Common Structural Elements in the Chromophore Binding Pocket of the Pfr State of Bathy Phytochromes†
Abstract
Phytochromes are bimodal photoreceptors which, upon light absorption by the tetrapyrrole chromophore, can be converted between a red-absorbing state (Pr) and far-red-absorbing state (Pfr). In bacterial phytochromes, either Pr or Pfr are the thermally stable states, thereby constituting the classes of prototypical and bathy phytochromes, respectively. In this work, we have employed vibrational spectroscopies to elucidate the origin of the thermal stability of the Pfr states in bathy phytochromes. Here, we present the first detailed spectroscopic analysis of RpBphP6 (Rhodopseudomas palustris), which together with results obtained for Agp2 (Agrobacterium tumefaciens) and PaBphP (Pseudomonas aeruginosa) allows identifying common structural properties of the Pfr state of bathy phytochromes, which are (1) a homogenous chromophore structure, (2) the protonated ring C propionic side chain of the chromophore and (3) a retarded H/D exchange at the ring D nitrogen. These properties are related to the unique strength of the hydrogen bonding interactions between the ring D N-H group with the side chain of the conserved Asp194 (PaBphP numbering). As revealed by a comparative analysis of homology models and available crystal structures of Pfr states, these interactions are strengthened by an Arg residue (Arg453) only in bathy but not in prototypical phytochromes.
Introduction
Phytochromes are light-sensing proteins found in plants and microorganisms, composed of an N-terminal sensory module with the PAS-GAF-PHY domains, and a C-terminal regulatory or catalytic module 1-4. Upon light absorption by the linear methine-bridged tetrapyrrole chromophore, which can be biliverdin IXα, phycocyanobilin or phytochromobilin, the photosensor unit is converted between a red-absorbing state (Pr) and a far-red-absorbing state (Pfr), corresponding to a ZZZssa and ZZEssa chromophore configuration, respectively. The phototransformations between the two parent states in both directions follow similar albeit not identical mechanistic patterns. Common to both routes is the photoisomerization of the tetrapyrrole, followed by a series of chromophore and protein relaxation steps and a secondary structure change of the highly conserved tongue region which adopts a β-sheet and an α-helix structure in Pr and Pfr, respectively 5. This structural change is thought to be linked to the (de)activation of the output module. The coupling between chromophore and protein structural changes is mediated via proton translocation steps which are likely to be different for the Pr-to-Pfr and Pfr-to-Pr conversion 6, 7. In most phytochromes, Pr is the thermodynamically stable state such that Pfr can also revert to the Pr state in the dark 1-4. In contrast to these prototypical phytochromes, the thermodynamic stability of the parent states is reversed in some bacterial representatives of the phytochrome family termed bathy phytochromes, in which Pfr is the most stable state 8, 9.
Most of the studies on bathy phytochromes focused on PaBphP, the single phytochrome from Pseudomonas aeruginosa, and Agp2 from A. tumefaciens, for which detailed structural analyses of the chromophore binding pocket (CBP) in the Pfr state were carried out 6, 10 on the basis of the crystal structure of the photosensory core module (PCM) including the protein domains (PAS-GAF-PHY) from PaBphP 11 (Fig. 1). These studies identified several characteristic structural properties of chromophore binding pocket in the Pfr state of PaBphP and Agp2. First, the biliverdin IXα chromophore displays a highly homogeneous structure in contrast to the Pfr states of prototypical phytochromes 10. In the latter case, two distinct ZZEssa substates were identified displaying slightly different structural parameters of the A-B (bond angle) and C-D (torsional angle) methine bridges. The equilibrium between these conformers was suggested to be functional for opening a thermal isomerization pathway which is blocked in the Pfr state of bathy phytochromes. Second, the Pfr states of PaBphP and Agp2 display an unusual H/D exchange pattern at the pyrrole nitrogens, resulting from an extremely slow exchange rate of the ring D N-H group 6. Furthermore, the ring C propionic side chain was found to be protonated due to an unprecedented high pKa. These latter two findings point to a unique hydrogen bond network in the Pfr state of PaBphP and Agp2.
