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The role of the propionic side chains for the photoconversion of bacterial phytochromes Maria Fernandez Lopez, Anh Duc Nguyen, Francisco Velazquez Escobar, Ronald González, Norbert Michael, Zaneta Nogacz, Patrick Piwowarski, Franz Bartl, Friedrich Siebert, Inge Heise, Patrick Scheerer, Wolfgang Gärtner, Maria Andrea Mroginski, and Peter Hildebrandt Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00526 • Publication Date (Web): 26 Jul 2019 Downloaded from pubs.acs.org on August 1, 2019
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Biochemistry
The role of the propionic side chains for the photoconversion of bacterial phytochromes Maria Fernandez Lopez†, ‡, Anh Duc Nguyen†,‡, Francisco Velazquez Escobar†, Ronald González†, Norbert Michael†, Żaneta Nogacz, Patrick Piwowarski, Franz Bartl, Friedrich Siebert§, Inge Heise⌠, Patrick Scheerer⌡, Wolfgang Gärtner⌠,₸, Maria Andrea Mroginski†*, and Peter Hildebrandt†* †
Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straße des 17. Juni 135, D-
10623 Berlin, Germany. Humboldt
Universität zu Berlin, Institut für Biologie, Biophysikalische Chemie, Invalidenstr.
42, D-10115 Berlin, Germany. §Albert-Ludwigs-Universität
Freiburg, Institut für Molekulare Medizin und Zellforschung,
Sektion Biophysik, Hermann-Herderstr. 9, D-79104 Freiburg, Germany. ⌠
Max Planck Institut für Chemische Energiekonversion, Stiftstr. 34-36, D-45470 Mülheim,
Germany. ⌡
Group Protein X-ray Crystallography and Signal Transduction, Institute of Medical Physics
and Biophysics, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
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₸
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Institut für Analytische Chemie, Universität Leipzig, Linnéstr. 3, D-04103 Leipzig, Germany.
ABSTRACT: Bacteriophytochromes harbouring a biliverdin IX (BV) chromophore undergo photoinduced reaction cascades to switch between physiologically inactive and active states. Employing vibrational spectroscopic and computational methods, we analysed the role of propionic substituents of BV for the transformations between the parent states Pr and Pfr in prototypical (Agp1) and bathy (Agp2) phytochromes from Agrobacterium fabrum. Both proteins form adducts with BV monoesters (BVM), esterified at the propionic side B (PsB) or C (PsC) but in each case only one monoester adduct is reactive. In the reactive Agp2-BVM-B complex (esterified at ring B) the Pfr dark state displays the structural properties characteristic of bathy phytochromes, including a protonated PsC. As in native Agp2, PsC is deprotonated in the final step of the Pfr phototransformation. However, the concomitant -helix/-sheet secondary structure change of the tongue is blocked at the stage of unfolding the coiled loop region. This finding and the shift of the tautomeric equilibrium of BVM towards the enol form are attributed to the drastic changes of the electrostatic potential. The calculations further suggest that deprotonation of PsC and the protonation state of His278 control the reactivity of the enol tautomer, thereby accounting for the extraordinary slow thermal reversion. Although strong perturbations of the electrostatic potential are also found for Agp1-BVM, the consequences for the Pr-to-Pfr phototransformation are less severe. Specifically, the structural transition of the tongue is not impaired and thermal reversion is even accelerated. The different response of Agp1 and Agp2 to monoesterification of BV points to different photoconversion mechanisms.
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Biochemistry
Introduction Phytochromes represent a class of photoreceptors that were originally discovered in plants but meanwhile are also found in cyanobacteria, bacteria, and fungi.1–6 Canonical phytochromes consists of a photocore/photosensor module (PCM), composed of a PAS (Per/Arnt/Sim-), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA-) and PHY (phytochrome-specific-) domain triad carrying the covalently bound light-absorbing tetrapyrrole chromophore, and an output module which is frequently a histidine kinase (HK) or a HK-related module.2,7 The type of chromophore and its binding site differs among the phytochromes of different origin. In plant phytochromes, phytochromobilin is attached to a Cys in the GAF domain (alike the binding situation in canonical cyanobacterial phytochromes except of the use of phycocyanobilin as chromophore) whereas in bacterial phytochromes a Cys residue of the PAS domain forms a thioether linkage to the biliverdin IX (BV) chromophore (Figure 1). Common to all phytochromes is the photo-induced reaction sequence that starts with the photoisomerisation of the methine bridge double bond between the pyrrole rings C and D, followed by a series of chromophore relaxation steps and eventually structural changes of the protein.2,8 These events constitute the photoswitch between the parent states Pr and Pfr in which the chromophore adopts a ZZZssa and ZZEssa configuration, respectively. The functional structural change of the protein that couples the photoswitch with the output module is a secondary structure transition of the socalled tongue segment of the PHY domain from an extended -sheet (Pr) to a largely -helical structure (Pfr).9 Despite the availability of crystallographic structures of the parent states,9,10,19,20,11–18 and even of an early and a late intermediate of the photocycle,20,21 the underlying molecular mechanism of generating the biological signal is still not understood.
