Target sequence recognition by a light-activatable bZIP factor

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Target sequence recognition by a light-activatable bZIP factor, Photozipper Samu Tateyama, Itsuki Kobayashi, and Osamu Hisatomi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00995 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 5, 2018

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Biochemistry

Target sequence recognition by a light-activatable bZIP factor, Photozipper Samu Tateyama, Itsuki Kobayashi and Osamu Hisatomi* Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.

KEYWORDS. bZIP, LOV, DNA-binding, aureochrome, Quartz crystal microbalance (QCM).

ABSTRACT

Photozipper (PZ) is a light-activatable basic leucine zipper (bZIP) protein composed of a bZIP domain and a light-oxygen-voltage-sensing domain of aureochrome-1. Blue light induces dimerization and subsequently increases the affinity of PZ for the target DNA sequence. We prepared site-directed PZ mutants in which Asn131 (N131) in the basic region were substituted with Ala and Gln. N131 mutants showed spectroscopic and dimerization properties almost identical to wild-type PZ, as well as an increase of helical content in the presence of the target sequence. Quantitative analyses by electrophoretic mobility shift assay and quartz crystal

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microbalance (QCM) measurements demonstrated that the half-maximal effective concentrations of N131 mutants to bind to the target sequence were significantly higher than those of PZ. QCM data also revealed that N131 substitutions accelerated the dissociation speed without affecting the association speed, suggesting that a base-specific interaction of N131 occurred after the association between PZ and DNA. Activation of PZ by illumination decreased both the standard errors and the unstable period of QCM data. Optical control of transcription factors will provide new knowledge of the recognition of the target sequence.

Introduction Light is essential for most life on Earth, not only as an energy source but also as a mediator of information. Living organisms have, therefore, evolved various photoreceptor molecules. The light-oxygen-voltage-sensing (LOV) domain is one of the photoreceptor domains found in plants and microorganisms.1 The LOV domain consists of about 100 amino acids and a chromophore, flavin mononucleotide (FMN). FMN absorbs blue light (BL) and subsequently forms a covalent adduct with a highly conserved cysteine residue in the light (S390) state.1–3 The cysteinyl photoadduct spontaneously dissociates and FMN reverts to that in the dark (D450) state with an individual time constant. The adduct formation induces a reversible conformational change of the LOV domain, which was believed to regulate various biological activities.4 A new family of LOV domain-containing proteins (aureochromes) was found from a stramenopile algae, Vaucheria frigida.5 Aureochromes contain a LOV domain and a basic leucine zipper (bZIP) domain at the N-terminal side of the LOV domain. The bZIP domain is an α-helical DNAbinding motif found among the eukaryotic transcription factors.6,7 Aureochrom-1 of V. frigida


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Biochemistry

(VfAUREO1) has been suggested to be responsible for the BL-induced branching response of this organism through transcriptional regulation.5,8 We made a synthetic gene (opZL), which encoded an N-terminally truncated AUREO1 containing bZIP and LOV domains, and called the recombinant protein Photozipper (PZ).9, 10 PZ showed an absorption band (λmax at 447 nm) with a triplet vibrational structure characteristic for LOV domains. The band rapidly decreased upon BL illumination, and regenerated with a half reaction time (τ1/2) of ~ 7.5 min in the dark.10,11 Dynamic light scattering (DLS) and size exclusion chromatography (SEC) analyses demonstrated that PZ is monomeric in the dark and forms a dimer upon BL illumination. 9,10 The BL-induced dimerization increased the affinity of PZ for the target DNA sequence, TGACGT. 5,9,12 Structural analyses of VfAUREO1 and PtAUREO1a from Phaeodactylum tricornutum have revealed that LOV domains of aureochromes have similarities to those of other LOV domaincontaining proteins.13-15 FTIR studies have suggested the conformational changes of Jα and A’α helices in the LOV domain of PtAUREO1a,16,17 and an EPR study has clarified the distance between FMNs of VfAUREO1 LOV dimer in the light state.18 In contrast, only a few studies on the bZIP domain of aureochromes have been reported.14,19 The basic region of the bZIP domain is largely unstructured in the absence of DNA, but adopts a helical structure upon DNA binding.20-24 Crystallographic analyses suggested that an asparagine residue (corresponding to Asn131 of PZ) in the basic region of bZIP transcription factors plays a role in the recognition of the target DNA sequence.6,7,25 Moreover, Asn131 (N131) of VfAUREO1 has been suspected to be responsible for the spectral shift in FTIR spectra upon binding to DNA.26 To elucidate the role of N131 on DNA binding, we prepared the N131 mutants of PZ, in which N131 was replaced with Ala (N131A) or Gln (N131Q).