To check whether or not these structural properties are characteristic of all bathy phytochromes, this work extends the spectroscopic analyses to another bathy phytochrome, RpBphP6, from Rhodopseudomas palustris 12. By increasing the experimental data basis, complemented by molecular homology modeling, this study aims at identifying possible correlations between structural features of the CBP and the thermal stability of the Pfr state as the main phenomenological property of bathy phytochromes.
Materials and Methods
Sample preparation. Protein expression, chromophore assembly and protein purification of PaBphP and Agp2 are described elsewhere 13, 14.
For the expression of the PAS-GAF and PAS-GAF-PHY (PCM) domains of RpBphP6, the amino acid sequence of RpBphP6 was adopted from NCBI reference sequence NP_946341.1 (now re-annotated: WP_011156523.1) as published by Larimer et al. 15. The genomic DNA sequence for the RpBphP6 PAS-GAF domain comprising codons for amino acids 1-311 was generated by commercial gene synthesis (GeneArt, Life Technologies, Regensburg, Germany) using the GeneOptimizer® algorithm to optimize codon usage for expression in Escherichia coli cells. The cDNA was excised by BamHI and NotI and ligated into a modified pQE81L-Amp vector (Qiagen, Hilden, Germany) carrying an ampicillin resistance gene and an engineered NotI restriction site. The resulting protein sequence had an N-terminal 6xHis tag (N-terminal sequence MRGSHHHHHHTDPAT preceding the start methionine of the RpBphP6 sequence). Similarly, the cDNA of the RpBphP6 PHY domain (comprising codons for amino acids 312-495) was obtained by artificial gene synthesis and subcloned in frame into the RpBphP6-PAS-GAF/pQE81L-Amp construct described above (sequences available upon request). All DNA sequences were verified by sequencing (Eurofins MWG Operon, Ebersberg, Germany).
In order to accomplish biliverdin cofactor insertion into phytochromes already upon protein expression in E. coli, NEB Turbo cells (New England Biolabs) were co-transformed with a 1:1 mixture of pQE81L-Kan plasmid harboring the cDNA for human heme oxygenase 2 (hHox2) and one of the pQE81L-Amp plasmids with RpBphP6 sequences. Subsequent protein expression and purification by affinity chromatography were performed as described previously 16.
All samples were prepared in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, 5 mM EDTA) with the pH(D) adjusted to 7.8 by addition of aliquots of HCl (DCl).
Resonance Raman spectroscopy. All spectra were recorded at 80 K (Linkam cryostat, Resultec) using a Fourier transform (FT) Raman RFS-100/S (Bruker) spectrometer equipped with NdYAG laser (1064 nm). 2000–3000 single scans were averaged for each RR spectrum. Further details are given elsewhere 6.
Infrared spectroscopy. IR spectra were recorded at ambient temperature using an IFS-28 (Bruker) FT-IR spectrometer. “Pr” minus “Pfr” difference IR spectra were obtained from the spectrum recorded after far-red light irradiation (LED array 780 nm), using the dark spectrum as a reference. After back conversion with 670 nm LED irradiation and subsequent equilibration for ca. 15 min in the dark, the measurements including 200 single scans were repeated several times to improve the signal-to-noise-ratio.
Spectra treatment and global component analysis. All vibrational spectra were recorded and initially processed using the OPUS 5.5 or higher software. Component analysis of RR spectra was performed as described previously 6, 17.
Structural homology models. Based on the crystal structures of the Pfr states of PaBphP (PDB entry 3C2W) 11 and the DrBphP (Deinococcus radiodurans) photosensory module (PDB entry 5C5K) 18, homology models were constructed for the thermally stable Pfr states of bathy phytochromes Agp2 8 and RpBphP6 12, 15, the “bathy-like” phytochrome XccBphP (Xanthomonas campestris) 19, and for the activated Pfr states of the prototypical phytochromes Agp1 (A. tumefaciens) 8, CphB (cyanobacterial phytochrome) 20 and RpBphP2 12, 15. Pfr homology models were also compared to the available RpBphP1 Pfr structure (PDB entry 4GW9) 21. Protein sequences were obtained from the GenBank®. Unless noted otherwise, amino acid numbering refers to PaBphP in case of bathy phytochromes and DrBphP for prototypical phytochromes. Using the ClustalX program suite, each protein sequence was aligned to the corresponding peptide sequence of the reference structure 22. Subsequently, based on alignment of sequences homology modeling of protein three-dimensional structures were generated using the MODELLER 9.15 suite 23. From five potential conformations, only the lowest energy structure was selected. All Pfr state homology structures were superimposed to the chromophore moiety. All figures were prepared with the VMD 1.9 program package 24.
