Spectroscopic and DNA-binding characterization of the isolated heme-bound basic helix–loop–helix-PAS-A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with circadian rhythms Yuji Mukaiyama 1 , Takeshi Uchida 2 *, Emiko Sato 1 , Ai Sasaki 1 , Yuko Sato 1 , Jotaro Igarashi 1 , Hirofumi Kurokawa 1 , Ikuko Sagami 1 †, Teizo Kitagawa 2 and Toru Shimizu 1 1 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan 2 Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Japan In animals, biological rhythms are coordinated in adaptive synchrony by the brain, specifically by the suprachiasmatic nucleus of the hypothalamus. The suprachiasmatic nucleus is a major coordinator of internal circadian organization and is itself synchron- ized with the day–night cycle by direct neural input from specialized retinal photoreceptors [1–6]. In its simplest form, the molecular clockwork consists of autoregulatory transcriptional and translational feed- back loops that have both positive and negative ele- ments [1–6]. The positive components are two transcription factors, CLOCK and mouse brain and Keywords circadian rhythms; DNA binding; heme- sensor protein; PAS domain; resonance Raman spectroscopy Correspondence T. Shimizu, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan Fax: +81 22 217 5604 ⁄ 5390 Tel: +81 22 217 5604 ⁄ 5605 E-mail: shimizu@tagen.tohoku.ac.jp Present address *Division of Chemistry, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Japan † Graduate School of Agriculture, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto, Japan (Received 23 February 2006, accepted 4 April 2006) doi:10.1111/j.1742-4658.2006.05259.x Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm-associated transcription factor with two heme-binding sites on two PAS domains. In the present study, we compared the optical absorption spectra, resonance Raman spectra, heme-binding kinetics and DNA-binding characteristics of the isolated fragment containing the N-terminal basic helix–loop–helix (bHLH) of the first PAS (PAS-A) domain of NPAS2 with those of the PAS-A domain alone. We found that the heme-bound bHLH-PAS-A domain mainly exists as a dimer in solution. The Soret absorption peak of the Fe(III) complex for bHLH-PAS-A (421 nm) was located at a wave- length 9 nm higher than for isolated PAS-A (412 nm). The axial ligand trans to CO in bHLH-PAS-A appears to be His, based on the resonance Raman spectra. In addition, the rate constant for heme association with apo-bHLH-PAS (3.3 · 10 7 mol )1 Æs )1 ) was more than two orders of magni- tude higher than for association with apo-PAS-A (< 10 5 mol )1 Æs )1 ). These results suggest that the bHLH domain assists in stable heme binding to NPAS2. Both optical and resonance Raman spectra indicated that the Fe(II)–NO heme complex is five-coordinated. Using the quartz-crystal microbalance method, we found that the bHLH-PAS-A domain binds spe- cifically to the E-box DNA sequence in the presence, but not in the absence, of heme. On the basis of these results, we discuss the mode of heme binding by bHLH-PAS-A and its potential role in regulating DNA binding. Abbreviations bHLH, basic helix–loop–helix; BMAL1, mouse brain and muscle Arnt-like 1; CooA, CO-sensing heme-bound transcriptional regulator from Rhodospirillum rubrum; Ni-NTA, Ni 2+ -nitrilotriacetic acid; NPAS2, neuronal PAS domain protein 2; QCM, quartz-crystal microbalance method. 2528 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS muscle Arnt-like 1 (BMAL1), both of which contain a basic helix–loop–helix (bHLH) domain and two PAS domains [7–10]. These two transcription factors form a heterodimer that binds to the E-box sequence, which drives the transcription of three Period genes (designa- ted mper1, mper2 and mper3 in the mouse) and two cryptochrome genes (mcry1 and mcry2). The mPER and mCRY proteins appear to act as negative compo- nents of the feedback loop [1–10]. The mammalian forebrain expresses neuronal PAS domain protein 2 (NPAS2), which is homologous to CLOCK [7–10]. NPAS2 plays an important role in maintaining circadian behaviors in normal light–dark and feeding conditions, and it is critical for adaptation to food restriction [11–14]. NPAS2 also forms a het- erodimer with BMAL1, and the heterodimer activates the transcription of per and cry, whereas it represses BMAL1 gene expression. Like CLOCK and BMAL1, NPAS2 has bHLH, PAS-A and PAS-B domains in its N-terminal region, but in contrast to CLOCK, both the PAS-A and PAS-B domains of NPAS2 contain a heme-binding site, and CO binding to the heme inhib- its the DNA-binding activity of the NAPS2–BMAL1 heterodimer [15]. The bHLH-PAS proteins are critical regulators of gene expression networks underlying a variety of essen- tial physiological and developmental processes [7,16]. In many cases, bHLH proteins dimerize to form func- tional DNA-binding complexes, whereas bHLH-PAS proteins are distinct from other members of the broader bHLH superfamily due to the dimerization specificity conferred by their PAS domains. The bHLH-PAS proteins tend to be ubiquitous latent sig- nal-regulated transcription factors that often recognize variant forms of the classic E-box enhancer sequence bound by other bHLH proteins [7,16]. Because CO binding to the heme causes dissociation of NPAS2 from BMAL1 and the E-box sequence, the bound heme itself may affect the DNA-binding properties of NPAS2 by interacting with the bHLH region in a direct or indirect manner. Therefore, it is worth study- ing how the bHLH domain contributes to heme bind- ing to the PAS-A domain in NPAS2 and how the bound heme participates in the binding of NPAS-2 to the E-box DNA sequence. In the present study, we investigated the role of the bHLH domain by characterizing the isolated heme- bound bHLH-PAS-A and heme-bound PAS-A domains of NPAS2 using optical absorption spectros- copy, resonance Raman spectroscopy, and heme-bind- ing kinetic analyses. We found that the bHLH domain significantly affects the spectra of the heme-bound PAS-A domain and appears to assist in stable heme binding, stabilizing the protein molecule. We also dem- onstrated specific binding of the bHLH-PAS-A protein to the E-box of DNA using the quartz-crystal micro- balance (QCM) method. Results Protein expression and purification We attempted to express the isolated bHLH-PAS-A (amino acids 1–240) and PAS-A (amino acids 78–240) domains of NPAS2 as described previously (see Experimental procedures) [22]. The bHLH-PAS-A domain was properly overexpressed and folded in Escherichia coli and displayed sufficient heme-binding ability. SDS ⁄ PAGE followed by staining with Coo- massie Brilliant Blue revealed that the purified His- tagged bHLH ⁄ PAS-A and PAS-A domains of NPAS2 were more than 95% homologous (supplementary Fig. S1). To remove the His tag, the His-tagged bHLH-PAS-A and PAS-A domains were treated with thrombin and then purified using a Ni 2+ -nitrilotriace- tic acid (Ni-NTA) agarose column. The yields of the bHLH-PAS-A and PAS-A domains were 8.0 and 9.0 mgÆL )1 of culture, respectively. Size exclusion chromatography Gel filtration analysis of a solution of bHLH-PAS-A protein using Superdex 75 (Amersham Biosciences, Uppsala, Sweden) revealed that the solution contains one major peak (more than 70%) and two minor peaks (supplementary Fig. S2A). The heme-reconstitu- ted bHLH-PAS-A domain was the major species and had a molecular mass of nearly 60 kDa (supplement- ary Fig. S2B). Because the monomer has a predicted molecular mass of 28 kDa, this suggests that the major species of the bHLH-PAS-A domain exists as a dimer. We collected only the major fraction and reapplied it to the Superdex 75 column. The chromatographic pro- file was the same, suggesting that the fraction was in equilibrium between monomer, dimer (majority) and trimer forms. Optical absorption spectra of Fe(III), Fe(II) and Fe(II)–CO complexes To understand the heme environment of the bHLH- PAS-A domain of NPAS2, we obtained optical absorption spectra of the overexpressed and purified bHLH-PAS-A domain. Optical absorption spectra of the Fe(III), Fe(II) and Fe(II)–CO complexes of the bHLH-PAS-A domain of NPAS2 are shown in Y. Mukaiyama et al. Characterization of bHLH-PAS-A of NPAS2 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS 2529 Fig. 1A and are summarized in Table 1. For the Fe(III) form, the absorption maxima were located at 421 nm and 543 nm at pH 8.0. The absorption max- ima of the Fe(II) species were at 426, 530 and 559 nm, and those of the Fe(II)–CO complex were at 422, 538 and 566 nm. On the other hand, optical absorption spectra of the Fe(III), Fe(II) and Fe(II)–CO complexes of the PAS-A domain of NPAS2 were different from those of the bHLH-PAS-A domain (Fig. 1B and Table 1). Namely, in the Fe(III) form, the absorption maxima were located at 412 and 538 nm at pH 8.0. The absorption maxima of the Fe(II) species were at 423, 530 and 558 nm, and those of the Fe(II)–CO complex were at 420, 530 and 568 nm. It is likely that Fe(II) and Fe(II)–CO complexes of the bHLH-PAS-A domain have mostly six-coordinate low-spin heme, whereas the Fe(III) complex has five-coordinate high- spin heme as a minor component in addition to the major six-coordinate low-spin heme [17]. Effect of pH on the Fe(III) complex To identify the axial ligand of the bHLH-PAS-A domain of NPAS2, we examined the effect of modula- ting pH on the Fe(III) complex. We did not find any remarkable pH-dependent spectral changes between pH 6 and pH 11. Resonance Raman spectra of Fe(III), Fe(II) and Fe(II)–CO complexes To further examine the nature of the heme environ- ment of the bHLH-PAS-A domain of NPAS2, we ana- lyzed the resonance Raman spectra of the Fe(III), Fe(II) and Fe(II)–CO complexes. The spectra of the Fe(III) and Fe(II) complexes in the high-frequency region are shown in Fig. 2 and summarized in Table 2. 