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Quantifications of DNA-binding for transcription factors have mainly been performed by electrophoretic mobility shift assay (EMSA), circular dichroism (CD) and fluorescence quenching.12,14,19-24 In this report, we quantitatively compared the DNA binding of PZ and N131 mutants by EMSA and quartz crystal microbalance (QCM). QCM can detect a small mass change attached to the electrode on the quartz crystal by changes of resonant frequency, and be used to detect the interaction of biomolecules in solution.27-31 Besides the half-maximal effective concentration (EC50) of complex formation, QCM has advantages for the measurement of the kinetic parameters, the association and dissociation speed constants of the complex.29-31 In this study, we report the EC50 and speed constants of PZ and its mutants, and indicate the role of N131 on the DNA binding of bZIP transcription factors.

Experimental procedures Preparation of recombinant proteins To prepare N131 mutants of PZ (N131A and N131Q), the expression vector containing opZL gene encoding G113–K348 of VfAUREO1 with C162S and C182S substitutions (Fig. 1A)11,35 was mutated with a PrimeSTAR mutagenesis kit (Takara Bio) and primer sets; PZ_N131A-F and PZ_N131A-R for N131A, PZ_N131Q-F and PZ_N131Q-R for N131Q (Fig. S1). Sequences of all constructs were confirmed using the Thermo SequenaseTM Dye Primer Manual Cycle Sequencing Kit (GE Healthcare), with an SQ-5500 DNA sequencer (Hitachi Hitech), and the expression vector for each PZ mutant was then introduced into BL21 (DE3) cells (Invitrogen). E. coli cells expressing recombinant proteins were harvested by centrifugation and disrupted by sonication as described previously.9,11 After the cell debris was removed by centrifugation, recombinant proteins were purified twice by binding to and elution from a Ni Sepharose 6 Fast


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Biochemistry

Flow column (GE Healthcare) and a HiTrap Heparin HP column (GE Healthcare), according to the manufacturer’s instructions. The recombinant proteins were stored at 4°C in loading buffer (400 mM NaCl, 20 mM Tris-HCl, pH 7.0) containing 1 mM dithiothreitol (DTT) and 0.2 mM phenylmethylsulfonyl fluoride. Concentrations of the recombinant proteins were determined from absorbance at 447 nm using an extinction coefficient of 13,000 M-1cm-1.9

Spectroscopic measurements Recombinant proteins were diluted to 4 µM in loading buffer containing 1 mM DTT. Ultraviolet–visible absorption spectra were measured using a V550 spectrophotometer (JASCO). Spectral changes accompanying the regeneration of the D450 state were monitored at intervals during incubation in the dark after initial BL illumination (30 W/m2, λmax at 470 nm for 1 min) at 25°C.9

DLS DLS of the protein solutions was measured with a Zetasizer-µV system (Malvern Instruments) in automatic mode at 25°C, and the z-average molecular sizes expressed as RH(app) in solution were determined using Zetasizer Software (version 6.20) as previously described.9,10,32 In brief, after removal of the aggregates by centrifugation, DLS of the recombinant proteins (30–60 µM) was measured several times in the dark (D state), immediately after the termination of BL illumination for 1 min (L state), and after regeneration for 1 h in the dark (LD state). RH(app) values of the proteins were plotted versus the monomer concentrations ([monomer]0) applied to the solutions, and RH values were obtained from the extrapolated data at a 0 µM protein


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concentration.9,10,32 Standard deviations of all RH(app) values obtained from the multiple measurements were less than 0.05 nm.

SEC SEC was performed using an ÄKTA purifier column-chromatography system (GE Healthcare) with a Superdex 75 30/10 column (GE Healthcare) and loading buffer at 25 ± 1°C, with a flow rate of 0.5 mL/min.9,10 To analyze proteins in the D state, reaction mixtures (containing 4 µM protein) were incubated for 20 min at 25°C in the dark, and 150 µL aliquots were subjected to SEC. To analyze proteins in the L state, reaction mixtures were illuminated for 2 min with BL, and applied to the column under continuous illumination with an LED light. To analyze proteins after regeneration (LD state), BL-illuminated samples were kept in the dark for 1 h prior to SEC.