Results
Chromophore structure of the Pfr state in RpBphP6
The Pfr states of PaBphP and Agp2 have been analyzed in detail previously 6, 10, using RR spectroscopy and quantum-mechanics/molecular mechanics (QM/MM) hybrid calculations on the basis of the crystal structure of PaBphP 11. The chromophore displays a homogeneous structure with a ZZEssa configuration of the methine bridges and all pyrrole nitrogens being protonated. The nearly identical RR spectra obtained from the Pfr states of PaBphP and Agp2 (Fig. 2A,B) allow concluding that the structure of the chromophore and its immediate interactions with the protein environment are very similar 10.
This conclusion can be extended to the bathy phytochrome RpBphP6 since in its Pfr state, the RR spectrum reveals an excellent agreement with the corresponding spectra of PaBphP and Agp2 in the most structure-sensitive spectral regions (Fig. 2). These are the methine bridge stretching modes between 1560 and 1640 cm−1, indicating the gross chromophore configuration, the N-H in-plane bending (ip) mode at 1549 cm−1, a marker for the protonation of the pyrrole rings B and C, the bands between 1250 and 1350 cm−1, involving coordinates of the chromophore substituents, the torsional modes below 750 cm−1, which are sensitive to conformational details of the chromophore, and the unique, very intense C-H out-of-plane mode at 810 cm−1, which responds to changes of the C-D methine bridge torsion. Within the experimental accuracy, all these spectral regions are congruent for the three species. This is in contrast to the spectra of the Pfr states of prototypical phytochromes as exemplarily documented for Agp1 (Fig. 2) 25. As shown previously, the spectral differences in Agp1 compared to bathy phytochromes are due to a structural heterogeneity associated with small conformational differences at the methine bridges A-B and C-D 10, 25.
Chromophore protonation in the Pfr state in RpBphP6
In PaBphP and Agp2, the RR spectra display a weak band at 1753 and 1751 cm−1, respectively (Fig. 2). On the basis of isotopic labeling, this band was assigned to the C=O stretching of the protonated propionic side chain of ring C, which displays a pKa larger than 11 6. This band is also observed in the spectrum of RpBphP6 but it is missing in the spectra of all prototypical phytochromes (e.g. Agp1; Fig. 2). Also in terms H/D exchange behavior, RpBphP6 displays the same characteristics as PaBphP and Agp2. Although the four pyrrole nitrogens are protonated in the Pfr states of both classes of phytochromes, rapid H/D exchange at all N-H groups of the chromophore is only possible for prototypical phytochromes. As for PaBphP and Agp2 6, H/D exchange in RpBphP6 occurs on two different time scales for the pyrrole rings as reflected by the RR spectra. Here, we specifically consider the N-H ip mode of rings B and C and the methine bridge stretching modes (Fig. 3A). These modes primarily involve the respective C=C stretching, but also small contributions of the N-H ip coordinates of neighboring pyrrole rings (Supporting Information, Table S1). Immediate H/D exchange in RpBphP6 causes the disappearance of the N-H in-plane bending (rings B, C) at 1549 cm−1 (downshift to ca. 1060 cm−1; vide infra) and the 7 cm−1 downshift of the B-C stretching (7% N-H ip ring C), reflecting the deuteration at the rings B and C (Fig. 3B).