4.0 3.0 2.0 1.0 0 . 0 Absorbance 00700600 5 0 0 4003 )mn( h tgnelev aW 3× )III(eF )II(eF OC-)II(eF 8.0 6 .0 4 .0 2.0 0.0 Absorbance 0070 0 600500400 3 )mn( h tgnelevaW 5× ) III(eF )II(eF OC-)II( e F A B Fig. 1. Optical absorption spectra of the isolated heme-bound basic helix–loop–helix (bHLH)-PAS-A (A) and PAS-A (B) domains. Fe(III) (solid line), Fe(II) (dotted line) and Fe(II)–CO (dashed line) com- plexes are shown. Spectra were obtained in a buffer consisting of 100 m M Tris (pH 7.5). All proteins were His-tag-free. Table 1. Optical absorption spectra of isolated basic helix–loop– helix (bHLH)-PAS-A and PAS-A domains of neuronal PAS domain protein 2. bHLH-PAS-A PAS-A Soret (nm) Visible (nm) Soret (nm) Visible (nm) Fe(III) 421 543 412 538 Fe(II) 426 530, 559 423 530, 558 Fe(II)–CO 422 538, 566 420 530, 568 Intensity 007100610051004100310 0 21 t fihs nam a R (mc 1- ) 1640 1579 1504 1631 1471 1359 1493 1555 1618 1605 )III(eF )II(eF 1582 1492 1374 1623 1629 Fig. 2. Resonance Raman spectra of the high-frequency region of the Fe(III) (lower) and Fe(II) (upper) complexes of the basic helix– loop–helix (bHLH)-PAS-A domain. Characterization of bHLH-PAS-A of NPAS2 Y. Mukaiyama et al. 2530 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS Bands at 1374 and 1359 cm )1 for the Fe(III) and Fe(II) complexes were assigned as the redox-sensitive m 4 band [17,32]. For the Fe(III) complex, the spin-coordination and coordination-state marker bands (m 3 ) were located at 1470, 1492 and 1504 nm, corresponding to the six- coordinate high-spin, five-coordinate high-spin and six-coordinate low-spin states, respectively [17,32]. The shoulder at 1492 cm )1 was ascribed to a five-coordinate high-spin complex observed as a minor component in the Fe(III) complex. For the Fe(II) complex, on the other hand, m 3 bands were observed at 1471 and 1493 cm )1 , representing the five-coordinate high-spin and six-coordinate low-spin states, respectively. Because of the sensitivity of the Fe–CO and C–O stretching frequencies to the heme environment (i.e. electrostatic and steric interactions with surround- ing groups), spectra of CO adducts of heme proteins provide valuable information about the heme pocket [17,32]. Low-frequency and high-frequency regions of the resonance Raman spectra of the Fe(II)–CO complex of the bHLH-PAS-A domain are shown in Fig. 3A,B. Isotope-sensitive lines were observed at 486 cm )1 for the Fe–CO stretching mode (m Fe–CO ) and at 1919 cm )1 for the C–O stretching mode (m C–O ) when we used 13 C 18 O. Finally, we assigned the 495 and 1962 cm )1 bands to m Fe–CO and m C–O , respectively (Table 3). The inverse relationships between the m Fe–CO and m C–O frequencies are used to determine the axial ligand of the Fe(II) heme iron (Fig. 4) [17,32]. The frequen- cies for the bHLH-PAS-A domain corresponded to Fe–His but not Fe–S coordination. Because the m Fe–CO stretching frequency (495 cm )1 ) is lower than those of heme complexes in a polar environment [17,32], it appears that the CO is located in a somewhat hydro- phobic environment. Optical absorption and resonance Raman spectra of the Fe(II)–NO complex Since NO may be involved in transcription control, we next obtained optical absorption and resonance Table 2. Resonance Raman spectra of the basic helix–loop–helix (bHLH)-PAS-A domain of neuronal PAS domain protein 2. Excitation was at 413.1 nm. 5cHS, five-coordinate high spin; 6cLS, six-coordi- nate low spin. Complex m 2 (cm )1 ) m 3 (cm )1 ) Coordination Fe(III) 1552 1492 5cHS 1579 1504 6cLS Fe(II) 1555 1471 5cHS 1582 1493 6cLS mc( 1– ) Intensity 00700 6 0050040 0 3 mc (tfi h Sn a m a R 1– ) 500 482 495 486 A Intensity 0 50 2 00 0 2 0 5 9 1 0 09 1 0 58 1 0 0 8 1 0571 tfihSnamaR 1919 1962 1919 1962 B Fig. 3. Effects of isotopically labeled CO molecules on resonance Raman spectra of the Fe (II)–CO complexes of basic helix–loop–helix (bHLH)-PAS-A in the low-frequency (A) and high-frequency (B) regions. The bottom lines show the 12 C 16 O complex, the middle lines the 13 C 18 O complex, and the top lines the difference spectra between the 12 C 16 O and 13 C 18 O complexes. Y. Mukaiyama et al. Characterization of bHLH-PAS-A of NPAS2 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS 2531 Raman spectra of the Fe(II)–NO complex. As shown in Fig. 5, the Fe(II)–NO complex had a Soret absorp- tion peak at 394 nm, which is characteristic of a five- coordinated Fe(II)–NO heme complex [23–27,32]. In addition, the characteristics