Regulatory Implications of Structural Changes in Tyr201 of the Oxygen

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Regulatory Implications of Structural Changes in Tyr201 of the Oxygen Sensor Protein FixL Takeo Yamawaki, Haruto Ishikawa, Misao Mizuno, Hiro Nakamura, Yoshitsugu Shiro, and Yasuhisa Mizutani Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00405 • Publication Date (Web): 01 Jul 2016 Downloaded from http://pubs.acs.org on July 2, 2016

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Biochemistry

Regulatory Implications of Structural Changes in Tyr201 of the Oxygen Sensor Protein FixL

Takeo Yamawaki†, Haruto Ishikawa†, Misao Mizuno†, Hiro Nakamura‡, Yoshitsugu Shiro‡, Yasuhisa Mizutani†* †

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan. ‡

RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.

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ABSTRACT FixL is a heme-based oxygen-sensing histidine kinase that induces the expression of nitrogen fixation genes under hypoxic conditions. Oxygen dissociation from heme iron in the sensor domain of FixL initiates protein conformational changes that are transmitted to the histidine kinase domain, activating autophosphorylation activity. Conversely, oxygen binding inhibits FixL kinase activity. It is essential to elucidate the changes that occur in the protein structure upon this oxygen dissociation for understanding of the allosteric transduction mechanism. We measured ultraviolet resonance Raman spectra of FixL and its mutants for deoxy, oxy, and carbonmonoxy forms to examine the changes in protein structure upon oxygen dissociation. The observed spectral changes indicated that Tyr201 and its neighboring residues undergo structural changes upon oxygen dissociation. Kinase assays showed that substitution of Tyr201 significantly decreased the inhibition of kinase activity upon oxygen binding. These data mean that weakening of the hydrogen bond of Tyr201 that is induced by oxygen dissociation is essential for inhibition of kinase activity. We also observed spectral changes in Tyr residues in the kinase domain upon oxygen dissociation from FixL, which is the first observation of oxygen-dependent structural changes in the kinase domain of FixL. The observed structural changes support the allosteric transduction pathway of FixL which we proposed previously [Yano, S., Ishikawa, H., Mizuno, M., Nakamura, H., Shiro, Y., and Mizutani, Y. (2013) J. Phys. Chem. B 117, 15786-15791].


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INTRODUCTION Allosteric regulation of protein function is achieved by structural changes induced by association and dissociation of ligand molecules. These structural changes are transmitted to other parts of the protein that are distant in space from the ligand binding sites. A striking example of such an allosteric regulation is found in PAS kinases.1, 2 These kinases consist of a sensor domain and a histidine kinase domain. A PAS kinase undergoes structural changes when external stimuli such as oxygen, light, and redox potential are perceived by the sensor domain. Subsequently, the structural changes are transmitted to the kinase domain resulting in regulation of the kinase activity. FixL is one of the best characterized PAS kinases. This protein responds to low O2 concentration and regulates the expression of nitrogen-fixation genes by phosphorylating the response regulator FixJ.3, 4 The O2-dependent regulation of its kinase activity is initiated by the dissociation of O2 from the sensor domain of FixL, resulting in conformational changes of the protein.5, 6 These changes are transmitted to the histidine kinase domain, thereby regulating the kinase activity.7-10 The deoxy FixL is autophosphorylated on a histidine residue in the kinase domain, using ATP as the phosphoryl donor. Although the crystal structure of the deoxy and oxy sensor domains of FixL have been reported,11-15 an understanding of how the O2 dissociation initiates structural changes is still limited. As for the kinase domain, even crystal data for neither full length protein nor truncated kinase domain have been reported. Thus, the allosteric mechanism for the O2-dependent regulation of the kinase activity of FixL is poorly understood. The sensor domain of FixL is a good prototype for the PAS structural motif, which is widely involved in a number of sensory proteins.16 The PAS structural motif is characterized by its threedimensional structure composed of antiparallel, five-strand β-sheets and an α-helix. Figure 1 shows a crystal structure of the truncated sensor domain of FixL in the oxy form.12 Residues


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discussed in the present paper are shown with labels. The kinase domain of FixL contains regions that are highly conserved among other histidine kinases.17 The histidine kinase domain is further divided into two subdomains: a catalytic ATP-binding domain and a histidine phosphotransfer domain. Because of the high conservation of these two domains, FixL can be used as a model system for structural and functional studies of PAS histidine kinases. In this study, we investigated the pathway of structural changes that regulate the kinase activity of FixL using ultraviolet resonance Raman (UVRR) spectroscopy and phosphate affinity SDSPAGE (Phos-tag SDS-PAGE). UVRR spectroscopy makes it possible to probe protein structure by selectively enhancing Raman bands of Tyr, Phe, and Trp residues. Phos-tag SDS-PAGE separates phosphorylated from non-phosphorylated proteins based on mobility differences in a polyacrylamide gel and thus it is applicable to a kinase assay.18-20 However, the Phos-tag reagent is labile to the reductant that is necessary for reduction of FixL, and thus Phos-tag SDS-PAGE has never been applied to the kinase assay of the ferrous form of FixL. We successfully optimized experimental conditions of Phos-tag SDS-PAGE for kinase assay of the ferrous form of FixL. By combining the data of UVRR spectroscopy and Phos-tag SDS-PAGE, structural changes that regulate the kinase activity of FixL were elucidated. In particular, we showed that weakening of the hydrogen bond of Tyr201 in the sensor domain upon O2 dissociation is essential to inhibition of the kinase activity. We also observed spectral changes in Tyr residues in the kinase domain upon O2 dissociation from FixL. This is the first observation of O2-dependent structural changes in the kinase domain of FixL The observed structural changes support the allosteric transduction pathway of FixL which we proposed previously.10