The prominent band at 1599 cm−1 represents an overlap of the strong C-D stretching (<5% N-H ip ring D) and the somewhat weaker A-B stretching mode (<5% N-H ip ring A), which are sensitive to H/D exchange at rings D and A, respectively. The band undergoes an immediate downshift by 3 cm−1 and a further 6 cm−1 downshift corresponding to the exchange at all pyrrole nitrogens on the time scale of hours (vide infra) or, alternatively, immediately after running through a complete photocycle via Pr back to Pfr (Fig. 3C). In view of the nature of the underlying modes and their relative intensities (see Table S1), the instantaneous downshift is attributed to the exchange at ring A, affecting only the relatively weak A-B mode, whereas the subsequent exchange refers to ring D. This assignment is in fact in line with previous calculations of the Raman spectra for different protonation pattern of the chromophore in Agp2 6. Also, the reversal of the exchange (D→H) confirms this conclusion. At first, we note the immediate re-appearance of the N-H in-plane bending (B, C) at 1549 cm−1 (Fig. 3D). While the position of the strongest band at 1590 cm−1 band remains unchanged, a distinct shoulder at 1605 cm−1 is observed, implying that the C-D (1590 cm−1) and A-B (1605 cm−1) stretching modes do no longer coincide. This can only be rationalized for the D→H exchange at ring A but not at ring D. Again, complete D→H exchange to afford the spectrum in Fig. 3A is achieved only after long times in the dark (vide infra) or after a full photocycle. Thus, we conclude that, in analogy to PaBphP and Agp2 6, RpBphP6 exhibits a retarded H/D (D/H) exchange at ring D in the Pfr state.
The four isotopomers of the Pfr chromophore which can be obtained in pure form should assist the vibrational assignment. Unfortunately, the N-H ip coordinates of rings A and D are distributed among many modes (Fig. S1). Thus, in contrast to the corresponding coordinates of rings B and C which dominate the composition of two modes, there are no marker bands that may trace specific hydrogen bond interactions of the N-H groups of rings A and D. However, as a consequence of the delocalization of the ring A and ring D NH ip coordinates, they also contribute to the C=O stretching modes of rings A and D, respectively, which can be detected in the IR difference spectra (“Pr minus Pfr”) (Fig. 3E-H). In line with normal mode analyses (Table S1) and assignments previously made for Agp2 6, the signal pair at 1709 (Pr) and 1698 cm−1 (Pfr) in the fully protonated form of RpBphP6 (Fig. 3E) originates from both C=O stretching modes. Upon immediate H/D exchange, taking place at ring A but not at ring D, the negative peak (Pfr) at 1684 cm−1 represents the downshifted C=O stretching of ring A, whereas the residual minimum at 1698 cm−1 reflects the superposition of the unchanged (negative) signal of the ring D C=O stretching of Pfr and the downshifted positive signals of both C=O stretchings of Pr (Fig. 3F). Note that in Pr, H/D exchange at ring D is not retarded. Complete H/D exchanges in Pfr removes the 1698 cm−1 peak and enhances the signal at 1684 cm−1 since now both C=O stretching modes of Pfr overlap again (Fig. 3G). In a similar way, one may rationalize the spectrum after immediate D/H re-exchange (Fig. 3H). Furthermore, the negative band at 1751 cm−1, originating from the C=O stretching of the propionic side chain of ring C (Pfr, H2O, Fig. 3E), has no positive counterpart implying that this carboxyl function is deprotonated in Pr, consistent with previous findings for Agp2 6. In Pfr, the proton of this carboxyl group is immediately exchanged as shown by the 8 cm−1 downshift in Fig. 3F, in line with the calculations (Table S1). In addition, the spectrum in Fig. 3E shows a weak positive band at 1731 cm−1 which was assigned to a protonated carboxyl of a Glu or Asp residue in Pr 6. Its negative counterpart (Pfr) might be obscured by the strong (positive) C=O stretching modes of the rings A and D.