of the resonance Raman spectra of the Fe(II)–NO complex (Fig. 6) were very similar to those of the five-coordinated Fe(II)–NO complex [24,32]. Isotope-sensitive resonance lines were observed at 514 cm )1 for the Fe–NO stretching mode (m Fe–NO ) and at 1670 cm )1 for the N–O stretching mode (m N–O ) when we used 15 N 16 O. Heme-binding kinetics In order to understand the heme-binding character of the bHLH-PAS-A domain, we examined the association rate constants (k on ) for binding of the Fe(II)–CO heme complex to the apo-bHLH-PAS-A and apo-PAS-A domains of NPAS2, using a modification of the methods described by Hargrove et al. [18]. Fe(II)–CO heme (1.0 lm) was mixed with 2, 3 or 4 lm bHLH-PAS-A domain or 16 lm apo- PAS-A domain using a stopped-flow apparatus at 25 °C. There was a concomitant increase in the absorbance at 421 and 419 nm and a decrease at 408 nm, which correspond to the binding of Fe(II)– CO heme to the apo-bHLS-PAS-A domain and the apo-PAS-A domain, respectively. This shows that Fe(II)–CO heme bound strongly to the apo-bHLH- PAS-A domain (Fig. 7A). The time-dependent increase in absorbance accompanying Fe(II)–CO heme binding to the apo-bHLH-PAS-A domain was composed of only a single phase (Fig. 7A, inset), and the rate of association was dependent on the apoprotein concentration (Fig. 7C). As summarized in Table 4, the rate constant for association with the apo-bHLH-PAS-A domain was 3.3 · 10 7 mol )1 Æs )1 . In contrast, Fe(II)–CO heme binding to the apo- PAS-A domain was very slow and did not saturate under our experimental conditions (Fig. 7B). There- fore, the k on value of the PAS-A domain should be less than 10 5 mol )1 Æs )1 . We also determined the rate constant for the dissoci- ation of heme from the holo-bHLH-PAS-A domain of NPAS2 by mixing it with an excess of the H64Y ⁄ V68F apomyoglobin mutant. The reaction was monitored by following the increase in absorbance at 410 nm (Fig. 8), which accompanies the formation of Fe(III) heme-bound myoglobin. The observed k off was 5.3 · 10 )3 s )1 for the bHLH-PAS-A domain. Table 4 summarizes k off as well as the calculated K d values for the bHLH-PAS-A domain and those of other heme- binding proteins. The K d value of heme was estimated to be 1.6 · 10 )10 m for the bHLH-PAS-A domain. Analysis of DNA binding by the QCM method The QCM is a very sensitive device for the detection of DNA–protein and protein–protein interactions in solution, which are monitored by the linear decreases of the emitted frequency with increasing mass present 01. 0 80.0 60.0 40 . 0 2 0.0 0 0.0 Absorbance 00700600500400 3 )mn(htgelevaW 3× )II(eF ON-)II(eF Fig. 5. Optical absorption spectra of the Fe(II)–NO (bold line) and Fe(II) (thin line) heme complexes of basic helix–loop–helix (bHLH)- PAS-A. The position of the Soret band for the Fe(II)–NO complex (394 nm) suggests that it is a five-coordinated NO–heme complex. Table 3. Resonance Raman spectra of Fe(II)–CO complexes of the basic helix–loop–helix (bHLH)-PAS-A and PAS-A domains of neuron- al PAS domain protein 2. m Fe–CO (cm )1 ) m C–O (cm )1 ) References bHLH-PAS-A 495 1962 this work PAS-A 496 1962 [22] Fig. 4. Inverse correlations between frequencies of m C–O and m Fe–CO of resonance Raman spectra for the Fe (II)–CO complex. The axial ligand trans to CO of basic helix–loop–helix (bHLH)-PAS-A appears to be His. Characterization of bHLH-PAS-A of NPAS2 Y. Mukaiyama et al. 2532 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS on the QCM electrode [19,20]. It appeared to be worth examining the interaction between the bHLH-PAS-A domain and DNA. To understand whether the bHLH- PAS-A domain of NPAS2 can bind to DNA with the E-box sequence, we injected the heme-bound bHLH- PAS-A domain onto the E-box-bound sensor chip, and the decrease in frequencies was observed with time (Fig. 9A). This confirms that, under the experimental conditions used here, the heme-bound bHLH-PAS-A domain bound to the DNA containing the E-box sequence. To examine whether the heme is required for binding to the E-box sequence, we injected heme-free bHLH-PAS-A onto the E-box-bound sensor chip. In this case, a decrease in the frequencies was not observed (Fig. 9B). To confirm that the binding to the E-box sequence was specific, we examined the binding of a mutant E-box sequence (wild type: CACGTG: mutant GACGTC). Essentially no frequency shift was observed when either the heme-bound or heme-free domains were applied to the mutant E-box (Fig. 9C,D). Similarly, the PAS-A domain without the bHLH domain did not bind to the E-box sequence. Also, the heme-bound bHLH-PAS-A domain did not bind to a random DNA sequence (not shown). Collec- tively, these results show that the bHLH-PAS-A domain specifically binds to DNA containing an E-box sequence under the