MATERIALS AND METHODS


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Protein Expression and Purification. Recombinant 6×His-tagged soluble wild-type (WT) FixL from Sinorhizobium meliloti (V131-D505) and its site-directed mutants were expressed in the E. coli strain JM109 with C301A mutation to prevent aberrant disulfide bond formation at Cys301.7 Site-directed mutagenesis was performed using PrimeStar Max methods (Takara Bio). The cells were grown in TB medium containing ampicillin (final concentration, 100 μg/mL) and subsequent JM109 cells harboring the plasmid were grown to an OD600 of 0.7 in a 37 °C incubator, followed by addition of 50 μM isopropyl 1-thio-β-D-galactopyranoside. After 10 h of induction at 25 °C, cells were harvested by centrifugation at 4 °C. The His-tagged recombinant FixL was purified using a cOmplete His-Tag Purification Column (Roche Diagnostics). The purified FixL was dialyzed against 50 mM sodium phosphate buffer at pH 7.0 containing 5% glycerol. The purity of the recombinant FixL was confirmed by SDS-PAGE. The protein concentration used for the UVRR measurements was 30 μM in 50 mM sodium phosphate containing 300 mM sodium perchlorate as an internal intensity standard and 5% glycerol at pH 7.0. The deoxy, carbonmonoxy, and oxy forms of FixL were prepared according to protocols described previously.8 The oxy and carbonmonoxy forms of FixL for all measurements were prepared under 1 atm of O2 and CO, respectively. Resonance Raman Measurements. UVRR spectra were obtained as described previously.10 Briefly, probe pulses at 233 nm were the fourth harmonics of the output of an Nd:YLF-pumped Ti:sapphire laser (Photonics Industries, TU-L). The laser power was set to 0.5 mW. Visible resonance Raman spectra of the deoxy form were obtained using the 441.6-nm line of a He-Cd laser (Kimmon Electric, IK4401R-D). Visible resonance Raman spectra of the oxy and carbonmonoxy forms were obtained using a 405-nm line of a diode laser (Innovative Photonics Solutions, I0405SD0045B). Scattered light was detected with a liquid-nitrogen-cooled CCD


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camera (Roper Scientific, Spec-10:400B/LN) attached to a custom-made prism prefilter (Bunkokeiki) equipped with a single spectrograph (Horiba, iHR550). Raman shifts were calibrated with Raman bands of cyclohexane and carbon tetrachloride. Sample solutions were contained in an airtight 10 mmϕ NMR tube, spun at 3200 rpm at room temperature. The integrity of the samples against possible denaturation due to exposure to laser light was carefully checked by inspecting the change in the visible absorption spectrum before and after the resonance Raman measurements. Assay of FixL Phosphorylation. The FixL autophosphorylation reaction was performed in 3 mL of 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.2 mM MnCl2, 2.0 mM ATP, and 5.0 μM FixL at 23 °C. The deoxy, oxy, and carbonmonoxy forms of FixL samples were contained in an air tight absorption cell. To stop the reaction, 2.0 μL of the reaction mixture was taken out and added to 2.0 μL of an SDS-PAGE loading buffer (0.22 M Tris-HCl (pH 6.8), 8.9% w/v SDS, 10% v/v glycerol, 0.10 M DTT, and 0.10% w/v BPB) for the desired incubation period (15 min), The remaining sodium dithionite for the preparation of deoxy, oxy, and carbonmonoxy forms of FixL prevented proper migration in Phos-tag SDS-PAGE gel, because the reductants may react with the Phos-tag reagent. To oxidize sodium dithionite, we added 1 μL of 100 mM potassium ferricyanide solution. Phos-tag SDS-PAGE was performed at 4 °C using a 12.5% acrylamide gel with 50 μM of the Phos-tag reagent (Wako Pure Chemical Industries) and two equivalents of MnCl2 to the Phos-tag reagent. The band densities of Coomassie brilliant blue-stained (CBB) gels were detected using a gel imager (Bio-Rad, ChemiDoc™ XRS+). The boiling treatment of sample with the SDSPAGE loading buffer was omitted for the Phos-tag SDS-PAGE analysis because we could not detect the phosphorylated band for the FixL proteins for the boiled sample. The phosphorylated histidine in the FixL protein would be very unstable in the SDS-PAGE loading buffer.


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RESULTS UVRR Spectra of WT FixL. UVRR spectra of the deoxy (trace a), oxy (trace b), and carbonmonoxy forms (trace c) of WT FixL excited at 233 nm are shown in Figure 2. The spectra contain UVRR bands due to Tyr (labeled Y) and Phe (labeled F) residues. The mode assignments of the Tyr and Phe bands made by Harada and Takeuchi21 were adopted: Y8a, Y7a, Y9a, and Y1 modes are the ring stretch, Cring–Cβ stretch; CH in-plane bend, and the ring symmetric stretch coupled with the Cring–Cβ and the Cring–O stretch of Tyr, respectively. The F12 mode is the ring deformation mode of Phe. Excitation at 233 nm selectively enhances the Raman bands of Trp residues in addition to those of Tyr and Phe residues in proteins. However, no Raman band due to a Trp residue was observed in the spectra in the present study since FixL does not contain any Trp residues. The band at 934 cm–1 that is marked by an asterisk is due to perchlorate ion that was added to the sample solution. Spectral contributions of the buffer, quartz, and oxygen gas were subtracted as an internal intensity standard. To examine spectral changes upon ligand dissociation, difference spectra between the deoxy and liganded forms were calculated. Trace (d) in Figure 2 is the difference spectrum between the deoxy and oxy forms [(a)-(b)] whereas trace (e) is the difference spectrum between the deoxy and carbonmonoxy forms [(a)-(c)]. These difference spectra were calculated after correction of the self-absorption effect on Raman scattering using the perchlorate band at 934 cm–1. Raman intensities of the difference spectra are magnified by a factor of 2 to more clearly show spectral changes upon ligand dissociation. The difference spectra indicate that the band positions were not significantly changed upon ligand dissociation. However, the intensity of the Y8a, Y7a, and Y9a bands decreased upon O2 dissociation. The intensity decrease upon O2 dissociation was larger than


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that upon CO dissociation, indicating that the spectral change reflected specific structural changes of FixL upon O2 dissociation. Decreases in intensity of Y8a, Y7a, and Y9a bands upon ligand dissociation and smaller intensity changes with CO dissociation than with O2 dissociation were also observed for the truncated sensor domain of FixL as we previously reported.10 Thus, the O2 specific structural change of Tyr residue(s) in FixL was observed regardless of whether the kinase domain was present or not. The intensity of the F12 band that is due to Phe residues increased following both O2 and CO dissociation. UVRR Spectra of FixL Mutants. To identify which Tyr residue(s) is (are) responsible for the observed intensity changes in the UVRR spectra, we constructed FixL mutants, in which each single Tyr residue was replaced with a Phe residue, and compared their UVRR difference spectra between the deoxy and oxy forms with that of WT FixL. Substitution of a specific Tyr residue was assumed to eliminate the contribution of that specific Tyr to the UVRR intensity. Full-length FixL contains four Tyr residues in the sensor domain at positions 172, 190, 197, and 201, and three Tyr residues in the kinase domain at positions 297, 379, and 496. We therefore constructed Y172F, Y190F, Y197F, Y201F, Y201H (vide infra), Y297F, Y379F, and Y496F FixL mutants. The absorption spectra of the eight mutants resembled those of WT, indicating that the substitution of Tyr with Phe or His did not largely change the heme environment (see Figure S1 in Supporting Information). Figure 3 shows the UVRR difference spectra of FixL WT and the seven Phe mutants between the deoxy and oxy forms. For comparison, the UVRR spectrum of the oxy form is shown at the top. The difference spectra were calculated after the intensities of the spectra of the mutants were normalized by using the 934-cm−1 perchlorate band so that band intensities among the Phe mutants could be compared.