H/D exchange kinetics
To determine the kinetics of the slow exchange at ring D, phytochrome samples of Agp2 were converted into the Pfr state in H2O (pH 7.8), followed by 3–4 times rebuffering in D2O completed in ca. 1 h which defines the time zero of the kinetic experiments. The exchange was carried out at ambient temperature (298 K) in the dark but the extent of the exchange at given delay times was monitored by RR spectra measured at 80 K. These spectra were analyzed on the basis of the complete component spectra which for the H→D exchange included the chromophore states deuterated at all pyrrole nitrogens (Fig. 4, blue line; see Fig. 3C) and deuterated only at rings A, B and C (Fig. 4, magenta line, see Fig. 3B). In the analogous way, we analyzed the back exchange D→H, here using the component spectra of the states protonated at all pyrrole nitrogens (Fig. 4, red line; see Fig. 3A) and protonated only at rings A, B and C (Fig. 4, green line, see Fig. 3D).
The data for the D→H exchange at ring D seem to follow a biphasic exponential behavior (Figure 5). Due to the “dead time” of the experiment (1 h, vide supra), the half time of the “fast” component could not be determined and thus is estimated to be <10 h, whereas the “slow” component is associated with a half time (τD→H) of ca. 150 h. The H→D exchange at ring D is even much slower since at t = 150 h only a fraction of 20% of the ring D protons were exchanged and the corresponding half time (τH→D) is estimated to be ca. 3 times larger.
Role of the PHY domain
To probe the specific effect of the PHY domain on the structure of the CBP, we investigated a RpBphP6 construct comprising only the PAS-GAF (PG) domain thereof, denoted as RpBphP6-PG. Assembly of the holoprotein was achieved upon addition of biliverdin in the ZZZ configuration. Thus, the smaller extent of Pfr formation for the truncated RpBphP6-PG compared to the wild-type version RpBphP6-PCM (PAS-GAF-PHY) indicates that the PHY domain supports the thermal ZZZ→ZZE isomerization (Fig. S5). Accordingly, the residual contribution of the Pr state had to be subtracted from the RR spectrum of RpBphP6-PG for a comparison with the Pfr state of the complete sensor module (Fig. 6).
The RR spectrum of RpBphP6-PG displays characteristic features of the Pfr state including the N-H ip of rings B and C at 1550 cm−1, the strong C-H out-of-plane mode at 822 cm−1 which, like the C-D stretching at 1604 cm−1 is, however, significantly upshifted compared to RpBphP6-PCM. These frequency shifts point to an increased torsion of the C-D methine bridge 10. The lack of the PHY domain has also a strong impact on the hydrogen bond network in the CBP since, unlike to RpBphP6-PGP, there is no evidence for a protonation of the propionic side chain of ring C. Furthermore, H/D exchange at ring D occurs concomitantly to the other rings as indicated by the one-step 8 cm−1 downshift of the prominent C-D mode that parallels the disappearance of the ring B/C N-H ip mode (Fig. 6).
Role of the substitution pattern of the chromophore
PaBphP has been shown to assemble biliverdin derivatives that differ with respect to the substitution pattern of the propionic side chains 14. In δ-BV, the vinyl and propionic substituents of rings D and B are interchanged as shown in Fig. 7. Although binding of δ-BV to the apoprotein is less efficient compared to the native chromophore, the resultant holoprotein shows a Pfr-like absorption spectrum 14. Moreover, RR spectroscopy revealed the characteristic features of the Pfr chromophore geometry in the entire spectral region (Fig. 7, Fig. S2) indicating that even details of the ZZEssa geometry are essentially the same as for the native chromophore. Also the retarded H/D exchange at the ring D nitrogen is preserved as shown by the stepwise downshift of the C-D stretching mode from 1600 to 1596 cm−1 upon exchange in the dark and eventually to 1591 cm−1 after a full photocycle (Fig. 7).