experimental conditions used. Because the bHLH-PAS-A forms mainly a dimer in solution (supplementary Fig. 2S), it may bind to the E-box as a dimer. Discussion The findings from the current study suggest that the bHLH domain assists and stabilizes heme binding by the isolated bHLH-PAS-A domain of NPAS2. In addi- tion, specific binding of the isolated bHLH-PAS-A domain to the E-box was observed only when it was bound to heme. The optical absorption spectra revealed that Fe(III), Fe(II) and Fe(II)–CO complexes of bHLH-PAS-A and PAS-A were six-coordinate low spin. The Soret peaks of Fe(III), Fe(II) and Fe(II)–CO complexes of bHLH- PAS-A, however, were red-shifted compared to those of PAS-A, suggesting that there are direct or indirect interactions between the bHLH domain and the heme environment in the PAS-A domain. In addition, the Soret absorption of PAS-A had a shoulder at approxi- mately 370–380 nm, which probably corresponds to free heme. Therefore, it appeared that the affinity of the isolated PAS-A domain for heme is lower than that of the bHLH ⁄ PAS-A domain. This idea was sup- Intensity 0 07 0060 05 004 003 tfihSnamaR(mc 1– ) 675 523 585 348 514 A B Intensity 0 0 71 0 06 10 05 100 41 0 031 tfihS n amaR(mc 1– ) 1361 1376 1508 1584 1648 1606 1670 1642 Fig. 6. Effects of isotopically labeled NO molecules on resonance Raman spectra of the Fe(II)–NO complexes of basic helix–loop–helix (bHLH)-PAS-A in low-frequency (A) and high-frequency (B) regions. The bottom lines show the 14 N 16 O complex, the middle lines the 15 N 16 O complex, and the top lines the difference spectra between the 14 N 16 O and 15 N 16 O complexes. Y. Mukaiyama et al. Characterization of bHLH-PAS-A of NPAS2 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS 2533 ported by the kinetic studies of heme binding, which revealed that heme binding to the isolated PAS-A is very slow and did not saturate under our experimental conditions (Fig. 7, Table 4). The resonance Raman spectra showed that the heme coordination states of Fe(III) complexes of the isolated bHLH-PAS-A and the PAS-A domains were a mixture of five-coordinated high spin and six-coordinated low spin. Similarly, for the Fe(II) complexes, both are a mixture of five-coordinated high spin and six-coordina- Table 4. Association and dissociation rate constants and equilib- rium parameters for heme binding to the basic helix–loop–helix (bHLH)-PAS-A and PAS-A domains and other heme proteins. Proteins k on (mol )1 Æs )1 ) k off (s )1 ) K d (M) References bHLH-PAS-A 3.3 · 10 7 5.3 · 10 )3 1.6 · 10 )10 This work PAS-A < 10 5 This work PAS-B a 7.7 · 10 5 3.2 · 10 )3 [21] <10 5 3.0 · 10 )4 HRI 1.1 · 10 7 1.5 · 10 )3 1.4 · 10 )10 Unpublished observations b SOUL 1.9 · 10 6 6.1 · 10 )3 3.2 · 10 )9 [17] P22HBP 1.0 · 10 8 4.4 · 10 )3 4.4 · 10 )11 [17] Sw Mb 7.6 · 10 7 8.4 · 10 )7 1.3 · 10 )14 [18] a Spectral changes observed for both association and dissociation were composed of two phases. b Data for heme-regulated eIF2a kinase (HRI) were taken from the PhD thesis of J. Igarashi (Tohoku University, Sendai, Japan). 03. 0 02.0 0 1 .0 Absorbance 0640440 2 400 4 083 )mn(ht gnele v a W A B 62 .0 42.0 22. 0 Absorbance at 410 nm 00040003000200010 ) s (e m iT Fig. 8. Optical absorption spectral changes accompanying heme dissociation from basic helix–loop–helix (bHLH)-PAS-A and associ- ation to H64Y ⁄ V68F apomyoglobin mutant (A) and the spectral change upon Fe(III)–myoglobin formation as monitored at 410 nm with a mixture of Fe(III) bHLH-PAS-A and H64Y ⁄ V68F apomyoglobin (B). B C 01x08 3 - 06 04 02 Absorbance Absorbance 0 84 06404402400 4 08 3 A 0 1 x 5 5 3 - 05 5 4 04 5 3 Absobance at 421 nm 0 . 2 5. 1 0 . 1 5.00 .0 ) s ( emiT 0 1 x 0 8 3 - 06 04 0 2 04403402401400409 3 Wavelength (nm) Wavelength (nm) 0 7 06 0 5 04 03 02 01 0 k obs (s -1 ) 01 x 0. 2 6 - 5 .1 0.15 . 00 .0 ) M(] A-S AP/HLHb[ Fig. 7. Optical absorption spectral changes accompanying assoc- iation of Fe(II)–CO heme with heme-free basic helix–loop–helix (bHLH)-PAS-A (A) and PAS-A (B) after mixing using a stopped-flow spectrometer. The inset in (A) shows the spectral change at 421 nm, which was composed of only a single phase. The correl- ation between k obs and the concentration of heme-free bHLH- PAS-A is shown in (C). Characterization of bHLH-PAS-A of NPAS2 Y. Mukaiyama et al. 2534 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS ted low spin (Table 2). In addition, the inverse correla- tion between m Fe–CO and m C–O frequencies revealed that the ligand trans to CO in the bHLH-PAS-A domain and PAS-A domain is His. These spectral findings are the same as those reported for the isolated PAS-A domain [22]. In contrast to the effect of CO, the effect of NO on the transcription activity of the NPAS2–BMAL1 heterodimer has not been