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There was no large variation in the degree of the intensity changes of the F12 band according to the mutants, whereas intensity changes of specific Tyr bands were distinctly different among the mutants. The most dramatic intensity change of the Tyr bands was observed for the Y201F mutant, suggesting that Tyr201 is a main contributor to the decreased intensity of Tyr bands upon O2 dissociation. The intensities of the negative Tyr bands in the difference spectra of Y172F, Y190F, Y192F, Y379F, and Y496F mutants were smaller than that of WT, suggesting that Tyr172, Tyr190, Tyr192, Tyr379, and Tyr496 provide smaller contributions to the intensity decrease of Tyr bands upon O2 dissociation. The difference spectrum of the Tyr297 mutant showed that Tyr297 barely contributes to the intensity decrease. The large contribution of Tyr201 to the intensity decreases indicate that Tyr201 and its environment undergo evident rearrangement upon O2 dissociation. A large intensity change of the Tyr bands was also observed for the FixL Y201H mutant, as shown in Figure S2 (see Supporting information). Visible resonance Raman spectra of FixL Y201 mutants. Figure 4 shows visible resonance Raman spectra for the deoxy, oxy, and carbonmonoxy forms of WT, Y201F and Y201H FixL mutants. The spectra of the mutants are similar to those of WT FixL. In particular, the frequency of the iron-oxygen stretching [ν(Fe−O2)] mode at 570 cm–1 of the oxy form and that of the ironcarbon monoxide stretching [ν(Fe−CO)] mode at 496 cm–1 of the carbonmonoxy form are identical within experimental error. Assignment of the 570-cm–1 band to the ν(Fe−O2) mode was confirmed by the isotope shift between 16O2- and 18O2-derivatives (see Figure S3 in Supporting Information). It is known that ν(Fe−O2) and ν(Fe−CO) frequencies are sensitive to the structure and the environment of the distal heme pocket. High similarity of the stretching frequencies clearly shows that the substitution of Tyr201 has a negligible effect on the distal heme pocket. In the deoxy forms, the frequency of the iron-histidine stretching [ν(Fe−His)] mode at 211 cm–1 was identical for the


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WT and the Y201F and Y201H mutants. The frequency of the ν(Fe−His) band is known to be sensitive to the structure of the proximal side of the heme pocket. The finding of identical frequencies demonstrates that there is no noticeable structural difference in the proximal side among WT, Y201F and Y201H mutants. The intensity ratio of the two propionate bending modes observed at 360-390 cm–1 in the Y201H mutant is different from those of the deoxy forms of the WT and Y201F mutants, suggesting that the orientation of the heme propionates would be changed upon Y201H mutation. Visible resonance Raman spectra in the 1050-1800 cm–1 region show remarkably high similarity among WT, Y201F, and Y201H mutants as shown in Figure S4 (see Supporting information). Phosphorylation Assay. The Phos-tag molecule interacts with a phosphorylated amino acid side chain, resulting in slower migration of the phosphorylated protein in SDS-PAGE.19 Phos-tag SDS-PAGE analysis has been used for the detection of phosphorylated proteins.20, 22 In the case of FixL, the Phos-tag assay was previously applied to the ferric form, which was used as a model of the deoxy form.18 An application to the ferrous form is essential to investigate the ligand-dependent regulation of the kinase activity of FixL. We here expanded this method for analysis of the phosphorylation of the deoxy, oxy, and carbonmonoxy forms of FixL. Figure 5A shows a CBB stained Phos-tag SDS-PAGE gel used for analysis of the phosphorylation of the deoxy, oxy, and carbonmonoxy forms of FixL. Phosphorylation reactions were performed in the presence or absence of 2.0 mM ATP for 15 min. The deoxy form of WT FixL migrated as two bands on the gel in the presence of ATP. The upper band was the phosphorylated form of FixL that slowly migrated through the gel due to its interaction with the Phos-tag reagent that was bound to the acrylamide gel.18 This result showed that the Phos-tag SDSPAGE method can be applied to phosphorylation analysis of the ferrous form of FixL. The upper


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band of the lane of the oxy form of WT FixL was fainter than that of the deoxy form in the presence of ATP. This is because binding of the O2 molecule to FixL inhibits its kinase activity and therefore the amount of phosphorylated FixL is smaller. Two bands were observed on the gel following Phos-tag SDS-PAGE analysis of the carbonmonoxy form of WT FixL in the presence of ATP, and this result was very similar to that for the deoxy form of WT FixL. This result suggested that inhibition of autophosphorylation of FixL by the CO molecule was negligible. These observations were consistent with a previous radioactive phosphorylation assay for WT FixL.7 We quantitatively analyzed these gel bands using gel imager densitometry and calculated the density of the phosphorylated band divided by the sum of the density of phosphorylated and nonphosphorylated bands, which was then expressed as a percentage. Figure 5B shows a bar graph of the percentages of deoxy, oxy, and carbonmonoxy forms of WT, Y201F, and Y201H FixL mutants that are phosphorylated. The percentages of deoxy forms of WT, Y201F, and Y201H FixL mutants that were phosphorylated were estimated to be 60%, 57%, and 51%, respectively, indicating that the effects of amino acid substitution at the position 201 on the kinase activity of the deoxy form were small. Binding of O2 decreased the percentage by approximately 30% for WT FixL, while it is estimated that the binding decreased by about 15% and less than 5%, respectively, for the Y201F and Y201H mutants. The ratios of the percentage of the oxy form to that of the deoxy form were 52%, 76% and 93% in WT FixL, Y201F, and Y201H FixL mutants, respectively. The ratios of the percentage of the carbonmonoxy form to that of the deoxy form were 104%, 96% and 101% in WT FixL, Y201F, and Y201H FixL mutants, respectively. It was confirmed that formation of the met form due to autoxidation was negligible based on absorption and visible resonance Raman spectra. The addition of CO for autophosphorylation inhibition was within experimental error for


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all samples. Thus, mutation of Tyr201 causes malfunction of the kinase inhibition that results from O2 binding to FixL.