Homology models
Solely based on the protein sequence, multiple alignment of previously clustered prototypical and bathy phytochromes did not reveal any unambiguous indication of a different overall structure of the PCMs (PAS-GAF-PHY) in these two groups of phytochromes. Therefore, we generated three-dimensional (3D) homology models with the program MODELLER for bathy phytochromes Agp2 and RpBphP6 and the bathy-like phytochrome XccBphP taking the crystal structure of the PaBphP photosensory module as initial starting model for the 3D alignment. In addition, we compared these models with the RpBphP1 crystal structure 21. Using the Pfr crystal structure of DrBphP-PCM as reference 18, we constructed crude homology models of the PCMs for Agp1, RpBphP2 and CphB in the structurally yet unknown Pfr states of these prototypical phytochromes. The superimposed PCM homology models and specifically the CBP revealed an overall high degree of similarity regarding the backbone structure of the PAS and GAF domains. In contrast, we found several key differences in the PHY domain between prototypical and bathy phytochromes.
Among the two opposed structural elements of the so-called PHY-tongue, the three-fold α-helix is largely preserved in the Pfr state, whereas the N-terminal random coil element revealed substantial variations (Fig. 8, Fig. S3). In bathy phytochromes, the random coil element forms an extended loop near ring A of the chromophore, the CBP, and the Asp-Ile-Pro (DIP) motif. In addition, it has a large contact area with the PAS domain (Fig. 8A,C). Conversely, prototypical phytochrome Pfr homology models lack this type of interaction, since here the random coil element moves toward the GAF domain, shortening the tongue loop region between ring A and the conserved DIP motif around the highly conserved Asp194 (numbering refers to the PaBphP sequence) (Fig. 8B,D).
The distinct contacts between the PHY-tongue and the chromophore binding domain lead to different interaction patterns of ring D, the DIP-aspartate and the Pro-Arg-x-Ser-Phe (PRxSF) motif. In bathy phytochromes, there is an additional arginine (Arg453 in PaBphP; PDB entry 3NHQ) (Fig. 8C) displaying a relative strong hydrogen bond interaction network with the carboxylate side chain of the adjacent aspartate (Asp194), the hydroxyl group of Ser459 of the PRxSF motif and Ser193. In prototypical phytochromes (e.g. the crystal structure of DrBphP in its Pfr state, PDB entry 5C5K) only Ser468 (here numbering refers to DrBphP) of the PRxSF motif interacts with the conserved aspartate of the DIP motif, whereas the additional arginine is not conserved in prototypical phytochromes. This position is often occupied by an additional aspartate instead of an arginine which is directed away from the DIP motif. On the one hand, the shorter loop in prototypical phytochromes impairs a stronger interaction between residues of the PHY domain with the DIP motif. On the other hand, the crystal structure of DrBphP in its Pfr state and our Pfr state homology models of other prototypical phytochromes showed a hydrophobic residue (Leu or Ile) instead of an arginine conserved in bathy phytochromes in the vicinity of the DIP motif (Fig. 8B,D). However, this hydrophobic residue (Leu or Ile) is highly conserved and is located two amino acid positions preceding the PRxSF motif in both prototypical and bathy phytochromes. Conceivably, this hydrophobic position contributes to correct packing and positioning of ring A in both prototypical and bathy phytochrome Pfr states. In general, the Pfr crystal structure and homology models of prototypical phytochromes partly reveal different interaction patterns between ring D, the DIP-aspartate and the PRxSF motif compared to the Pfr state of bathy phytochromes. In contrast, all bathy phytochrome crystal structures and homology models showed a well-preserved interaction cluster around the ring D.
Interestingly, positioned in a slightly different fold of the RpBphP1 structure, another additional arginine from the variable x position of the PRxSF motif faces toward the DIP-aspartate.
Discussion
The present vibrational spectroscopic analysis of the Pfr state of RpBphP6 reveals essentially the same homogenous chromophore structure as determined previously for PaBphP and Agp2 10, including the protonated ring C propionic side chain, and a retarded H/D exchange at the ring D nitrogen 6. These properties are, hence, identified as characteristic structural descriptors for bathy phytochromes in general. In fact, XccBphP, which due to its thermal equilibrium between Pr and Pfr states bears characteristics of both bathy and prototypical phytochromes, displays a RR spectrum of the Pfr state different to those of PaBphP, Agp2 and RpBphP6 and is therefore classified as “bathy-like” phytochrome 19. Thus, it is tempting to conclude that all three descriptors listed above account for the phenomenological property of bathy phytochromes, the thermal stability of the Pfr state.