previously reported. We obtained a typical optical absorption spectrum with a Soret band at 394 nm and resonance Raman spectra corresponding to the five-coordinated NO–Fe(II) heme complex of bHLH-PAS-A (Figs 5 and 6). Some of the heme-sensor proteins, including soluble guanylate cyclase [23], CO-sensing heme-bound transcriptional regulator from Rhodospirillum rubrum (CooA) [24], cystathionine b-synthase [25], heme-regulated inhibitor [26], and cytochrome c¢ [27], form five-coordinate NO– heme complexes, and this modifies their function. Thus, it seems likely that NO binding to the heme affects the DNA-binding properties of NPAS2. The rate constant for heme association (k on ) with the isolated bHLH-PAS-A domain of NPAS2 was of the same order as those for heme association with the heme-regulated kinase inhibitor (unpublished observa- tions) and sperm whale myoglobin [18]. Also, the rate constant for the association of heme with isolated bHLH-PAS-A of NPAS2 was much higher than that for the isolated PAS-A domain. This further supports the idea that the bHLH region assists with the stable binding of heme to the PAS-A domain in the isolated bHLH-PAS-A protein. We also determined the rate constant for the dissoci- ation of heme (k off ) from the isolated holo-bHLH-PAS- A domain. We found that the rate constant was similar to those of other heme proteins (Table 4). On the basis of these association and dissociation rate constants, we estimated the heme dissociation equilibrium constant (K d ). The apparent K d value of heme for the isolated bHLH-PAS-A domain was much higher than that of sperm whale myoglobin, but comparable to that of heme-regulated kinase inhibitor (unpublished observa- ∆ F (Hz) ∆ F (Hz) ∆ F (Hz) ∆ F (Hz) s)(emiTs)(emiT s)(emiT s)(emiT 2SAPN-oloh 2SAP N-oloh 2 S APN-oloh 2SAPN-ol oh 2SAPN-oloh A SB 2SAPN-opa ASB 2SAPN-opa 2SAPN-opa 2SAPN-opa 2SAPN-opa B A 2SA PN-oloh 2 SAPN-o l oh 0 0 01- 005- 0 0 001 0 004 005300030052000 2 0051000 1 2SAP N -ol o h 2SAP N -ol o h 00 5 A S B 2SA P N-oloh C 00 0 1 - 005- 0 00 5 00 01 00 5 20002 0 0 51 000 1 A S B 2SAPN-opa 2SAPN-opa 2SAPN-opa 2 S AP N - o p a 2SAPN-opa D Fig. 9. Quartz crystal microbalance (QCM) analyses for the binding of holo (heme-bound)-basic helix–loop–helix (bHLH)-PAS-A to the E-box sequence (A), apo (heme-free)-bHLH-PAS-A to the E-box sequence (B), holo-bHLH-PAS-A to the mutant E-box sequence (C), and apo-bHLH- PAS-A to the mutant E-box sequence (D). Aliquots (5 lL) of holo-bHLH-PAS-A (2.55 lgÆlL )1 ) or apo-bHLH-PAS-A (1.80 lgÆlL )1 ) were added stepwise to 2 mL of buffer bathing the chip, allowing time for the frequency change to stabilize between each step. Addition of the PAS-A domain lacking the bHLH domain did not change the frequency. The DNA sequence containing the E-box was 5¢-GGGGCGC CACGTGA GAGG-3¢, and that containing the mutant E-box was 5¢-GGGGCGC GACGTCAGAGG-3¢ (E-box regions are underlined). Y. Mukaiyama et al. Characterization of bHLH-PAS-A of NPAS2 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS 2535 tions). Because the heme-regulated kinase inhibitor responds to the heme concentration in cells by switching the kinase reaction on or off, heme must bind to the pro- tein reversibly. Therefore, it is possible that in NPAS-2, heme reversibly binds to the PAS-A domain. Note that the spectral change accompanying both association and dissociation of heme for the isolated PAS-B domain of NPAS2 was composed of two phases (Table 4). The QCM data demonstrated that the isolated bHLH-PAS-A domain binds to the E-box DNA sequence under specific conditions. In contrast, the bHLH-truncated PAS-A domain did not bind to the E-box, and the isolated bHLH-PAS-A domain did not bind to the mutated E-box, indicating that the binding of the isolated bHLH-PAS-A domain to the E-box sequence is specific. In addition, a previous study showed that full-length human NPAS2 alone, without a partner such as BMAL1 or ARNT, is unable to bind to the E-box sequence [13] and that mouse NPAS2 alone does not activate circadian rhythm-associated transcription [7]. Thus, truncation of the C-terminal domain and the binding of heme may affect the DNA- binding properties of the bHLH-PAS-A fragment. We further examined the binding of CO to the Fe(II) bHLH-PAS-A domain (5 lm heme) of NPAS2, but the increase of the Soret absorption upon CO binding was composed of more than two phases (data not shown). Also, the initial phase was not dependent on the CO concentration at high concentrations (200–500 lm). Taking into account only initial CO-dependent (10– 90 lm CO) CO binding, which is a straight line, we estimated that the CO binding rate is 0.13 lmol )1 Æs )1 . This value is slightly smaller than that reported by Dioum et al. (0.37 lmol )1 Æs )1 ) [15]. Resonance Raman