DISCUSSION Structural changes in FixL and the functional importance of Tyr201.

The UVRR

measurements of WT and Tyr mutants of FixL showed that Tyr201 and its neighboring residues undergo structural changes upon O2 dissociation from FixL. The Phos-tag assay measurements showed that substitution of Tyr201 decreased kinase inhibition upon O2 binding of FixL. Both experimental results indicate that structural change of Tyr201 and its neighboring residues is essential to the inhibition of FixL kinase activity upon O2 binding. Substitution of Tyr201 affects the structure of neither the distal nor the proximal sides of the heme pocket as shown by the fact that the iron-ligand stretching frequencies [ν(Fe−O2), ν(Fe−CO), and ν(Fe−His)] of the Tyr201 mutants were the same as those of WT. This finding indicates that the observed decrease in kinase inhibition is not due to modification of the heme pocket but is due to disruption of the interaction of Tyr201 with neighboring residue(s). It is known that the intensities of Tyr bands such as Y7a, Y8a, and Y9a bands decrease when the Tyr residue is within a more hydrophilic environment23, 24 or when its hydrogen bond strength is weakened.25,

26

Accordingly, the observed decrease in intensity of the Tyr bands of Tyr201

suggests that dissociation of O2 induced a structural change so that the environment of Tyr201 became more hydrophilic and/or its hydrogen bond strength was weakened. Figure 1 shows a cartoon representation of the crystal structure of the truncated sensor domain of FixL from Bradyrhizobium japonicum (BjFixL) in the oxy form that was previously reported.27 We compared the structures of the sensor domain of the oxy and deoxy forms of FixL based on


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the crystal structures of BjFixL because the crystal structure of FixL from Sinorhizobium meliloti (SmFixL) in the oxy form has not been reported. BjFixL is a homologue of the SmFixL that we used in this study. It is known that the structures of the deoxy and met forms of BjFixL are very similar to those of SmFixL.15 SmFixL and BjFixL sequences share 50% identity and 66% similarity. The RMSD between the crystal structures of the two FixL’s in the deoxy (PDB ID: 1EW015 and 1XJ314) and met forms (PDB ID: 1D0615 and 2VV611) are 0.59 and 0.61 Å, respectively. The indicated numbers of the amino acids in BjFixL in Figure 1 have corresponding amino acid numbers in SmFixL. The crystallographic data show that Tyr201 forms a hydrogen bond with Glu234 in the Hβ sheet. Figure 6 compares the crystal structures of the oxy and deoxy forms of BjFixL. The comparison shows that the positions of the Gβ sheet, the FG loop and the Fα helix are distinctly different between the oxy and the deoxy forms. It can be assumed that the structural change observed for the truncated sensor domain would occur in the same way in full-length FixL because similar spectral changes assignable to Tyr201 were observed both for the truncated sensor domain10 and full-length FixL. Tyr201 is positioned at the C-terminal side of the Fα helix where a structural difference was observed between the crystal structures of the oxy and deoxy forms. The displacement of the Fα helix can change the distance between the Fα helix and the Hβ sheet and thus weaken the hydrogen bond strength between Tyr201 and Glu234. The assumed change in the hydrogen bond upon the O2 dissociation is supported by weakened intensities of the Y7a, Y8a, and Y9a bands. Furthermore, the weakened inhibition of the kinase activity in the Y201F mutant indicated that the hydrogen bond of Tyr201 is required for the signal transduction. Accordingly, we propose a model that the hydrogen bond between Tyr201 and Glu234 transmits the structural change induced by O2 dissociation.


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Substitution of Tyr201 with a His residue weakened the inhibition of the kinase activity even though a His residue can form a hydrogen bond with a Glu residue. This result would be due to the fact that the side chain of a His residue is less bulky than that of a Tyr residue and thus it cannot form a stable hydrogen bond with Glu234. Or it may be due to that the N-H group of the histidine ring is not oriented to form a stable hydrogen bond. However, weakening of this hydrogen bond is not the sole factor that explains the decreased inhibition of kinase activity because the O2 binding of the Y201H mutant showed weaker inhibition than that of the Y201F mutant. The van der Waals volumes of Tyr, Phe and His residues are 141, 135, and 118 Å3, respectively.28, 29 The side chains of Tyr and Phe residues are similar in structure and in van der Waals volume and therefore, the steric effect of the residue at the 201st position does not greatly change by mutation of Tyr to Phe. However, the imidazoyl ring of a His residue has a much smaller van der Waals volume than that of the phenol ring of a Tyr residue. The mutation of Tyr201 to a His residue therefore weakens the steric interaction with Glu234. Accordingly, the Y201H mutant shows weakened inhibition of kinase activity both because of this smaller steric interaction and because of the weakened hydrogen bond to the Glu234 at a distant position. The F12 band of Phe showed an intensity change upon O2 dissociation from FixL. This intensity change of the F12 band can be attributed to a change in the environmental polarity. Indeed, the F12 Raman band of Phe at 1000 cm–1 has been reported to be enhanced by up to twice its original value by a decrease in environmental polarity when excited at 220–240 nm.24, 30 The full-length FixL contains as many as twelve Phe residues; there are six residues in each domains. Since the intensity change of the F12 band was not observed for truncated sensor domain, it is likely that the Phe residue(s) responsible for the spectral change locates in the kinase domain and/or the domain interface.


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Structural changes in the sensor domain.