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Figure 1. Top, simplified reaction scheme of phytochromes with the black arrows indicating the photoinduced reaction sequences. In prototypical and bathy phytochromes, Pr and Pfr are the stable dark states, respectively. The gray arrows refer to the thermal reversion routes from Pfr to Pr and Pr to Pfr in prototypical and bathy phytochromes, respectively. Bottom, structural formulas of the biliverdin monomethylesters with the ester function (highlighted in blue) at the propionic side chain of ring B (BVM-B, left) and ring C (BVM-C, right). The chromophores are shown in the ZZZssa configuration of the Pr state; in Pfr, the chromophore adopts the ZZEssa configurations generated by rotation around the C(15)=C(16) double bond (red). The chromophore is covalently bound to the protein via addition of a Cys side chain to the vinyl function of ring A (highlighted in yellow).
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Biochemistry
A particular challenge in phytochrome research is to determine the role of individual amino acid residues or functional groups of the chromophore pocket and the interplay between chromophore and protein that has been addressed by site-directed mutagenesis,22,23 and assembly with chemically-modified or –locked chromophores.24–27 Substitution of highly conserved amino acids in the chromophore pocket frequently impaired the Pr/Pfr phototransformations and thus provided only limited information about the mechanistic role of the respective residues.22,23 Appropriate chemical modifications of the chromophores, albeit synthetically demanding, were shown to be an instructive alternative as they allow for less drastic alterations of the chromophore and its immediate surroundings.27 Thus, a variety of modifications were made at ring D leading to photoactive constructs after assembling with plant phytochrome phyA. The ring D substitutions primarily affected the optical properties of the parent states without notable perturbations of the general reaction mechanism. More severe effects were noted upon modifying the propionic side chains of rings B and C.25,26,28–30 Early experiments by Song and coworkers demonstrated that esterification of both side chains of the phytochromobilin chromophore impaired assembly with the phyA, whereas a single free propionic side chain, albeit not specified if ring B or C, was reported to be essential for photoconversion.28 Similar results were obtained for phyB demonstrating the specific importance of the ring C propionic side chain for the Pr-to-Pfr transition.25,26 In a comparative study on the cyanobacterial phytochrome Cph12 and the bacterial phytochrome DrBphP from Deinococcus radiodurans, carrying a phycocyanobilin (PCB) and biliverdin (BV) chromophore in their respective native states, Lagarias and coworkers employed chromophores with single amidated side chains to determine the role of the two propionic side chains separately.29,30 The authors found that covalent binding, albeit less efficient, occurred in either case, but the effect of side chain
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functionalization was different for the Cph12 and DrBphP adducts with the respective PCBand BV derivatives. For DrBphP both propionic side chains were found to be important for the proper incorporation of the chromophore in the protein and the respective photoconversion, since amidation of either side chain induced structural heterogeneity of the parent states, reflected by dual absorption bands in the UV-vis spectra. However, the molecular nature of the structural heterogeneity was not clear. Inspired by these studies, we have now employed UV-vis absorption and vibrational spectroscopies together with quantum-mechanics/molecular-mechanics (QM/MM) methods to acquire a molecular description of the impact of the propionic chains on the structure of the chromophore and its photoinduced reaction in bacterial phytochromes. The investigations were based on Agp1 and Agp2 from Agrobacterium fabrum as representatives of the groups of prototypical and bathy phytochromes, respectively.5,31,32 Whereas in prototypical phytochromes the Pr state persists in the dark, Pfr is the thermodynamically stable state in bathy phytochromes (Figure 1). Correspondingly, there are, in addition to the photochemical transformations between Pr and Pfr, thermal conversion routes that are essentially unidirectional from Pfr to Pr and from Pfr to Pr in prototypical and bathy phytochromes, respectively. For the dark states of Agp1 (Pr) and Agp2 (Pfr) as well as for the functional intermediate Meta-F of an Agp2 variant, crystal structures were determined previously.17,20 These 3D-structures served as a basis for computations and the interpretation of spectroscopic data in the present work. Here we have studied Agp1 and Agp2 assembled with BV derivatives in which one propionic side chain was esterified (biliverdin IX monomethylester - BVM; Figure 1). Based on this combined experimental-theoretical approach we show that the local electrostatics in the chromophore binding pocket is a key parameter for controlling the mechanism and dynamics of the
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Biochemistry
photoinduced reaction sequences of phytochromes. The consequences of introducing a BVM are particularly severe for Agp2 since the secondary structure change of the tongue is impaired and thermal reversion of the final product of the phototransformation is drastically slowed down. These findings can be rationalized on the basis of QM/MM calculations that indicate the structural and mechanistic importance of the local electrostatic potentials and their modulation by the deprotonation of the propionic side chain C. The less severe sensitivity of Agp1 towards substituting BV by BVM further supports the view of different mechanisms for the Pfr Pr and Pr Pfr phototransformations in bathy and prototypical phytochromes, respectively.