CD measurements CD measurements were carried out using a J-720W spectropolarimeter (JASCO) with a temperature-controlling system under N2 gas flow. The ellipticities of PZ and N131 mutants (10 µM) in CD buffer (200 mM NaCl, 2 mM EDTA, 1 mM DTT, 20 mM Tris-HCl, pH 7.0) were measured within the wavelength region of 200–250 nm, using a 1 mm path length quartz cell at 25°C. For measuring CD spectra in the L state, a scan was immediately measured after the termination of BL illumination for 1 min. For measurements in the presence of DNA, BLilluminated protein solutions were kept in the dark for 1 hour and mixed with final concentration of 5 µM double-stranded Apo (dsApo) containing the target sequence TGACGT or Cpo (dsCpo) without the target sequence (Fig S1), and incubated for 20 min at 25 °C.11,33 Each scan was completed within 1 min and repeated 10 times to average the spectrum in each state. Helicity


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Biochemistry

change of proteins by binding to DNA was determined by the observed ellipticity around 222 nm.34

EMSA EMSA were carried out as described in Nakatani and Hisatomi (2015).12 Briefly, 2 nM Alexa Fluor 647-labeled dsApo (647dsApo) was incubated with various concentrations of N131 mutants in 20 µL reaction mixtures (140 mM KCl, 5% glycerol, 0.1 mg/mL BSA, 2 mM DTT, 50 mM Tris-HCl, pH 7.0) in the presence of 2.5 mM MgCl2 at 25 ± 1°C for 20 min in the dark.12,15,35 An aliquot (9 µL) of reaction mixture was loaded onto a 5% polyacrylamide gel containing 0.5×TBE buffer in the presence of 2.5 mM MgCl2 and separated in the same running buffer at a constant voltage of 100V for 50–60 min at 25 ± 1°C. To investigate DNA binding by N131 mutants in the L state, the reaction mixture was illuminated for 5 min with BL, and separated by electrophoresis under gel illumination with BL (5 W/m2). The fluorescence signals from 647dsApo in the gels were recorded on a Fluor-S MAX image analyzer (Bio-Rad) equipped with a band-pass filter (692 nm × 40 nm; Edmund optics) under illumination by laser diodes (DL-3247-165; λmax at 650 nm; DL-3247-165, Sanyo). The fluorescence intensity of each DNA band was quantified with ImageJ software, with subtraction of the background intensity in each lane. The fractions of dsApo bound to N131 mutants were determined from the ratio of free dsApo to total dsApo, normalized by the amount of free single-stranded Apo, and plotted against the monomer concentrations of N131 mutants ([monomer]0).12 In each case, EC50 was estimated from curve fitting by the Hill equation using the maximum of the bound fraction (Bmax) as follows:

1 Fraction bound  1 (EC50 / [monomer]0 )n Bmax


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QCM measurements 27MHz-QCM (Single-Q, As one) used in this study has a sensitivity of 0.6 ng/cm2 of mass change per Hertz of frequency decrease in the air. Five microliters of neutral avidin solution (100 µg/ml, Wako) were placed on the Au electrode of a QCM sensor and incubated in a wet chamber for 2 days at 4 °C. The QCM sensor was set in the chamber and rinsed twice with QCM buffer (140 mM KCl, 2 mM EDTA, 0.4% BSA, 50 mM Tris-HCl, pH 7.0). Five microliters of biotinylated dsApo or dsCpo solution (5 µM) were added to 450 µL of the QCM buffer, and allowed to anchor double-stranded oligonucleotides to the electrode. The amount of dsApo attached to the electrode was estimated to be less than 1 pmol, which was significantly less than that of PZ in the solution under the experimental conditions. The biotinylated oligonucleotide solutions in the chamber were replaced with the QCM buffer (450 µL), and vibrational frequency of the electrode was monitored at intervals of 1 second to stabilize the resonance frequency (defined as the zero position) with a stirrer rate of 240 rpm at 25°C. To analyze proteins in the D state, 1-50 µM protein solutions were incubated for 20 min at 25°C under a yellow LED light (λmax at 590 nm), and an aliquot of 5 µL was added to the solution in the chamber for each injection. To analyze DNA-binding in the L state, 1-50 µM protein solutions were illuminated for 2 min with BL, and an aliquot (5 µL) was applied to the solution continuously illuminated with a blue LED light (λmax at 470 nm, 5W/m2 at the sensor position; LEDGFPMS, Optocode). The frequency changes (ΔF) by binding of PZ and its mutants to the oligonucleotides were plotted against [monomer]0. In each case, EC50 was estimated from curve fitting by the Hill equation using the maximum of frequency change (ΔFmax) as follows.

