lys-183 nor Ser-297 is responsible for the inability to form the Pfr state in RpBphP3–521. Hypothesis i is therefore not supported: Pr/Pfr photoconversion can occur even in the presence of additional stabilizing polar residues in the 15Za pocket.

Fig. 3.

UV-visible absorption spectra of wild-type and selected mutants of RpBphP3–521 (a) and RpBphP2–505 (b). Spectra are in order of appearance in the text from the top down. Spectra were measured in solution in the dark-adapted state (solid lines) and light-illuminated state (dashed lines). The corresponding mutants in RpBphP3 and RpBphP2 are shown side by side. Spectra on the bottom of each panel are from RpBphP3-CBD (1–337) and RpBphP2-CBD (1–321). Numbers in parentheses represent the estimated half-time of dark reversion for each sample.

Hypothesis ii suggests that the different photoconversion behaviors of RpBphP3 and RpBphP2 arise from differences in the 15Ea pocket. Strikingly, illumination at 690 nm of the single mutant L207Y in the 15Ea pocket of RpBphP3–521 resulted in the appearance of a new absorption band ≈750 nm, characteristic of the Pfr state (Fig. 3a). This Pfr state can undergo photoconversion back to the Pr state upon illumination at 750 nm (data not shown). Thus, introduction of a single tyrosine residue into the 15Ea pocket was sufficient to replace the unusual photoconversion of wild-type RpBphP3 (Pr/Pnr) by the classical photoconversion exemplified by RpBphP2 (Pr/Pfr). Formation of the Pfr state in RpBphP3–521 was further enhanced in the double mutant (L207Y/D209G) and the quadruple mutant (L207Y/D209G with K183M/S297A) (Fig. 3a). When the corresponding reverse mutations were introduced into RpBphP2–505 (the single mutant Y193L, the double mutant Y193L/G195D, and the quadruple mutant Y193L/G195D with M169K/A283S), the extent of photoconversion from the Pr to the Pfr state was greatly reduced compared with wild type (Fig. 3b). These results are consistent with hypothesis ii: introduction of Tyr-207 in the 15Ea pocket (a bulky, aromatic, and polar residue compared with Leu-207) promotes Pr/Pfr photoconversion.

The single mutant D209G did not enable the formation of Pfr in RpBphP3 (Fig. 3a); however, the double mutant (L207Y/D209G) shows higher Pr/Pfr photoconversion efficiency than the single L207Y mutant. Also, the single mutants in RpBphP2, G195D and G195A, significantly reduce the formation of the Pfr state compared with wild type. These data support hypothesis iii: Gly in the 15Ea pocket facilitates Pr/Pfr photoconversion, probably by providing sufficient flexibility of the region.

Photoconversion and the PHY Domain.

Efficient Pr/Pfr and Pr/Pnr photoconversion in Bphs depends on the presence of the PHY domain (7), as our experiments on RpBphP3–521 and RpBphP2–505 confirm. In our RpBphP3-CBD structure residues in the 15Ea pocket (207–212) together with two conserved residues, Asp-216 and Tyr-272, form a continuous patch on the surface of the GAF domain (Fig. 4). As noted earlier, the branched side chain of Asp-216 may interact with the carbonyl group of ring A (3.49 Å) and with the side chain of Tyr-272 (3.36 Å) (Fig. 2a). We speculated that certain residues in or adjacent to the 15Ea pocket modulate photoconversion by directly interacting with both the chromophore and the PHY domain.

Fig. 4.

VDW surface of the RpBphP3-CBD structure. Residues Tyr-272, Asp-216, and Leu-207-Phe-210-Phe-212 in the 15Ea pocket (colored in maroon) form a continuous surface patch and shield ring D of the chromophore (in cyan) in the GAF domain.

We therefore examined the mutants D216A in RpBphP3–521 and the corresponding D202A in RpBphP2–505 and found that both exhibit minimal photoconversion, as do the corresponding mutants D207A in Cph1 (17) and D197A in Agp1 (18), as well as RpBphP3-CBD and RpBphP2-CBD, which completely lack the PHY domain (Fig. 3). In Agp1, the conserved Asp-197 was proposed to stabilize the protonated chromophore in the Pr state. Alternatively, this aspartate in the PASDIP motif may play an important role in coupling the chromophore to the surface and engaging the PHY domain. Its main-chain carbonyl oxygen coordinates with the pyrrole nitrogens (Fig. 2a), and its conserved side chain contributes to the surface patch (Fig. 4). We also examined the single-residue substitutions, Y272F in RpBphP3–521 and the corresponding Y258F in RpBphP2–505. Strikingly, photoconversion is barely detectable in Y272F: in steady-state experiments minimal bleaching is observed in the Pr region (≈700 nm) and the Pnr/Pfr states are not formed (Fig. 3a). However, Y258F demonstrated photoconversion behavior comparable to wild type, in which the Pr state is efficiently converted to the Pfr state (Fig. 3b). The contrasting phenotypes between Y272F in RpBphP3–521 and Y258F in RpBphP2–505 indicate that other residues in the 15Ea pocket and/or in the PHY domain are likely to be involved in engaging the GAF and PHY domains for photoconversion. We note that an Asn residue in the 15Ea pocket of PsBphP replaces the conserved Leu-208 in RpBphP3 and other Bphs. Interactions with the PHY domain may explain the photoconversions observed in PsBphP (19) and similarly in Cph2 (20), although no polar residues are present in positions corresponding to Leu-207/Phe-210/Phe-212 (Fig. 2c).

