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Original Paper	Pdf file on Synergy OPEN omments
Acta Biochim Biophys Sin 2008, 40: 783-789
doi:10.1111/j.1745-7270.2008.00462.x

In vitro observation of the molecular interaction between NodD and its inducer naringenin as monitored by fluorescence resonance energy transfer

Fengqing Li¹, Bihe Hou¹, Lei Chen², Zhujun Yao², and Guofan Hong¹*

¹ State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

² State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China

Received: April 21, 2008

Accepted: May 11, 2008

The work was supported by grants from the National Basic Research Program of China (No. G2002CB713808) and the PanDeng Plan of China

*Corresponding author: Tel, 86-21-54921223; Fax, 86-21-54921011; E-mail, [email protected]

At initial stages in the Rhizobium legume symbiosis, most nodulation genes are controlled by NodD protein and plant inducers. Some genetic studies and other reports have suggested that NodD may be activated by its direct interaction with plant inducers. However, there has been no molecular evidence of such an inducing interaction. In this paper, we used fluorescence resonance energy transfer technique to see whether such an interaction exists between NodD and its activator, naringenin, in vitro. The tetracysteine motif (Cys-Cys-Pro-Gly-Cys-Cys) was genetically inserted into NodD to label NodD with 4',5'-bis(1,3,2-dithioarsolan-2-yl) fluorescein (FlAsH). Naringenin was labeled with fluorescein by chemical linking. In the fluorescence resonance energy transfer experiments in vitro, the fluorescence intensity of one acceptor, NodD(90R6)-FlAsH, increased by 13%. This suggests that NodD may directly interact with inducer naringenin in vitro and that the reaction centre is likely near hinge region 1 of NodD.

Keywords NodD; naringenin; fluorescence resonance energy transfer (FRET)

The development of nitrogen-fixing plant root nodules by rhizobia requires an exchange of signals between the two partners, and hence the nodulation process exhibits high host specificity and coordination. Legume roots secrete specific flavonoid or isoflavonoid compounds that induce the transcription of bacterial nodulation (nod, nol and noe) genes. During this process, NodD acts as both a sensor of the plant inducer and an activator of nod gene transcription [1]. In Rhizobium leguminosarum biovar viciae, NodD proteins are localized in the cytoplasmic membrane [2], where the inducing flavonoid, naringenin, also accumulates [3]. It was proposed that the un-ionized form of naringenin accumulates in the cytoplasmic membrane and activates, in a metabolically unaltered form, the NodD protein [3]. Direct binding of inducers to NodD has not been demonstrated due to technical difficulties, as flavonoids stick to all kinds of materials, including proteins (K. Recourt, unpublished data). However, results with mutant nodD genes [4-7], analysis of inducible nod gene transcription in an isogenic background with nodD genes from various sources [8,9], and an enhanced binding of nod box DNA by a 35 kDa protein in the presence of flavonoid inducers [10] together strongly suggest that NodD functions as a specific receptor for flavonoids. Our previous study also showed that NodD interacts directly with naringenin in vitro when naringenin was at 4.0 mM, a concentration thousands of times higher than in physiological conditions [11]. In R. meliloti, both NodD1 and NodD2 interact with plant flavonoids with the help of GroESL in vitro [12], but whether the interaction exists in vivo remains unclear. Fluorescence resonance energy transfer (FRET) is a quantum phenomenon occurring between two dye molecules. When the fluorescence spectrum of one fluorophore (the donor) overlaps with the excitation spectrum of another fluorophore (the acceptor), the excitation of the donor induces fluorescence of the acceptor, while its own fluorescence decreases. The extent of FRET is extremely sensitive to the distance between the donor and the acceptor and is inversely proportional to the sixth power of the distance. For FRET to occur, the donor and acceptor molecules must be within 1-10 nm of each other. This phenomenon has been exploited to study intermolecular and intramolecular relationships in biophysical systems and cell biology [13-17]. In this study, FRET technique was employed to observe the interactions between NodD and naringenin in vitro. We used naringenin (YA6006) as the fluorescence donor and NodD-FlAsH as the fluorescence acceptor to investigate the FRET between labeled NodD and naringenin (Fig. 1). We found that NodD may interact with inducer naringenin in vitro and the reaction centre is likely near hinge region 1 of NodD.