1 F  Fmax 1 (EC50 / [monomer]0 )n


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Biochemistry

Time courses of DNA binding of 200–600 nM of PZ or its mutants were measured by the same system with a stirring rate of 120 rpm. Biotinylated oligonucleotides attached to the QCM electrodes were incubated in 396–423 µL of the QCM buffer. After stabilizing the vibrational frequency of the sensor, 3.3 or 5 µM of protein solutions (27–54 µL) in the QCM buffer were injected into the chamber, and ΔF was recorded under a yellow safety light (D state). When the frequencies were stabilized in equilibria between dsApo and the PZ•dsApo complexes in the dark (dark equilibrium), solutions were illuminated with a blue LED light (λmax at 470 nm, 5W/m2) and the ΔF were recorded under continuous illumination (DL state). To analyze speed constants in the L state, 27–54 µL of 3.3 or 5 µM protein solutions were pre-illuminated for 2 min with BL and injected into the chamber, and ΔF were measured under continuous illumination with the blue LED light. As each normalized ΔF(t) can be fit by a single exponential after the unstable period (τ), we normalized ΔF(τ)=0 and ΔF(∞)=ΔFmax (dark or light equilibrium) and calculated the speed constant k by fitting with the following equation. ΔF(t-τ)/ΔFmax = exp(-k(t-τ)) - 1 Apparent kon(app) and koff(app) values for monomeric PZ and its mutants were obtained from the following equation.30 k = kon(app) [monomer]0 + koff(app)


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Figure 1. (A) Domain structure of PZ, and amino acid sequences of PZ and N131 mutants in the basic region. Spectral changes during dark regeneration of N131A (B) and N131Q (C) at 25°C. The spectra were measured in the initial dark state (solid line), and immediately (dashed line), 2, 4, 8, 16 (thin solid lines) and 32 min (dotted lines) after BL illumination.

Results Spectroscopic properties of N131 mutants Fig. 1A shows the domain structure of PZ and amino acid sequences of N131 mutants (N131A and N131Q) in the basic region. Similar to PZ, each recombinant N131 mutant appeared as a single band with a molecular mass of about 30 kDa (Fig. S2A). Figure 1B and 1C show the absorption spectra of N131A and N131Q, respectively, in the dark (thick solid lines), just after BL illumination (dashed lines), during dark regeneration (thin solid lines), and 32 min in the dark


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Biochemistry

(dotted lines). These mutants have spectral peaks at 447 nm with triplet vibrational structures in the dark states similar to PZ.10,11 Moreover, the τ1/2 of regeneration (7.5–7.6 min) are almost identical to that of PZ (Fig. S2B), suggesting that mutations in the basic region have no effect on the spectroscopic properties of the LOV domain.

Dimerization properties of N131 mutants The hydrodynamic radii (RH) of N131 mutants were measured by DLS analyses. Fig. 2 shows the apparent hydrodynamic radii (RH(app)) against the monomer concentrations applied to the solution with regression lines for (A) PZ, (B) N131A and (C) N131Q in the dark (D state, closed circles and a solid line) and light (L state, open circles and a dashed line) states, and after the dark regeneration (LD state, closed triangles and a dotted line). RH(app) increase according to protein concentration and the RH were estimated by extrapolation of protein concentrations to zero.9,10,31 The RH of PZ and N131 mutants were ca. 2.9 in the D states, which increased to ca. 3.7 nm in the L states within this concentration range (Fig. 2D). In the LD states, the RH returned to the same values as those in the D states. The volume changes calculated from the RH between the D and L states were 2.0–2.1, suggesting that N131 mutants underwent reversible dimerization upon illumination similar to PZ.9,10


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Figure 2. The apparent hydrodynamic radii, RH(app) of PZ (A), N131A (B) and N131Q (C), plotted against the monomer concentrations ([monomer]0) applied to the solutions with regression lines, in the D state (closed circles and a thin solid line), L state (open circles and a dashed line), and LD state (closed triangles and a dotted line). The standard deviations of each RH(app) value are less than 0.05 nm. (D) Hydrodynamic radii (RH) of PZ and N131 mutants estimated by extrapolation of protein concentration to zero. L/D and (L/D)3 represent RH change and volume change between the D and L states.

