Crystal structure of the chromophore binding domain of an unusual bacteriophytochrome, RpBphP3, reveals residues that modulate photoconversion Xiaojing Yang*, Emina A. Stojkovic´*, Jane Kuk*, and Keith Moffat*†‡ *Department of Biochemistry and Molecular Biology and †Institute 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 June 11, 2007 (received for review February 27, 2007)
biliverdin 兩 red-light photoreceptor
P
hytochromes 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 C15AC16 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 IX␣ (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 Cterminal histidine kinase domain (Fig. 1a). Despite 52% sewww.pnas.org兾cgi兾doi兾10.1073兾pnas.0701737104
quence 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). 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 fulllength 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 [Pro-
Author contributions: X.Y. and E.A.S. contributed equally to this work; X.Y. initiated and designed the RpBphP2/RpBphP3 project, collected diffraction data, and solved, refined, and analyzed structure; E.A.S. designed primers, cloned and purified proteins, carried out mutagenesis and spectroscopic work, and participated in data collection; J.K. purified protein and grew crystals; K.M. initiated photoreceptor projects; and X.Y., E.A.S., and K.M. interpreted structure, analyzed spectra, and wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Abbreviations: BV, biliverdin IX␣; Bph, bacteriophytochrome; P⌽B, phytochromobilin; CBD, chromophore binding domain; PDB, Protein Data Bank. Data deposition: The atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2OOL). ‡To
whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/ 0701737104/DC1. © 2007 by The National Academy of Sciences of the USA
PNAS 兩 July 24, 2007 兩 vol. 104 兩 no. 30 兩 12571–12576
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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 nearred (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 IX␣. 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.
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 Fo ⫺ Fc omit map in the region of chromophore. The chromophore is shown as 2(S),3(E)-P⌽B covalently linked to Cys-28.
tein 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 -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 -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 -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 Å2 (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). 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 12572 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0701737104
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 Fo ⫺ Fc 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 Fo ⫺ Fc 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 (P⌽B), does not fully account for all positive densities. Our Fo ⫺ Fc maps of RpBphPCBD 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)-P⌽B; 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)-P⌽B and 2(S),3(E)-P⌽B (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 Yang et al.
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 His269). 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 C15AC16 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 Yang et al.
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 C14OC15 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-183Ser-297-His-299 as the ‘‘15Za pocket.’’ It is believed that the primary photochemical event in Bphs is Z/E isomerization about the C15AC16 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 C15AC16 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 PNAS 兩 July 24, 2007 兩 vol. 104 兩 no. 30 兩 12573
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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).
12574 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0701737104
a
b RpBphP2-505 WT (3min)
K183M/S297A (25min)
M169K/A283S (30min)
L207Y (27min)
Y193L (