To date, several hypotheses regarding the

mechanism of the inhibition of kinase activity and the O2 selectivity of FixL have been proposed. Crystal structures of the sensor domains of FixL12, 13, 15, 27, 31 have provided some notable findings regarding differences between the liganded and deoxy forms. Structures of the sensor domain bound to CN– and O2 show that ligand binding results in a structural change in the FG loop of the protein, termed the FG loop switch.12,

13, 27

The crystal structure showed that this change is

accompanied by a reorientation of the Arg214 side chain (corresponding to Arg220 in BjFixL) in the heme pocket, and the formation of a hydrogen bond with the bound ligand. In contrast, despite the fact that the carbonmonoxy form adopts a hexacoordinated heme complex similar to that of the CN– and oxy complexes, its structure was reported to be nearly identical to that of the met and deoxy forms of BjFixL.13, 27 Based on these findings, Hao et al. proposed the following regulatory mechanism based on structural changes in the FG loop of the protein. In this mechanism, ligand binding is accompanied by displacement of the heme propionate groups. Displacement of the carboxylate of heme propionate 7 weakens its salt bridge to the side chain of Arg214, prompting replacement of this group at the propionate with the side chain of Arg200 (corresponding to Arg206 in BjFixL). Arg214 then moves into the heme pocket where it forms a hydrogen bond with the bound oxygen, creating a steric clash with Ile209 (corresponding to Ile215 in BjFixL) and producing a ~2 Å shift in the FG loop. The greater sensitivity of FixL to O2 and CN− than to other ligands can be explained by this model, since O2 and CN− are capable of holding the released Arg214 in the heme pocket. Nonetheless, other factors must be involved in the partial inhibition of kinase activity in CO-, NO- and imidazole-bound forms of FixL, because, in those structures, movement of Arg214 into the pocket is not observed.32 Interestingly, in this context, the CN-bound


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R220A mutant of BjFixL retains some ability to weakly inhibit FixL in the absence of the FG loop switch.31 These finding suggests that Arg214 cannot be the sole factor in kinase regulation. Another mechanism proposed for ligand recognition is that the conformational change is driven by steric interaction between the bound ligand and distal hydrophobic residues. The ligand binding pocket of FixL contains three conserved hydrophobic side chains, termed the hydrophobic triad: Ile209, Leu230, and Val232 in SmFixL; and Ile215, Leu236, and Ile238 in BjFixL. Each of these side chains must be displaced for ligand binding, and this displacement gives rise to a trigger in the form of structural changes within the protein. The present results support the allosteric transduction model of FixL which we proposed previously. The model is also intimately connected to the models proposed previously by other groups. For example, the FG loop model assumes that Arg200 is moved upon O2 dissociation, and it was reported that Arg200 participates in switching of the FixL oxygen sensor.33 The movement of Arg200 would induce a shift of the Fα helix and Tyr201. Therefore, the observed change in Tyr201 is consistent with the FG loop model. This change in the position of Tyr201 would result in a change in the position of Glu234 in the Hβ sheet through the hydrogen bond between Tyr201 and Glu234. A shift in the Hβ sheet can also be induced by the displacement of Leu230 and Val232 as proposed by the hydrophobic triad model. The FG loop model assumes a shift of the Gβ sheet, which forms an anti-parallel β sheet with the Hβ sheet. Therefore, a mechanism of allosteric transduction that is mediated by concerted changes in the Fα helix, Gβ and Hβ sheets can be proposed. This model also explains the small intensity change of the Tyr bands upon CO dissociation from FixL, because the switch of Arg200 does not occur upon CO dissociation. This difference between CO and O2 dissociation results in O2 selectivity in the inhibition of kinase activity.


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Pathway of transduction of the structural changes to the kinase domain. The UVRR measurements of WT and Tyr FixL mutants showed that intensity decreases of Tyr bands upon O2 dissociation were also observed for Tyr379 and Tyr496 residues, which are in the kinase domain. This is the first observation of a structural change in the kinase domain that is induced by O2 dissociation from the sensor domain of FixL. Crystallographic data are not available either for full-length FixL or for the kinase domain of FixL. Instead, we discuss the transduction mechanism of the structural change from the sensor domain to the kinase domain based on the structure of the ThkA protein which is a PAS sensor histidine kinase from the thermophilic bacterium Thermotoga maritimas9 and on the structure of a blue light-activated histidine kinase from Erythrobacter litoralis HTCC2594 (EL346).34 The structural characteristics of the PAS domains of ThkA and EL346 are essentially the same as those of the sensor domain of FixL. Furthermore, the kinase domains of ThkA and EL346 have a high sequence homology to that of FixL and function as histidine kinases in a similar manner to FixL. The kinase domains of ThkA and FixL exhibited 27% amino acid identity and 45% similarity by the PAM-120 matrix. The kinase domains of EL346 and FixL exhibited 16% identity and 39% similarity by the PAM-120 matrix. For this reason, it is highly likely that the kinase domain of FixL has a similar structure to that of the ThkA and EL346 kinase domains. Accordingly, crystallographic data of ThkA and EL346 can provide an insight into understanding the transduction mechanism of FixL. Figure 7 compares the crystal structures of the BjFixL sensor domain, full-length ThkA and fulllength EL346. The PAS (sensor) domains and kinase domains are colored pink and blue, respectively. The structures of the proteins are drawn so that their PAS (sensor) domains are oriented in the same direction. The structure of the kinase domain of ThkA and EL346 is similar


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while their positions relative to the PAS domains are different. However, two strands that correspond to the Gβ and Hβ sheets in FixL contact the kinase domain in both ThkA and EL346. In ThkA, an interdomain, antiparallel β sheet was identified between the Gβ sheet in the PAS domain and a β sheet in the kinase domain.9 In EL346, it was reported that the Gβ and Hβ sheets form a sizeable interface with the kinase domain.34 It is therefore highly likely that the Gβ or the Hβ sheet, or both, in the sensor domain are in contact with the kinase domain in FixL. Accordingly, the model proposed in the previous section is consistent with the hypothesis that the Gβ or Hβ sheet or both forms the domain contact and transduces the structural changes in the sensor domain to the kinase domain in FixL. The corresponding positions of Tyr residues in the kinase domain of FixL are shown in red in Figure 7B and 7C. It is interesting that the corresponding positions of Tyr379 and Tyr496, which showed large intensity changes upon O2 dissociation from FixL, are close to the interdomain contact in ThkA. This situation might mean that the relative positions of the PAS and kinase domains of SmFixL are similar to those of ThkA. The present experimental data support the allosteric transduction pathway of FixL which we proposed previously.10 In this pathway, the dissociation of O2 from FixL induces changes in the orientation of Arg214 and Arg200. The latter change causes a shift in the Fα helix, which brings about a shift in the Hβ sheet through the hydrogen bond between Tyr201 in the Fα helix and Glu234 in the Hβ sheet. These structural changes induce a change in the Gβ sheet, which is sandwiched between the Fα helix and the Hβ sheet. The Gβ or Hβ sheet or both in the sensor domain are in contact with the kinase domain. Through this contact, the structural changes in the sensor domain induced by O2 dissociation from FixL are transmitted to the kinase domain, which regulates the kinase activity in the kinase domain of FixL.