Chromophore Conformation.

FTIR experiments (5) suggested that 15Za/15Ea isomerization occurs in RpBphP3, as in classical Bphs (13). If so, how does the chromophore conformation in the Pnr state of RpBphP3 differ from that in the Pfr state of classical Bphs? Structural analyses and modeling suggest that the hydrogen bonds to the carbonyl group of ring D in the 15Za pocket are disrupted when the C15C16 double bond undergoes isomerization via either one-bond-flip or hula-twist mechanisms (12, 21). Isomerization via the hula-twist mechanism minimizes the volume swept out by chromophore atoms during the primary photochemical processes of photoreceptors such as photoactive yellow protein and bacteriorhodopsin (22). In RpBphP3-CBD, isomerization via the hula-twist mechanism would involve both the C14C15 single bond and the C15C16 double bond of the chromophore, which would cause severe steric clashes with residues lining the cavity of ring D such as Asp-216, Tyr-272, Phe-212, and Tyr-185. In contrast, isomerization via the one-bond-flip mechanism is facilitated both by the offset angle of ring D from the plane of rings B and C and by the substantial cavity surrounding ring D that allows it to rotate with minimal hindrance around the C15C16 double bond in a counterclockwise fashion when looking down the rotation axis from C16 to C15 (Fig. 2b). In the RpBphP3-CBD structure, modeling of isomerization via the one-bond-flip mechanism suggests that Tyr-272 interacts with ring D as it rotates. The closest distance between the hydroxyl group of Tyr-272 and the pyrrole nitrogen of ring D is 2.8 Å, when ring D has rotated ≈100° around the C15C16 double bond from its original orientation in the Pr state. This suggests that Tyr-272 in RpBphP3 might be involved in stabilizing ring D in the 15Ea configuration, potentially via a hydrogen bond with the pyrrole nitrogen of ring D. In classical phytochromes such as DrBphP and RpBphP2, ring D may rotate further to adopt a conformation in which its carbonyl group is stabilized by residues in the 15Ea pocket when the PHY domain is present. Ring D appears to be a determinant for the Pnr spectral phenotype, because the Pnr-like spectral state was observed for Agp1 and Agp2 even when ring A was locked in either the 5Zs or 5Za configurations (23). The fact that the Pnr state has a substantially blue-shifted absorption maximum (≈650 nm) relative to the Pr state (≈690 nm) suggests that the extent of electron delocalization across rings A, B, C, and D is reduced (24). We speculate that ring D in the 15Ea configuration in RpBphP3 is stabilized by Tyr-272 in an orientation no longer conjugated to rings A, B, and C, thus generating the Pnr spectral phenotype. A similar effect was noted for plant phytochrome phyA assembled with synthetic open-chain tetrapyrroles with modified substitution patterns on ring D (25).

Ring A may also be involved in the formation of the Pfr state (23). NMR spectroscopic and ultrafast mid-infrared spectroscopic studies on the N-terminal domain of Cph1 indicate conformational changes in ring A during the Pr/Pfr photoconversion (26). However, no data support the direct involvement of ring A in forming the Pnr state of RpBphP3, nor it is clear how the chirality of ring A [as observed in 2(S),3(E)-PΦB] in the Pr state of RpBphP3 might play a role in determining the Pnr phenotype. More experimental evidence, such as crystal structures in the Pfr and Pnr states in the presence of the PHY domain, are needed to unambiguously establish the conformation of the chromophore in the different spectroscopic states.

Materials and Methods

Cloning, Expression, and Purification of RpBphP3-CBD.