Figure 3 shows the elution profiles of SEC for N131A (A) and N131Q (B) in D (solid lines), L (dashed lines) and LD (dotted lines) states. In the D state, all N131 mutants eluted at the same peak volume as PZ (9.9 ml, corresponding to 39 kDa).10 In the L state, the elution peak of each PZ mutant was detected at the same volume as that of PZ (8.8 ml, 62 kDa). When the samples were kept in the dark for 1 h after BL illumination (LD state), the elution peaks returned to 9.9


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Biochemistry

ml. We, therefore concluded that substitutions of N131 did not affect the conformational change and subsequent dimerization of PZ.

Figure 3. Elution profiles of N131A (A) and N131Q (B) in the D (black lines), L (dotted lines), and LD (thin dotted lines) states, monitored at 280 nm.

CD measurements The ellipticity at 222 nm (θ222) is indicative of the helicities of proteins and is used to estimate DNA-binding of bZIP transcription factors.20-23,34 Figure 4A shows CD spectra of 10 µM PZ solution in the D state (black solid line) and in the L state (black dashed line) in the absence of DNA. Similar to our previous studies, CD spectra of PZ showed negligible change upon BLillumination.11,32 Whereas θ222 decreased by the addition of 5 µM (final concentration) of dsApo containing the target sequence (Fig. S1), due to the formation of α-helix by binding to dsApo (gray solid line). Since EC50 value of PZ for dsApo (420 ± 50 nM) is significantly low even in the D state, most of the PZ fraction seems to bind to dsApo in the presence of 10 µM PZ and θ222


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were not changed by illumination (gray dotted line).12 When dsCpo without the target sequence (Fig. S1) was added instead of dsApo, θ222 was not changed (Fig. 4B, gray lines), suggesting that α-helix formation was induced by the specific interaction(s) with the target sequence. Similar changes in CD spectra were observed for N131A (Fig. 4C) and N131Q (Fig. 4D) by the addition of dsApo. CD spectra of N131 mutants were not changed by the addition of dsCpo (gray lines in Fig. S3). Our data suggested that α-helix formations of the basic region were induced by binding to the target sequence even in the absence of N131. As a basic region of a bZIP factor has been shown to bind non-specific DNA in a random coil configuration,36 PZ and N131 mutants possibly bind to dsCpo by electrostatic interactions.

Figure 4. CD spectra of PZ in the D (black solid line) and L (black dashed line) states in the absence of oligonucleotides, and in the presence of dsApo (A) and dsCpo (B) in the D (gray solid line) and L (gray dashed line) states. CD spectra of N131A (C) and N131Q (D) in the absence of


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Biochemistry

dsApo in the D (black solid lines) and L (black dashed lines) states, and in the presence of dsApo in the D (gray solid lines) and L (gray dashed lines) states.

EMSA EC50 values of N131 mutants for binding to dsApo were quantified by EMSA (Fig. S4). The dsApo fractions bound to the proteins were estimated from the reduction in fluorescence intensity of free dsApo compared with the protein-free sample, because protein–DNA complexes might dissociate during electrophoresis, affecting the levels of shifted bands.12 Figure 5 shows the relative bound fractions to dsApo plotted against the final monomer concentrations ([monomer]0) of N131A (A) and N131Q (B) applied to the solution. EC50 values of N131A and N131Q in the D states were estimated to be 1,000 ± 30 nM and 1,430 ± 210 nM, respectively (Table 1). In the L state, EC50 values of N131A and N131Q were estimated to be 110 ± 26 nM and 117 ± 20 nM, respectively. Although the N131 mutants increased the affinities for the target sequence upon illumination, the EC50 values of the N131 mutants were significantly higher than those of PZ in both D and L states (420 ± 50 nM and 39 nM ± 1 nM, respectively12).


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Figure 5. (A) The normalized dsApo fractions bound to N131A (A) and N131Q (B) against monomer concentrations ([monomer]0) obtained from EMSA data, with fitting curve in the D state (closed circles and solid lines) and L state (open circles and dotted lines).