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CONCLUSIONS In this study, we investigated the structure and structural changes of FixL using UVRR spectroscopy, and the kinase activity of FixL using Phos-tag SDS-PAGE. The UVRR spectra of the oxy, deoxy, and carbonmonoxy forms showed structural changes specific to O2 dissociation from FixL; weakening of the hydrogen bond between Tyr201 and Glu234 was observed to be induced by O2 dissociation. Phos-tag SDS-PAGE showed that Tyr201 is necessary for regulation of FixL kinase activity. Combining the measurements of protein structure and kinase activity, we showed that weakening of the hydrogen bond between Tyr201 and Glu234 upon O2 dissociation is essential to inhibition of FixL kinase activity. We also observed for the first time a structural change in the kinase domain induced by O2 dissociation from FixL. The observed structural changes support the allosteric transduction pathway of FixL which we proposed previously in which a shift of the Fα helix causes structural rearrangements of the Gβ and Hβ sheets, which results in a structural change in the kinase domain and an increase in the kinase activity of FixL.


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Figure captions. Figure 1. Crystal structure of the truncated sensor domain of FixL from Bradyrhizobium japonicum (BjFixL) in the oxy form together with a stick representation of the heme and amino acid residues (PDB ID: 1DP6). The indicated numbers correspond to the residue numbers of FixL from Sinorhizobium meliloti (SmFixL) that was used in this experiment. Val232 residue in SmFixL corresponds to Ile238 in BjFixL. Figure 2. UVRR spectra of (a) deoxy, (b) oxy and (c) carbonmonoxy forms of FixL. UVRR bands due to Tyr (labeled Y) and Phe (labeled F) residues are shown. To clearly show spectral change upon ligand binding, difference spectra were calculated using the perchlorate band (934 cm–1) as an internal intensity standard (asterisk) and the band intensity was doubled. Spectra (d) and (e) are the difference spectra of [(a) - (b)] and [(a) - (c)], respectively. Shown intensities of the difference spectra are enlarged by a factor of 2. The UVRR spectra were excited at a ~20-ns pulse of 233 nm. The repetition rate and energy of the pulse were 1 kHz and 0.5 μJ, respectively. Linear background and the Raman bands of water, oxygen gas, glycerol, and the quartz cell have been subtracted. The accumulation time for obtaining each spectrum was 120 min. Figure 3. Difference UVRR spectra between deoxy and oxy forms of WT and mutants of FixL. The difference spectra were calculated as in Figure 2. The spectrum of the oxy form of WT FixL is shown at the top for comparison; the intensity of this spectrum is multiplied by a factor of 0.5. Linear background and the Raman bands of water, oxygen gas, glycerol, and the quartz cell have been subtracted. The accumulation time for obtaining each spectrum was 120 min.


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Figure 4. Visible resonance Raman spectra of WT (black), Y201F (red) and Y201H (blue) mutants of FixL. Panels (A), (B) and (C) show spectra of deoxy, oxy and carbonmonoxy forms, respectively. Figure 5. Kinase activities of WT, Y201F and Y201H mutants of FixL. (A) Coomassie brilliant blue-stained Phos-tag gels. The lanes of each gel show deoxy, oxy, and carbonmonoxy (CO) forms of WT, Y201F and Y201H mutants of FixL. ATP (−) are samples without ATP addition, while ATP (+) are samples that underwent a phosphorylation reaction with ATP for 15 min. (B) Kinase activities of deoxy (blue), oxy (red) and carbonmonoxy (green) forms of WT, Y201F and Y201H mutants of FixL. Kinase activity was calculated as the percent of total FixL that was phosphorylated as assayed in a Phos-tag gel. N=4, Error bars show ±2 SE. Figure 6. Comparison of the crystal structures of the oxy (pink) and deoxy (blue, PDB ID: 1XJ3) forms of the sensor domain of BjFixL. The structure of the portion from the Fα helix to the Hβ sheet (residues 194–242 in BjFixL) is shown. Figure 7. Comparison of the structures of residues 154–256 in the BjFixL sensor domain (PDB ID: 1DP6), full-length ThkA (PDB ID: 3A0R) and full-length EL346 (PDB ID: 4R3A). (A) Crystal structure of the truncated sensor domain of oxy BjFixL. (B) Crystal structure of ThkA. The PAS and kinase domains are shown in pink and blue, respectively. Positions of Tyr residues that correspond to those in the kinase domain of SmFixL are shown in red. Residue numbers in corresponding positions of SmFixL are shown in parentheses. The position of the His residue used for autophosphorylation is shown in yellow. (C) Crystal structure of EL346. The PAS and kinase domains are shown in pink and blue, respectively. Positions of Tyr residues in the kinase domain that correspond to those in SmFixL are shown in red. Residue numbers in corresponding positions


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of SmFixL are shown in parentheses. The position of the His residue used for autophosphorylation is shown in yellow.

ASSOCIATED CONTENT Supporting Information. Absorption spectra of WT and eight mutants of FixL, difference UVRR spectra between deoxy and oxy forms of WT, Y201F and Y201F mutants of FixL, visible resonance Raman spectra of deoxy, oxy and carbonmonoxy forms of WT, Y201F and Y201F mutants of FixL. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Yasuhisa Mizutani, Tel.: +81-6-6850-5776. Fax: +81-6-6850-5776. E-mail: [email protected]. Funding This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Soft Molecular Systems” (No. 25104006) to Y.M. from The Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid for Education and Research to T. Y. from the Cross-Boundary Innovation Program of Osaka University.

Abbreviation BjFixL, FixL from Bradyrhizobium japonicum ; SmFixL, FixL from Sinorhizobium meliloti


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We are deeply grateful to Prof. Eiji Kinoshita of Hiroshima University for helpful discussions on Phos-tag SDS-PAGE.