Materials and Methods

Bacterial strains and plasmids

All the bacterial strains and plasmids used in this work and their relevant characteristics are listed in Table 1 [1824]. Escherichia coli strains were grown at 37 ºC in Luria-Bertani (LB) medium. Rhizobia were grown at 28 ºC in TY medium [25]. If appropriate, antibiotics were added at the following concentrations: for E. coli, 100 mg/ml of ampicillin, 50 mg/ml of kanamycin, and 20 mg/ml of chloramphenicol or tetracycline; for rhizobia, 100 mg/ml of streptomycin, 25 mg/ml of kanamycin, 20 mg/ml of tetracycline and 10 mg/ml of chloramphenicol.

Site-specific mutagenesis of NodD by overlapping extension polymerase chain reaction (PCR)

Site-specific mutagenesis by overlapping extension was used [26]. The primers used are listed in Table 2. To construct nodD(90R6), PCR was performed twice. Two fragments, D901 and 90D2, were obtained in the first PCR by using pIJ1518 plasmid as the PCR template and primer pairs BnodD and 90R6 as well as 90R6R and EnodD. In the second PCR, the annealing fragments of D901 and 90D2 were used as the PCR template and the primer pair BnodD and EnodD was used. The resulting products, nodD(90R6), nodD(294I6) and nodD(C-), were obtained in the same way but with primer pairs M294 and M294R as well I303 and I303R. These mutant nodD fragments were cloned into the plasmid pKT230 at BclI and EcoRI sites. The plasmids harboring these mutant nodD fragments were transferred by biparental conjugation from Escherichia coli S17-1 to the 8401 strain containing pMP221A. The resulting clones were sequenced by the Shenggong Campany (Shanghai, China).

FlAsH-EDT2 synthesis

FlAsH is fluorescein with As(III) substituents on the 4- and 5-positions. FlAsH is the first complementary membrane-permeant small dye. The rigid spacing of the two arsenics in FlAsH enables it to bind with considerable affinity and specificity to the tetracysteine motif transplanted into a variety of proteins. The synthesis of FlAsH-EDT2 (fluorescein arsenical helix binder, bis-EDT adduct) was as described by Adams et al [27].

Fluorescence labeling of naringenin

Using a direct and highly regioselective Mannich reaction recently developed in our own lab [28], we introduced an ethylene-glycol linker 4 to the C-6 position of the A ring of naringenin without any protection (Fig. 2). Subsequent Cu(I)-catalyzed Huisgen cycloaddition of azido compound 2 with the acetylene-bearing fluorescein derivative 3 afforded the corresponding fluorescently labeled naringenin derivative YA6006 (Fig. 2). To a mixture of compounds 2 (50 mg, 0.11 mmol) and 3 (43 mg, 0.11 mmol) in t-BuOH/H2O (4 ml, V/V=1:1), we added CuSO45H2O (2 mg, 0.008 mmol) and ascorbic acid (5 mg, 0.028 mmol). The mixture was stirred at room temperature for 24 h. EtOAc (50 ml) was added to dilute the reaction. The organic phase was washed by saturated brine, and dried over anhydrous Na2SO4. The crude product was purified by silica gel chromatography affording pure YA6006 (75 mg, 84%). Physical data for YA6006: IR (KBr): Vmax 3385, 2944, 1752, 1707, 1597, 1501, 1459, 1369, 1250, 1182, 1110, 1024, 836 cm-1. 1H NMR (acetone-d6, 300 MHz): d 8.12 (1H, s), 7.99 (1H, d, J=7.2 Hz), 7.78-7.71 (2H, m), 7.35 (2H, d, J=8.7 Hz), 7.26 (1H, d, J=7.5 Hz), 7.00 (1H, d, J=2.1 Hz), 6.88 (2H, d, J=8.4 Hz), 6.79-6.77 (1H, m), 6.74-6.69 (2H, m), 6.64 (2H, br), 5.78 (1H, s), 5.35 (1H, dt, J=12.9, 3.0 Hz), 5.23 (2H, s), 4.58 (2H, t, J=4.8 Hz), 3.89 (2H, d, J=4.8 Hz), 3.83 (2H, s), 3.66-3.55 (6H, m), 3.09 (1H, dd, J=17.1, 12.9 Hz), 2.83 (2H, t, J=5.4 Hz), 2.66 (1H, dd, J=17.1, 3.0 Hz), 2.39 (3H, s). 13C NMR (acetone-d6, 75 MHz): 195.7, 170.4, 168.6, 162.6, 161.2, 160.2, 159.7, 157.8, 153.0, 152.4, 152.4, 142.6, 135.2, 130.0, 129.9, 129.2, 129.1, 128.1, 126.9, 124.7, 124.5, 124.1, 115.3, 112.7, 112.4, 112.0, 110.5, 102.5, 101.7, 100.9, 100.4, 95.7, 82.7, 78.8, 70.1, 69.1, 67.2, 61.8, 55.7, 52.6, 49.9, 42.5, 40.3, 29.2. HRMS calcd for C46H42N4O12 (MH+): 843.2872, found 843.2897.