spectroscopy showed that the heme coordination state is a mixture of five-coordinate and six-coordinate and that the protein exists in solution in an equilibrium between the monomer, dimer and trimer. These factors may contribute to the complicated kinetic behavior. Further studies are required to address this issue. Based on resonance Raman spectral studies of His119fiAla, His138fiAla, His171fiAla and Cys170fiAla mutants of the isolated PAS-A domain, it has been suggested that His119 and Cys170 are the axial ligands for the Fe(III) complex, whereas His119 and His171 are the axial ligands for the Fe(II) complex [22]. Note that some sensor proteins, including PAS proteins, are known to have substantial flexibility [28–31]. In summary, the present study suggests that the bHLH domain plays an important role in assisting and stabilizing heme binding to the PAS-A domain in the isolated bHLH-PAS-A domain of NPAS2. The QCM data indicated that the isolated bHLH-PAS-A domain specifically binds to the E-box DNA sequence. Further studies using both NPAS2 and BMAL1 are needed to elucidate the mechanism of DNA binding by NPAS2. Experimental procedures Materials Oligonucleotides (18 bp; E-box, random) and 5¢-biotinylated oligonucleotides (18 bp) were synthesized by the Nippon Gene Institute (Sendai, Japan). Restriction enzymes and modification enzymes were purchased from Takara Bio (Otsu, Japan), Toyobo (Osaka, Japan), New England Bio- labs (Beverly, MA), and Nippon Roche (Tokyo, Japan). Other chemicals weer of the highest grade available and were purchased from Wako Pure Chemicals (Osaka, Japan). Plasmid construction To construct the plasmid coding for the N-terminal domain, the cDNA of NPAS2 was generated by RT-PCR using RNA isolated from mouse forebrain [21,22]. The sequences of the PCR products were confirmed by deter- mination of the nucleotide sequence by Sanger’s method using a DSQ-2000 L automatic sequencer (Shimadzu Co., Kyoto, Japan). The 6xHis-tagged isolated bHLH-PAS-A domain (amino acids 1–240) and isolated PAS-A domain (amino acids 78–240) of NPAS2 were created by subcloning into the NdeI and SalI sites of the of pET-28a(+) expres- sion vector (Novagen, Madison, WI). E. coli strain BL21 (DE3) codon plus RIL was transformed with the expression vectors pET28a-PAS-A or pET28a-bHLH-PAS-A. Protein expression and purification E. coli BL21 (DE3) cells harboring pET28a-PAS-A and pET28a-bHLH-PAS-A were incubated in Terrific Broth medium containing 50 lgÆmL )1 kanamycin and 50 lgÆmL )1 chloramphenicol at 37 ° C until the absorbance at 600 nm reached 0.6. Expression of the isolated bHLH- PAS-A and PAS-A domains of NPAS2 was then induced by treatment for 20 h at 20 °C with 0.05 mm isopropyl-1- b-thiogalactoside and mild shaking. The E. coli cells were then suspended in buffer A (50 mm sodium phosphate (pH 7.8), 50 mm NaCl, 10% glycerol, 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2 lgÆmL )1 aprotinin, 2 lgÆmL )1 leupeptin, 2 lgÆmL )1 pepstatin A, 2 mm 2-mercaptoethanol) and lysed by pulse sonication (three times for 2 min each with 2 min inter- vals) on ice using an Ultrasonic Disruptor UD-201 (Tomy Seiko, Tokyo, Japan). Hemin (100 lm final concentration) dissolved in 0.01 m NaOH was added to this lysate, and the mixture was allowed to equilibrate on ice for 30 min. After centrifugation at 35 000 g for 30 min, the superna- Characterization of bHLH-PAS-A of NPAS2 Y. Mukaiyama et al. 2536 FEBS Journal 273 (2006) 2528–2539 ª 2006 The Authors Journal compilation ª 2006 FEBS tant was adjusted to 20% saturated ammonium sulfate and incubated for 30 min on ice. After centrifugation at 35 000 g, the supernatant was adjusted to 70% saturated ammonium sulfate. Precipitates from the 70% saturated solution were collected by centrifugation and dissolved in buffer A. The excess ammonium sulfate was removed and the buffer was exchanged by applying the solution to Sephadex G-25 (100 mL) that had been pre-equilibrated with buffer B (50 mm sodium phosphate (pH 7.8), 50 mm NaCl, 10% glycerol, 2 mm dithiothreitol). The resulting solution was applied to an Ni-NTA column (Qiagen, Hil- den, Germany) pre-equilibrated with buffer C (50 mm sodium phosphate (pH 7.8), 50 mm NaCl, 2 mm 2-merca- ptoethanol). The column was washed stepwise with buffer C containing 0, 20 and 50 mm imidazole. The protein eluted at 100 and 150 mm imidazole was collected and concentrated. To remove the excess imidazole and to exchange the buffer with buffer D (100 mm Tris ⁄ HCl (pH 8.0) at 25 °C with 10% glycerol), the solution was applied to a HiTrap desalting column (Amersham Bio- sciences, Uppsala, Sweden). Removal of the His tag Purified His-tagged protein in 100 mm Tris ⁄ HCl buffer (pH 8.0) was equilibrated for 1 h on ice with 1–2 equivalents of hemin