Materials and methods Synthesis. Biliverdin IXα as starting material was synthesized from bilirubin IXα by oxidation with DDQ similarly as described previously.33 In brief, 200 mg (0.342 mmol) of bilirubin IXα, dissolved in dimethylformamide (DMF), were added at ambient temperature to 164 mg (0.72 mmol) of DDQ (2,3-dichloro-5,6-dicyano-1,4-benzochinone), dissolved in 320 mL DMF under an argon atmosphere yielding nearly immediately a dark-green colored solution. 1.6 L of icecooled water was added and the reaction mixture was repetitively extracted with chloroform, until the chloroform layer remained nearly colorless. The combined organic layers were dried over Na2SO4, followed by removal of the organic solvent via a rotatory evaporator, to afford a dark-green solid. Synthesis of biliverdin IXα monomethylester was carried out under Ar atmosphere and in the dark. Biliverdin IXα (BV) was dissolved in methanol and kept on ice, and concentrated sulfuric acid (2 mL) dissolved in methanol was slowly added. The progress of the reaction was followed
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by thin layer chromatography indicating that the reaction was completed within ca. 30 min. Then the reaction mixture was poured into a solution of NaHCO3 in 600 mL of ice water. The reaction product was extracted with dichloromethane, until the organic layer remained nearly colorless. After the first dichloromethane treatment, a saturated solution of NaHCO3 was added to the reaction mixture to neutralize the remaining acid. The organic layer was dried over Na2SO4, followed after filtration by the removal of the organic solvent. The reaction product was purified two times on a silica column yielding two blueish-green fractions. Characterization by NMR spectroscopy (methylester protons at 3.6 ppm) and mass spectrometry (Supporting Information, Figures S1 and S2) proved that the first fraction included the dimethylester (major component, ~ 70% of the sample) and the two BVMs carrying the ester function at the propionic side chain of ring B (BVM-B) or ring C (BVM-C) (Figure 1) with an overall yield of ca. 30%. However, BVM-B and BVM-C could not be separated such that they were used as a mixture for the assembly with apo-phytochromes (apo-Agp1 and apo-Agp2). All chemicals used for the syntheses were of highest purity grade available. Protein expression and assembly. The coding DNAs from Agp1 and Agp2 were cut to the length of the photosensor (PCM) modules (1-495 and 1-501 AA for Agp1 and Agp2, respectively),32 and cloned into a pET21b expression vector (Novagen) with C-terminal 6-His tags. The vector was transformed into E. coli strain DE3(BL21) (Novagen) and grown on M9-Glucose Medium with Ampicillin. All procedures were performed following standard methods.34 The cultures were induced with 100 µM IPTG and grown overnight at 20 oC. Cells were disrupted by a French press and soluble apo-phytochrome was purified by Ni2+ affinity chromatography. 10% excess of BV or BVM was dissolved in DMSO and diluted with an aqueous solution of 50 mM Tris (pH 7.8) and 300 mM NaCl, corresponding to a maximum of 10% DMSO. Under green safe
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light apo-phytochrome and chromophore solution were mixed and assembled at 5 oC overnight. Misfolded phytochrome and residual free chromophore was finally separated by SEC on Sephacryl S-200 HR (GE Healthcare) with 20 mM HEPES (pH 7.5) and 150 mM NaCl as moving phase. Spectroscopy. Whereas the spectroscopic experiments in this work were carried out with the photosensor modules of Agp1 and Agp2, the spectra of the Pr state of Agp2-BV, taken from previous studies, refer to the full-length protein since the fast thermal Pr Pfr reversion of the photosensor module does not allow a sound spectroscopic characterization of the Pr state. RR spectroscopic measurements were performed using a Bruker Fourier-transform Raman spectrometer RFS 100/S with 1064 nm excitation (Nd-YAG cw laser, line width 1 cm−1), equipped with a nitrogen-cooled cryostat from Resultec (Linkam). All spectra of the samples in frozen solution were recorded at ca. 90 K with a laser power at the sample of 780 mW with an accumulation time of typical one hour. In order to identify potential laser-induced damage of the phytochrome samples, RR spectra before and after a series of measurements were compared. In no case, changes between these control spectra were observed. Protein and buffer Raman bands were subtracted on the basis of a Raman spectrum of apo-phytochrome. IR spectroscopic measurements were carried out in the transmission mode at 20° C using a IFS66v/s Bruker FTIR spectrometer, equipped with a liquid nitrogen-cooled MCT (MercuryCadmium-Telluride) detector. IR spectra were recorded after 128 single scans with a spectral resolution of 2 cm1 in the range between 900 cm1 and 1800 cm1, using optical filter settings