Table 1. EC50 values of PZ, constitutively dimeric S2C mutant and N131 mutants for binding to dsApo or dsCpo estimated from EMSA and QCM data. Asterisks indicate the data from reference.12

EC50 estimated from QCM measurements The affinities of PZ for dsApo were then investigated by QCM measurements. Figure 6A and 6B show typical time courses of resonant frequency change (ΔF) by the addition of 5 µL PZ solution in the D and L states, respectively. ΔF decreased after each injection (arrows) toward the frequency in equilibria (ΔFeq) between dsApo and the PZ•dsApo complexes attached to the electrode. ΔFeq values were not affected by increasing the BL intensity. EC50 and maximum frequency change (ΔFmax, where [monomer]0 = ∞) were calculated by fittings assuming a Hill coefficient (n) equal to 1, because EC50 values showed only small changes when n was varied from 0.9 to 1.4. Panel 6C shows simple saturation curves of ΔFeq/ΔFmax for dsApo plotted against


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Biochemistry

the [monomer]0 of PZ. EC50 values for dsApo were calculated to be 400 ± 90 nM in the D state (closed symbols) and 50 ± 5 nM in the L state (open symbols). The affinities of PZ for dsCpo were also quantified and EC50 values were obtained to be 1,800 ± 100 nM and 840 ± 110 nM in the D and L states, respectively (Fig. S5A and S7A). The EC50 values of constitutively dimeric mutant (S2C) for dsApo were calculated to be ca. 10 nM irrespective of the light conditions (Fig. S5B and Fig. 6D), where the molar concentrations of the bZIP monomer were assumed to be [monomer]0 of S2C. The EC50 values are summarized in Table 1. These EC50 values are comparable with those previously reported from EMSA and FCS data.12 Similarly, the EC50 values of the N131A (Fig. S6A and Fig. 6E) and N131Q (Fig. S6B and Fig. 6F) for dsApo were quantified both in the D and L states (Table 1). The EC50 values of N131 mutants are consistent with those obtained from EMSA data, suggesting that N131 in PZ takes a pivotal role in stabilizing the PZ•dsApo complex.


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Figure 6. Time courses of ΔF for dsApo-attached QCM electrodes by adding 5 µL PZ solutions in the D (A) and L (B) states. Allow indicates the injection of each PZ solution. Saturation binding behavior (ΔFeq/ΔFmax) of dsApo-electrodes against [monomer]0 of PZ (C), S2C (D), N131A (E) and N131Q (F), in the D (closed symbols) and L (open symbols) states with regression curves.

Association and dissociation speed constants of the complex As PZ is in a monomer-dimer equilibrium, the apparent association (kon(app)) and dissociation (koff(app)) speed constants of the PZ•dsApo complex were evaluated against [monomer]0 of PZ and its mutants applied to each solution. Fig. 7A shows the time-courses of ΔF(t)/ΔFeq by injection of 300–600 nM [monomer]0 of PZ, which decrease after the injection (t=0) from 0 toward -1 (dark equilibrium) in the D state. As each ΔF(t)/ΔFeq can be fit by a single exponential when t is greater than 90 sec, we used unstable time τ=90 and estimated the speed constant k and ΔFeq by curve fitting. It should be noted that ΔF(t) showed a shift even with the addition of a protein free buffer (Fig. S7B, solid line). k and ΔFeq were similarly obtained for N131 mutants, and 200–400 nM of [monomer]0 were used for the constitutively dimeric S2C mutant. Fig. 7B shows k values plotted against [monomer]0 of PZ (black circles), N131A (blue squares), N131Q (red triangles) and S2C (green diamonds). k for each PZ and mutants appear to be proportional to [monomer]0. On assuming the simple binding reaction of the PZ monomer to a small amount of dsApo, kon(app) and koff(app) of PZ and its mutants in the D state were obtained as shown in Table 2 (upper). As the Kd value of the PZ dimer was 130 µM, the dimer concentrations of PZ and N131 mutants are very low in the D state.10 Cao et al. (2000) have reported that the monomer of the yeast transcriptional activator GCN4 recognizes its dimer binding DNA target sites without


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dimerization.37 The kon(app) for PZ and N131 mutants considerably differ from that of dimeric S2C, suggesting that the monomer mainly associates with the target sequence in the D state.