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Insert Table of Contents Graphic and Synopsis Here


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REFERENCES (1) Henry, J. T., and Crosson, S. (2011) Ligand binding PAS domains in a genomic, cellular, and structural context, Annu. Rev. Microbiol. 65, 261-286. (2) Gilles-Gonzalez, M.-A., and Gonzalez, G. (2004) Signal transduction by heme-containing PAS-domain proteins, J. Appl. Physiol. 96, 774-783. (3) Gilles-Gonzalez, M. A., Ditta, G. S., and Helinski, D. R. (1991) A haemoprotein with kinase activity encoded by the oxygen sensor of Rhizobium meliloti, Nature 350, 170-172. (4) Renalier, M. H., Batut, J., Ghai, J., Terzaghi, B., Gherardi, M., David, M., Garnerone, A. M., Vasse, J., Truchet, G., and Huguet, T. (1987) A new symbiotic cluster on the pSym megaplasmid of Rhizobium meliloti 2011 carries a functional fix gene repeat and a nod locus, J. Bacteriol. 169, 2231-2238. (5) David, M., Daveran, M.-L., Batut, J., Dedieu, A., Domergue, O., Ghai, J., Hertig, C., Boistard, P., and Kahn, D. (1988) Cascade regulation of nif gene expression in Rhizobium meliloti, Cell 54, 671-683. (6) Fischer, H. M. (1994) Genetic regulation of nitrogen fixation in rhizobia, Microbiol. Rev. 58, 352-386. (7) Akimoto, S., Tanaka, A., Nakamura, K., Shiro, Y., and Nakamura, H. (2003) O2-specific regulation of the ferrous heme-based sensor kinase FixL from Sinorhizobium meliloti and its aberrant inactivation in the ferric form, Biochem. Biophys. Res. Commun. 304, 136142. (8) Hiruma, Y., Kikuchi, A., Tanaka, A., Shiro, Y., and Mizutani, Y. (2007) Resonance Raman observation of the structural dynamics of FixL on signal transduction and ligand discrimination, Biochemistry 46, 6086-6096. (9) Yamada, S., Sugimoto, H., Kobayashi, M., Ohno, A., Nakamura, H., and Shiro, Y. (2009) Structure of PAS-linked histidine kinase and the response regulator complex, Structure 17, 1333-1344. (10) Yano, S., Ishikawa, H., Mizuno, M., Nakamura, H., Shiro, Y., and Mizutani, Y. (2013) Ultraviolet resonance Raman observations of the structural dynamics of rhizobial oxygen sensor FixL on ligand recognition, J. Phys. Chem. B 117, 15786-15791. (11) Ayers, R. A., and Moffat, K. (2008) Changes in quaternary structure in the signaling mechanisms of PAS domains, Biochemistry 47, 12078-12086. (12) Gong, W., Hao, B., and Chan, M. K. (2000) New mechanistic insights from structural studies of the oxygen-sensing domain of Bradyrhizobium japonicum FixL, Biochemistry 39, 3955-3962. (13) Hao, B., Isaza, C., Arndt, J., Soltis, M., and Chan, M. K. (2002) Structure-based mechanism of O2 sensing and ligand discrimination by the FixL heme domain of Bradyrhizobium japonicum, Biochemistry 41, 12952-12958. (14) Key, J., and Moffat, K. (2005) Crystal structures of deoxy and CO-bound bjFixLH reveal details of ligand recognition and signaling, Biochemistry 44, 4627-4635. (15) Miyatake, H., Mukai, M., Park, S.-Y., Adachi, S.-i., Tamura, K., Nakamura, H., Nakamura, K., Tsuchiya, T., Iizuka, T., and Shiro, Y. (2000) Sensory mechanism of oxygen sensor FixL from Rhizobium meliloti: Crystallographic, mutagenesis and resonance Raman spectroscopic studies, J. Mol. Biol. 301, 415-431.


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(16) Taylor, B. L., and Zhulin, I. B. (1999) PAS domains: Internal sensors of oxygen, redox potential, and light, Microbiol. Mol. Biol. Rev. 63, 479-506. (17) Stock, J. B., Ninfa, A. J., and Stock, A. M. (1989) Protein phosphorylation and regulation of adaptive responses in bacteria, Microbiol. Rev. 53, 450-490. (18) Kinoshita, E., Kinoshita-Kikuta, E., Matsubara, M., Yamada, S., Nakamura, H., Shiro, Y., Aoki, Y., Okita, K., and Koike, T. (2008) Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE, Proteomics 8, 2994-3003. (19) Kinoshita, E., Kinoshita-Kikuta, E., and Koike, T. (2009) Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE, Nat. Protocols 4, 1513-1521. (20) Fojtikova, V., Stranava, M., Vos, M. H., Liebl, U., Hranicek, J., Kitanishi, K., Shimizu, T., and Martinkova, M. (2015) Kinetic analysis of a globin-coupled histidine kinase, AfGcHK: Effects of the heme iron complex, response regulator, and metal cations on autophosphorylation activity, Biochemistry 54, 5017-5029. (21) Harada, I., and Takeuchi, H. (1986) Raman and ultraviolet resonance Raman spectra of proteins and related compounds, In Advances in Spectroscopy: Spectroscopy of biological systems (Clark, R. J. H., and Hester, R. E., Eds.), pp 113-175, Wiley, New York. (22) Kinoshita-Kikuta, E., Kinoshita, E., Matsuda, A., and Koike, T. (2014) Tips on improving the efficiency of electrotransfer of target proteins from Phos-tag SDS-PAGE gel, Proteomics 14, 2437-2442. (23) Chi, Z., and Asher, S. A. (1998) UV Raman determination of the environment and solvent exposure of Tyr and Trp residues, J. Phys. Chem. B 102, 9595-9602. (24) Takeuchi, H., Ohtsuka, Y., and Harada, I. (1992) Ultraviolet resonance Raman study on the binding mode of enkephalin to phospholipid membranes, J. Am. Chem. Soc. 114, 53215328. (25) Ludwig, M., and Asher, S. A. (1988) Ultraviolet resonance Raman excitation profiles of tyrosine: Dependence of Raman cross sections on excited-state intermediates, J. Am. Chem. Soc. 110, 1005-1011. (26) Takeuchi, H., Watanabe, N., and Harada, I. (1988) Vibrational spectra and normal coordinate analysis of p-cresol and its deuterated analogs, Spectrochim. Acta, Pt. A: Mol. Spectrosc. 44, 749-761. (27) Gong, W., Hao, B., Mansy, S. S., Gonzalez, G., Gilles-Gonzalez, M. A., and Chan, M. K. (1998) Structure of a biological oxygen sensor: A new mechanism for heme-driven signal transduction, Proc. Natl. Acad. Sci. USA 95, 15177-15182. (28) Counterman, A. E., and Clemmer, D. E. (1999) Volumes of individual amino acid residues in gas-phase peptide ions, J. Am. Chem. Soc. 121, 4031-4039. (29) Darby, N. J., and Creighton, T. E. (1993) Dissecting the disulphide-coupled folding pathway of bovine pancreatic trypsin inhibitor: Forming the first disulphide bonds in analogues of the reduced protein, J. Mol. Biol. 232, 873-896. (30) Cho, N., and Asher, S. A. (1996) UV resonance Raman and absorption studies of angiotensin II conformation in lipid environments, Biospectroscopy 2, 71-82. (31) Dunham, C. M., Dioum, E. M., Tuckerman, J. R., Gonzalez, G., Scott, W. G., and GillesGonzalez, M.-A. (2003) A distal arginine in oxygen-sensing heme-PAS domains is essential to ligand binding, signal transduction, and structure, Biochemistry 42, 77017708.