We first consider the unprecedented H/D exchange kinetics for the pyrrole nitrogens, which involves a fast exchange at the rings A, B and C and a much slower exchange at ring D. The fast exchange is beyond the resolution of the spectroscopic experiments. The structural models do not reveal any potential limitations for water or proton/deuteron access to the CBP such that the exchange of these proton may well occur on the time scale typical for fast-exchanging protons in proteins, that is within milliseconds 26. Correspondingly, also the retarded exchange at ring D can hardly be rationalized by a restricted accessibility. Instead, the unusually slow exchange points to extraordinarily strong hydrogen bonding interactions 27, which in the present case involve the carboxylate side chain of Asp194. Accordingly, the difference between the H→D and D→H exchange rates (τH→D ≈3·τD→H) must be primarily due to a larger hydrogen bond energy for the N-H (EHB) compared to the N-D group (EDB) of ring D. In fact, theoretical studies and experimental data indicate that the ratio EHB/EDB increases with increasing EHB 28, 29.
These considerations finally raise the question as to the structural basis for the particular strength of these hydrogen bonding interactions in bathy compared to prototypical phytochromes. In both cases, the carboxylate side chain of the highly conserved Asp194 (PaBphP numbering) serves as the primary proton/deuteron acceptor. However, as revealed by the present analysis of crystal structure and homology models, the specific differences refer to the additional interactions of Asp194 that forms a salt bridge or a hydrogen bond with Arg453 (PaBphP numbering) of the PHY domain in bathy phytochromes, whereas in the primary sequence of prototypical phytochromes an aspartate occupies this position instead of the arginine. Structurally, this aspartate faces away from the DIP motif and the chromophore pocket. Thus, we conclude that it is the specific Arg-Asp complex with ring D in bathy phytochromes which provides the high stability for the hydrogen bond of the ring D N-H group as reflected by the shorter N(ring D)–O(Asp) distances (Table S3). Most likely, the higher stability originates from the increased (negative) partial charges on the side chain oxygens of the DIP-aspartate in the presence of Arg453 as suggested by QM calculations (Table S2).
This interpretation is in line with the results obtained for the PAS-GAF domain of RpBphP6. The absence of the PHY domain and particularly of Arg453 removes the stabilization of the hydrogen bond such that the H/D exchange at ring D occurs concomitantly to the other N-H groups of the chromophore.
As a potential consequence of the stable hydrogen bond interaction network with ring D in bathy phytochromes, the entire chromophore structure is likely to be quite rigidly fixed, corresponding to the homogeneous conformation reflected by the RR spectra 10. It is now interesting to compare this conclusion with a recent two-dimensional photon echo spectroscopic study on the photoconversion of the Pfr state in PaBphP that revealed a functionally homogeneous Pfr chromophore, whereas the analysis of the excitation energies pointed to fluctuations of the surrounding protein environment 30. However, the impact of this ground-state heterogeneity on the chromophore structure is evidently too small to be reflected by the RR spectra 10. Conversely, RR spectroscopy can distinguish between “functionally” different ground-state conformers such as the fluorescent and nonfluorescent Pr conformers in RpBphP6 variants 16, 31 or the Pfr conformers of prototypical phytochromes that may be related to different thermal isomerization rates 10, 32. Here, the structural models indicate a significantly weaker hydrogen bond between ring D N-H and Asp207 (DrBphP) in the Pfr state of prototypical phytochromes. This may account for the formation of RR-detectable conformational substates differing with respect to the torsion around the C-D methine bridge 10. In bathy phytochromes, the PHY domain does not only stabilize the hydrogen bond of the ring D N-H group but also ensures the unprecedented high pKa value of the propionic side chain of ring C which is drastically decreased in the truncated version of RpBphP6. Conversely, modification of the substitution pattern of ring D in the δ-BV adduct of PaBphP does not affect the hydrogen bond interactions of the N-H group.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft, SFB1078 (IGK, B6 and C3), and by a fellowship from the Stifterverband für die Deutsche Wissenschaft (to T.F.).