Figure 7. Time courses of ΔF/ΔFeq for dsApo-attached QCM electrodes (A) by the addition of 300–600 nM [monomer]0 of PZ in the D state, (C) by BL illumination of PZ solutions in dark equilibria (DL state), and (E) by the addition of PZ in the L state. [monomer]0 of ΔF/ΔFeq with regression curves in each panel are 300 nM (thick pail purple with thin purple curve), 400 nM (thick pail blue and thin blue curve), 500 nM (thick pail green and thin green curve), and 600 nM (thick pail red and thin red curve). Unstable times are 90 sec for the D and L states and 30 sec for the DL state. Speed constants (k) of ΔF/ΔFeq versus [monomer]0 of PZ (black circles), N131A (blue squares), N131Q (red triangles) and S2C (green diamonds) in the D (B), DL (D) and L (F) states.


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Table 2. kon(app), koff(app), and koff(app)/kon(app) values for PZ, S2C, N131A and N131Q in D, DL and L states with standard errors. Samples in the dark equilibrium were then illuminated with BL and ΔF(t)/ΔFeq were recorded under continuous illumination with BL (designated as the DL state). When dsCpo without the target sequence was used instead of dsApo, ΔF was very small owing to the limited amount of PZ bound to dsCpo even in the presence of 600 nM [monomer]0 of PZ (Fig. S7B, broken line). Because each time course can be fit by a single exponential when t is greater than 30 sec, τ=30 was used for the DL state (Fig. 7C). k values were not changed by increasing the intensity of BL. k values for N131 mutants are very close in each concentration, but slightly greater than those of PZ (Fig. 7D). The calculated kon(app) of PZ and N131 mutants are close to one another, whereas koff(app) of N131 mutants are greater than that of PZ in the DL state (Table 2 lower). Fig. 7E shows time courses of ΔF(t)/ΔFeq after injection of light-adapted PZ in the sample solutions continuously illuminated with BL (L state). τ=90 was used to calculate the fitting parameters in the L state. Similarly, in the DL state regression lines are almost parallel but those of the N131 mutants are slightly higher than that of PZ and much higher than that of S2C (Fig. 7F). The


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kon(app) and koff(app) are summarized in Table 2. In each D, DL and L state, the kon(app) of PZ and N131 mutants are comparable to one another, but the kon(app) in DL and L states are two-fold greater than those in the D state and close to that of dimeric S2C. Further, koff(app) values of PZ and N131 mutants in the DL and L states are 3–4 fold smaller than those in the D state. The QCM data demonstrated that BL accelerates the association and decelerates the dissociation of the PZ•dsApo complex. Moreover, the koff(app) values of the N131 mutants are about twice that of PZ in each state, indicating that N131 stabilizes the PZ•dsApo complex by lowering the dissociation speed without affecting the association speed. Base-specific interactions of N131 likely occur after the association of PZ with dsApo.

Discussion In this study, we prepared N131 mutants and quantified the affinities of PZ and its mutants for the target DNA sequence. QCM measurements have an advantage to evaluate the speed constants of the complexes, in comparison with other methods to measure the static constants in a equilibrium state. Okahata et al. (1998) have reported kinetic studies of sequence-specific binding of GCN4-bZIP peptides to the CRE sequence by QCM measurements.30 Because Kd values of bZIP regions have been reported to be 10-7–10-8, they calculated kon and koff for bZIP dimer (kon(dim) and koff(dim)) to be 63×103 M-1s-1 and 2.1×10-3 s-1, respectively, in the presence of 10-7–10-6 M peptide. As the Kd of PZ dimer (PZ2) is 120 nM, 96–219 nM of PZ2 exists in the light state, where PZ [monomer]0 is 300–600 nM in the solution. Fig. S8 shows the k values of PZ and its mutants plotted versus dimer concentrations expected in DL and L states. Calculated kon(dim) and koff(dim) for PZ2 and N131 mutant dimers are about 45–50×103 M-1s-1 and 2.2–3.6×10-3 s-1, respectively, which are comparable to those of the GCN4-bZIP dimer. Moreover, kon(dim)