b-galactosidase activity assay

b-galactosidase activity was assayed as described by Miller [29], using 10 mM naringenin as the nodA gene inducer. Three independent experiments were performed for each strain at the same cells density (OD600=0.4).

Preparation of protein samples

A wet weight cell pellet (about 1 g) from a culture of 300 ml Rhizobium leguminosarum 8401 strain was resuspended in 10 ml TEG buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 5% glycerol] (100 mM NaCl). It was then sonicated 30 times (10 s per time) in an Ultrasonics W375 sonicator at 80% output power in a salt/ice bath. Phenylmethylsulfonyl fluoride was added to a 23 mg/ml final concentration. The lysate was centrifuged at 15,000 g for 10 min to remove unbroken cells and cell debris. The total protein concentration was determined by Bio-Rad protein assay kit with standard bovine serum albumin protein in TEG buffer.

Fluorescence spectroscopy

Fluorescence measurements were carried out with an F-3010 spectrofluorometer (Hitachi, Tokyo, Japan). All measurements were performed at room temperature in TEG buffer (100 mM NaCl). Then, 4 mM FlAsH and 6 mM naringenin (YA6006) were employed in each set of experiments. After the dyes were added, the samples were kept in the dark at 28 ºC for 30 min. Three independent experiments were performed for each protein extract at the same total protein concentration (4 mg/ml). An excitation wavelength of 514 nm and an emission wavelength of 537 nm were employed. The energy transfer efficiency was determined from the enhancement of the fluorescence intensity of acceptor, FlAsH.

Results

Three NodD mutants containing tetracysteine motif

NodD mutants were constructed by genetically inserting the tetracysteine motif (Cys-Cys-Pro-Gly-Cys-Cys) into NodD through overlapping extension PCR. The resulting three mutants were named as nodD(90R6), nodD(294I6) and nodD(C-) respectively. These nodD mutants were cloned into pKT230 plasmid, and the resulting plasmids were introduced into 8401/pMP221A to investigate their biological activities. The ability to activate nodA transcription of NodD(294I6), NodD(90R6), and NodD(C-) was about 2%, 50% and 78%, respectively, of native NodD (Table 3). NodD(294I6) had almost no biological activity. It therefore acted as a negative control.

FlAsH labeling of tetracysteine-tagged NodD proteins

Three tetracysteine-tagged nodD mutants were cloned into pKNDT plasmid [21], a high copy-expressing plasmid, and transferred into the 8401 strain by biparental conjugation. The resulting strains were named 8401/pU3D(90R6), 8401/pU3D(294I6) and 8401/pU3D(C-), respectively. These tetracysteine-tagged NodD proteins were fluorescent-labeled with FlAsH. The fluorescence intensity of FlAsH at various concentrations is listed in Table 4. Protein extracts from 8401/pKT230 were selected as a negative control, since 8401/pKT230 strain does not harbor the nodD gene on the symbiotic plasmid. The enhanced FlAsH fluorescence was likely caused by non-specific binding of FlAsH-EDT2 to endogenous cysteine-containing proteins [30]. At the same time, the fluorescence intensity of 8401/pKT230 protein extracts did not undergo much change with concentrations of FlAsH over 4 mM, suggesting that the endogenous cysteinecontaining proteins were saturated by 4 mM FlAsH-EDT2. So the fluorescence intensity of 8401/pKT230 protein extracts can be regarded as background fluorescence. Surprisingly, the protein extracts of 8401/pU3D(294I6) almost exhibited the same fluorescence intensity as that of 8401/pKT230 at all concentrations of FlAsH, suggesting that NodD(294I6) cannot be labeled with FlAsH. This may be caused by the tetracysteine motif being packaged inside the 3-D structure. In contrast, when the concentration of FlAsH increased to 6 mM, the fluorescence intensity did not change. This suggested that NodD proteins in the extracts are saturated by FlAsH. We chose 4 mM FlAsH to investigate the FRET between NodD-FlAsH and naringenin.