dissolved in 0.01 m NaOH. Excess hemin was then removed using a Bio-Gel P-6 column (Bio-Rad, Hercules, CA) with the same buffer. Thrombin protease (10 units ⁄ mg) was added to the heme-saturated His-tagged protein in 50 mm Tris ⁄ HCl (pH 8.0), 150 mm NaCl and 2.5 mm CaCl 2 , and incubated for 4 h at 16 °C. Next, the solution was applied to an Ni-NTA column pre-equilibrated with buffer D, and proteins were eluted with the same buffer. The purified protein was rapidly frozen in liquid nitrogen and stored at ) 80 °C. The concentration and purity were checked by Coomassie Brilliant Blue R250 dye binding. Gel filtration To determine the molecular mass, gel filtration was carried out using an AKTA liquid chromatography apparatus equipped with a Superdex75 HR 10 ⁄ 30 column (Amersham Biosciences). The buffer used for gel filtration was 100 mm Tris ⁄ HCl (pH 8.0) containing 0.5 mm EDTA. The molecu- lar mass was estimated by correlation between the molecu- lar mass and the elution volumes for the following standard proteins: albumin (67 kDa), ovalbumin (43 kDa), chymo- trypsinogen A (25 kDa), and ribonuclease A (13.7 kDa). Optical absorption spectra Spectral experiments were carried out under aerobic condi- tions using a Shimadzu UV-2500 spectrophotometer main- tained at 25 °C with a temperature controller. Anaerobic spectral experiments were conducted using a Shimadzu UV- 160 A spectrophotometer at 16 °C. When the heme was reduced by sodium dithionite, the sample solution was sat- urated with argon gas. Resonance Raman spectra The bHLH-PAS-A domain of NPAS2 (35 lm in 100 mm Tris ⁄ HCl (pH 8.0) and 10% glycerol) was placed in an air- tight spinning cell with a rubber septum and reduced by the addition of sodium dithionite (10 mm final concentration). 12 C 16 O, 13 C 18 O 14 N 16 Oor 15 N 16 O (Cambridge Isotope Laboratories, Andover, MA) gas was introduced into the Raman cell with an airtight syringe. Raman scattering was excited at 413.1 nm with a Kr ion laser (BeamLok 2060, Spectra-Physics, Mountain View, CA). The excitation light was focused into the cell at a laser power of 5 mW for the Fe(III) and Fe(II) complexes. For the CO–Fe(II) complexes, to avoid photolysis, the laser power was 0.1–0.2 mW. Raman spectra were detected with a Spec-10 N 2 -cooled CCD camera (Princeton Instruments, Trenton, NJ) attached to a SPEX750M single polychromator (Jobin Yvon, Longjum- eau, France). Raman shifts were calibrated with indene, acet- one, CCl 4 and an aqueous solution of ferrocyanide. Heme-binding kinetics Heme association experiments were carried out using an RSP-1000 stopped-flow apparatus (Unisoku, Osaka, Japan). The buffer contained 50 mm Tris ⁄ HCl (pH 8.0) and 50 mm NaCl and was purged with nitrogen gas for 30 min. The buffer was then saturated with CO gas. CO– Fe(II) hemin was mixed with the apoprotein at 25 °C. Reactions between the protein and CO–Fe(II) hemin were monitored at 421 nm [18]. Heme dissociation experiments were conducted using a Shimadzu UV-2500 spectrophotometer maintained at 25 °C with a temperature controller. Dissociation of heme from the Fe(III) bHLH-PAS-A domain of NPAS2 was examined as Fe(III) myoglobin formation by monitoring the increase in absorbance at 410 nm upon mixture of the Fe(III) bHLH-PAS-A domain of NPAS2 (3 lm) with a 10-fold excess of H64Y ⁄ V68F apomyoglobin (30 lm) in potassium phosphate buffer (pH 7.0) containing 0.6 m sucrose at 25 °C [17,18]. CO-binding kinetics To measure the CO association rates, the bHLH-PASA domain of NPAS2 (10 lm) was reduced with sodium dithionite in 50 mm Tris ⁄ HCl (pH 8.0) buffer. This solu- tion was then rapidly mixed with controlled CO-saturated buffer (c. 1mm CO) using a stopped-flow spectrophotom- Y. Mukaiyama et al. 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Taoka S & Banerjee R (20 01) Characterization of NO binding to human cystathionine b-synthase: possible implications of the effects of CO and NO binding to the human enzyme J Inorg Biochem 87, 24 5 25 1 Igarashi J, Sato A, Kitagawa T, Yoshimura T, Yamauchi S, Sagami I & Shimizu T (20 04) Activation of hemeregulated eukaryotic initiation factor 2a kinase by nitric oxide is induced by the formation of a five-coordinate . Spectroscopic and DNA-binding characterization of the isolated heme-bound basic helix–loop–helix -PAS- A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with. Fe(II) and Fe(II)–CO complexes of the bHLH -PAS- A domain of NPAS2 are shown in Y. Mukaiyama et al. Characterization of bHLH -PAS- A of NPAS2 FEBS Journal 27 3 (20 06) 25 28 25 39 ª 20 06 The Authors Journal. containing the N-terminal basic helix–loop–helix (bHLH) of the first PAS (PAS- A) domain of NPAS2 with those of the PAS- A domain alone. We found that the heme-bound bHLH -PAS- A domain mainly exists as a