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values are close to that of constitutively dimeric S2C mutant (42–43×103 M-1s-1). Using the transient grating method, Akiyama et al. (2017) suggested that the rate-determining step for DNA-binding of PZ is protein dimerization, and estimated the speed constant of dimer binding to be 77×103 M-1s-1.38 DNA binding may primarily be due to PZ2 in DL and L states. Our data suggested the substitution of N131 had a negligible effect on the dark regeneration kinetics of the LOV domain and on the subsequent dimerization of PZ. However, QCM and EMSA analyses showed that EC50 values of N131 mutants were significantly higher than those of PZ. The crystal structure of bZIP transcription factors suggested that Asn located at the center of the basic domain formed hydrogen bonds with specific DNA bases.6,7,25 Pu and Struhl (1991) have indicated from biological and biochemical assays that N235 was not essential for DNAbinding.39 They also reported from an in vivo study that N235A and N235Q derivatives of yeast GCN4 differ from wild-type GCN4 in their strong discrimination of the target sequence.40 Our results, indicating that N131 is responsible for lowering the dissociation speed constant of the PZ•dsApo complex without changing the association speed, are consistent with these studies. Okahata et al. (1998) have investigated the binding of GCN4-bZIP peptides to several oligonucleotides, and suggested that the sequence selectivity of bZIP is mainly determined by the dissociation process but not the binding process.30 We, therefore, propose the intermediate binding stage to explain our findings (Fig. 8). In the L state, PZ and PZ2 exist in equilibrium with a Kd of about 120 nM in solution (Fig. 8A). PZ2 primarily binds to the oligonucleotides attached to the QCM electrode by the non-selective interaction(s) (intermediate binding stage, Fig. 8B). The PZ2•DNA complex with base-specific hydrogen bonds of N131 is subsequently formed (proper binding stage, Fig. 8C).


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LO

LOV

ZIP ZI P

LOV

nAv

ZIP IP b Z

nAv

LOV

Intermediate binding stage

ZIP

ZIPP ZI

V

In solution

L OV

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L OV

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LOV Proper binding stage

QCM electrode

Figure 8. Binding scheme of PZ to the target DNA sequence in the L state. (A) PZ and PZ dimer (PZ2) are in equilibrium with Kd of about 120 nM in the solution. (B) A small fraction of PZ (primarily PZ2) non-specifically interacts with the oligonucleotides attached to the electrode. (C) The proper PZ2•dsApo complex is formed with the sequence-specific interaction of N131. LOV, LOV domain; ZIP, leucine zipper region; b, basic region; nAv, neutral avidin. Speed constants in DL states are almost identical to those in L states with smaller standard errors. Activation of PZ by illumination increased the accuracy and decreased the unstable period (τ) of QCM data. Optical control of transcription factors will provide new knowledge of the recognition of the target sequence.


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Supporting Information. List of oligonucleotides (Figure S1), SDS-PAGE and regeneration time course of N131 mutants (Figure S2), CD spectra in the presence of dsCpo (Figure S3), EMSA (Figure S4), QCM measurements for PZ with dsCpo and for S2C with dsApo (Figure S5), QCM measurements for N131 mutants with dsApo (Figure S6), Saturation binding behavior of PZ to dsCpo and ΔF of control experiments (Figure S7), Speed constants for the complex formation versus dimeric concentration (Figure S8), and kon(dim), koff(dim), and koff(dim)/kon(dim) values for PZ and mutants (PDF)

Corresponding Author * E-mail: [email protected]. ORCID Osamu Hisatomi: 0000-0003-2488-5299 Author Contributions Samu Tateyama carried out spectroscopic, DLS, EMSA and QCM measurements, and wrote the manuscript. Itsuki Kobayashi measured the CD spectra. Osamu Hisatomi performed site-directed mutagenesis, SEC and QCM measurements, and wrote the manuscript. Funding This work was partly supported by a Grant-in-Aid for Scientific Research (C) to O.H. (No. 26440077). Notes


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The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors thank Dr. Satoru Nakashima, Dr. Norio Hamada and Dr. Ryosuke Nakamura (Osaka University) and Dr. Hiroyuki Mino (Nagoya University) for helpful discussions.

ABBREVIATIONS PZ, Photozipper protein; bZIP, basic leucine zipper; LOV, light-oxygen-voltage-sensing; BL, blue light; QCM, quartz crystal microbalance; FMN, flavin mononucleotide; SEC, size exclusion chromatography; DLS dynamic light scattering; EMSA, electrophoretic mobility shift assay; EC50, half-maximal effective concentration; DTT, dithiothreitol; λmax, absorption maximum; RH, hydrodynamic radius; CD, circular dichroism; ΔF, resonant frequency change.


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(40) Tzamarias, D., Pu, W. T., and Struhl, K. (1992) Mutations in the bZIP domain of yeast GCN4 that alter DNA-binding specificity. Proc. Natl. Acad. Sci. USA. 89, 2007-2011.


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TOC graphic


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39x50mm (300 x 300 DPI)


Target sequence recognition by a light-activatable bZIP factor

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