The coding region for residues 1–337 of RpBphP3 was PCR-amplified from R. palustris strain CGA009 genomic DNA (American Type Culture Collection, Manassas, VA), cut by restriction enzymes XhoI and HindIII (New England Biolabs, Beverly, MA), and ligated into the corresponding sites of the expression vector pRSETB (Invitrogen, Carlsbad, CA). Escherichia coli BL21 (DE3) transformed with the constructed plasmid was grown aerobically at 37°C to 5 × 10⁸ cells per milliliter, then induced with 1 mM isopropyl-β-d-thiogalactopyranoside followed by growth at 16°C for 18–20 h. Cell pellets were suspended in sonication buffer [20 mM Tris·HCl, pH 8.0/50 mM NaCl/complete protease inhibitor EDTA-free (Roche, Indianapolis, IN)] and sonicated with pulses on ice. After centrifugation, the supernatant was incubated with 200 μM BV (Frontier Scientific, Logan, UT) for 30 min at 4°C, then applied to a Talon Co²⁺-IMAC resin column (Clontech, Mountain View, CA). The column was washed with 20 mM Tris·HCl (pH 8.0) and 1 M NaCl, then with cold sonication buffer. Protein was eluted with 20 mM Tris·HCl (pH 8.0), 50 mM NaCl, and 300 mM imidazole and concentrated on HiTrap Q (Amersham Pharmacia, Piscataway, NJ). Concentrated protein was loaded onto a Sephacryl S-200 HR gel filtration column (Amersham Pharmacia) and eluted in 10 mM Tris·HCl (pH 8.0) and 10 mM NaCl. Fractions containing protein were concentrated by using Amicon Ultra Centrifugal Filters (Millipore, Billerica, MA). The covalent attachment of bilin chromophores was monitored by zinc-induced fluorescence of the chromoproteins when subjected to SDS/PAGE.

Cloning, Mutagenesis, and Purification of RpBphP3–521 and RpBphP2–505.

The coding region for residues 1–521 in RpBphP3 and residues 1–505 in RpBphP2 were PCR-amplified from R. palustris genomic DNA strain CGA009 (American Type Culture Collection). Amplified RpBph3–521 and RpBph2–505 gene products were cut with NdeI/XhoI or NdeI/HindIII, respectively, and then introduced into the corresponding sites of the expression vector pET28c (Novagen, Madison, WI). For purification, RpBphP3–521 and RpBphP2–505 were coexpressed with heme oxygenase from plasmid pET11a in BL21 (DE3) cells at 16°C for 18 h using 1 mM isopropyl-β-d-thiogalactopyranoside and 0.5 mM δ-aminolevulinic acid (Sigma–Aldrich, St. Louis, MO). The subsequent purification procedure for wild-type and mutant RpBphP3–521 and RpBphP2–505 was the same as for RpBphP3-CBD. Site-directed mutagenesis was carried out on plasmids carrying RpBphP3–521 or RpBphP2–505 using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

UV-Visible Spectroscopy.

UV-visible spectra of purified RpBphP2–505 and RpBphP3–521 wild-type and mutant proteins were recorded at room temperature from 900 to 240 nm with a Shimadzu UV-1650 PC spectrophotometer. Spectra were recorded either in the dark or after 4-min illumination with light at 690 nm (red), 750 nm (far red), and 650 nm (near red) provided by interference filters with a 10-nm bandwidth (Andover, Salem, NH).

Crystallization and Data Collection.

Crystallization of purified RpBphP3-CBD was carried out at 20°C using the hanging-drop vapor diffusion method at a protein concentration of 12–15 mg/ml in 100 mM tri-sodium citrate (pH 5.6), 6% isopropanol (vol/vol), and 7% PEG 4000 (wt/vol). All steps in purification, crystallization, and cryoprotection were performed under green safety lights. Spectroscopy confirmed that the crystalline RpBphP3-CBD is in the Pr state. Crystals (0.15–0.2 mm in all three dimensions) were used for x-ray diffraction data collection at 100 K and yielded diffraction to a maximum resolution of 2.1 Å. Data were collected on an ADSC Q315 CCD detector at the BioCARS 14BM-C and SBC 19ID beam stations at the Advanced Photon Source, Argonne National Laboratory. All images were indexed, integrated, and scaled by using HKL2000 (27).

Structure Determination.

RpBphP3-CBD crystals are in space group P321 (a = b = 151.8 Å, c = 76.0 Å). The self-rotation function reveals a twofold noncrystallographic symmetry axis parallel to the crystallographic threefold axis. With two molecules per asymmetric unit, the solvent content is 66%. The crystal structure was determined with the molecular replacement method implemented in PHASER (28) using the crystal structure of DrBphP-CBD (PDB ID code 1ZTU) as search model (6). After solvent flattening and noncrystallographic symmetry averaging with Resolve (29), the two chains were built independently from scratch based on a figure-of-merit-weighted map. The crystal structure was initially refined with CNS (30) to correct gross errors in coordinates and finalized by Refmac5 (31), in which each molecule was treated as one TLS group. XtalView/Xfit (32) and Coot (33) programs were used for model building. Structural illustrations were generated with PyMol (http://pymol.org).

Abbreviations

BV

biliverdin IXα

Bph

bacteriophytochrome

PΦB

phytochromobilin

CBD

chromophore binding domain

PDB

Protein Data Bank.