Fluorescein-labeled naringenin YA6006

Fluorescein-labeled naringenin YA6006 was chemically synthesized. 8401/pIJ1518/pMP221A was constructed to examine the ability of YA6006 to activate nodA transcription. Fig. 3(A) shown that its biological activity was about 23% that of native naringenin. The fluorescence intensity of YA6006 increased directly in proportion to its concentration within 0-10 mM [Fig. 3(B)]. We chose 6 mM naringenin YA6006 to investigate the FRET between NodD-FlAsH and naringenin.

FRET in the NodD-naringenin complex

Naringenin (YA6006) was selected as the fluorescence donor and NodD-FlAsH was selected as the fluorescence acceptor. Since only acceptor emission at 537 nm was observed on excitation at 514 nm for the donor-acceptor pair, the energy transfer efficiency was determined from the enhancement of the acceptor emission at 537 nm, where the influence of the donor emission was negligible. The results are summarized in Table 5. Only the fluorescence intensity of 8401/pU3D(90R6) protein extracts was enhanced when 6 mM YA6006 was added. After excluding the influence of endogenous cysteine-containing proteins in the protein extracts, the fluorescence intensity of 8401/pU3D(90R6) protein extract was 257 DRFI (delta relative fluorescence intensity) without YA6006 and 291 DRFI with 6 mM YA6006. The fluorescence intensity of the acceptor was increased by 13%, showing a typical energy transfer.

Discussion

NodD (303 aa) is a regulatory protein belonging to the LysR family. NodD binds to nod-box in tetramer form [31]. A previous study involving CbnR (295 aa), another LysR family protein, showed that active tetramer CbnR is approximately 130 Å´70 Å´60 Å [32]. Therefore, the size of active tetramer NodD may be similar to active tetramer CbnR, and this size is suitable for FRET occurrence. In this study, FRET was observed in vitro only between NodD(90R6)-FlAsH and YA6006, but not between NodD(C-)-FlAsH and YA6006. This may have happened because FlAsH labeled on the C-terminal of NodD is too far away from the reaction center for FRET to occur with YA6006. However, FlAsH labeled at the hinge region 1 (aa residues 67-93) of NodD is close enough to the reaction center for FRET to occur with YA6006. Our results suggested that NodD can directly interact with naringenin in vitro and that the reaction center is near the hinge region 1 of NodD. Moreover, this is consistent with the proposal that NodD does not contain separate functional domains for DNA binding and flavonoid interaction [1].

Protein extracts from 8401 derivative strains harboring NodD mutants, rather than purified NodD protein, were employed in the experiments in vitro. As a result, two problems arose: the uncertain amount of NodD protein and the high background in fluorescence measurement. Three tetracysteine-tagged NodD were expressed with the same plasmid and introduced into the same strain, and the fluorescence intensity of protein extracts of 8401/pU3D(90R6) and 8401/pU3D(C-) were basically equal at the same concentration of FlAsH (Table 4); hence, it seemed likely that the amount of NodD proteins in the extracts of three 8401 derivative strains was basically equal. The fluorescence intensity of samples 8401/pU3D(90R6) and 8401/pU3D(C-) minus that of the negative control (8401/pKT230) is the specific signal of NodD-FlAsH. Using this data analysis, the high background could be partially excluded. However, further efforts to decrease the signal-to-noise ratio ought to be done.

Our results showed that naringenin (YA6006) has only 23% biological activity of native naringenin and that NodD(90R6) only has 50% biological activity of wild-type NodD. Therefore, in vitro observation of the interaction between impaired NodD and naringenin could not explain whether such interaction exists in vivo. To further investigate the interaction between NodD and naringenin in vivo, NodD and naringenin with higher biological activity should be employed in FRET measurements in vivo.

In conclusion, we labeled NodD and naringenin with suitable dyes and investigated the FRET between these molecules in vitro. We found that the fluorescence intensity of acceptor, NodD(90R6)-FlAsH, increased by 13%, suggesting that NodD may directly interact with inducer naringenin in vitro and that the reaction center is likely near the hinge region 1 of NodD.

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