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(32) Jain, R., and Chan, M. K. (2003) Mechanisms of ligand discrimination by heme proteins, J. Biol. Inorg. Chem. 8, 1-11. (33) Gilles-Gonzalez, M.-A., Caceres, A. I., Sousa, E. H. S., Tomchick, D. R., Brautigam, C., Gonzalez, C., and Machius, M. (2006) A proximal arginine R206 participates in switching of the Bradyrhizobium japonicum FixL oxygen sensor, J. Mol. Biol. 360, 8089. (34) Rivera-Cancel, G., Ko, W.-h., Tomchick, D. R., Correa, F., and Gardner, K. H. (2014) Fulllength structure of a monomeric histidine kinase reveals basis for sensory regulation, Proc. Natl. Acad. Sci. USA 111, 17839-17844.


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Yamawaki et al., Figure 1


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Figure 1. Crystal structure of the truncated sensor domain of FixL from Bradyrhizobium japonicum (BjFixL) in the oxy form together with a stick representation of the heme and amino acid residues (PDB ID: 1DP6). The indicated numbers correspond to the residue numbers of FixL from Sinorhizobium meliloti (SmFixL) that was used in this experiment. Val232 residue in SmFixL corresponds to Ile238 in BjFixL. 103x77mm (150 x 150 DPI)


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Figure 2. UVRR spectra of (a) deoxy, (b) oxy and (c) carbonmonoxy forms of FixL. UVRR bands due to Tyr (labeled Y) and Phe (labeled F) residues are shown. To clearly show spectral change upon ligand binding, difference spectra were calculated using the perchlorate band (934 cm–1) as an internal intensity standard (asterisk) and the band intensity was doubled. Spectra (d) and (e) are the difference spectra of [(a) - (b)] and [(a) - (c)], respectively. Shown intensities of the difference spectra are enlarged by a factor of 2. The UVRR spectra were excited at a ~20-ns pulse of 233 nm. The repetition rate and energy of the pulse were 1 kHz and 0.5 µJ, respectively. Linear background and the Raman bands of water, oxygen gas, glycerol, and the quartz cell have been subtracted. The accumulation time for obtaining each spectrum was 120 min. 102x95mm (600 x 600 DPI)


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Figure 3. Difference UVRR spectra between deoxy and oxy forms of WT and mutants of FixL. The difference spectra were calculated as in Figure 2. The spectrum of the oxy form of WT FixL is shown at the top for comparison; the intensity of this spectrum is multiplied by a factor of 0.5. Linear background and the Raman bands of water, oxygen gas, glycerol, and the quartz cell have been subtracted. The accumulation time for obtaining each spectrum was 120 min. 153x192mm (600 x 600 DPI)


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Figure 4. Visible resonance Raman spectra of WT (black), Y201F (red) and Y201H (blue) mutants of FixL. Panels (A), (B) and (C) show spectra of deoxy, oxy and carbonmonoxy forms, respectively. 211x389mm (600 x 600 DPI)


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Figure 5. Kinase activities of WT, Y201F and Y201H mutants of FixL. (A) Coomassie brilliant blue-stained Phos-tag gels. The lanes of each gel show deoxy, oxy, and carbonmonoxy (CO) forms of WT, Y201F and Y201H mutants of FixL. ATP (−) are samples without ATP addition, while ATP (+) are samples that underwent a phosphorylation reaction with ATP for 15 min. (B) Kinase activities of deoxy (blue), oxy (red) and carbonmonoxy (green) forms of WT, Y201F and Y201H mutants of FixL. Kinase activity was calculated as the percent of total FixL that was phosphorylated as assayed in a Phos-tag gel. N=4, Error bars show ±2 SE. 128x147mm (150 x 150 DPI)


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Figure 6. Comparison of the crystal structures of the oxy (pink) and deoxy (blue, PDB ID: 1XJ3) forms of the sensor domain of BjFixL. The structure of the portion from the Fα helix to the Hβ sheet (residues 194– 242 in BjFixL) is shown. 118x74mm (150 x 150 DPI)


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Figure 7. Comparison of the structures of residues 154–256 in the BjFixL sensor domain (PDB ID: 1DP6), full-length ThkA (PDB ID: 3A0R) and full-length EL346 (PDB ID: 4R3A). (A) Crystal structure of the truncated sensor domain of oxy BjFixL. (B) Crystal structure of ThkA. The PAS and kinase domains are shown in pink and blue, respectively. Positions of Tyr residues that correspond to those in the kinase domain of SmFixL are shown in red. Residue numbers in corresponding positions of SmFixL are shown in parentheses. The position of the His residue used for autophosphorylation is shown in yellow. (C) Crystal structure of EL346. The PAS and kinase domains are shown in pink and blue, respectively. Positions of Tyr residues in the kinase domain that correspond to those in SmFixL are shown in red. Residue numbers in corresponding positions of SmFixL are shown in parentheses. The position of the His residue used for autophosphorylation is shown in yellow. 203x91mm (150 x 150 DPI)


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Regulatory Implications of Structural Changes in Tyr201 of the Oxygen

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