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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
Author(s) -
Mukaiyama Yuji,
Uchida Takeshi,
Sato Emiko,
Sasaki Ai,
Sato Yuko,
Igarashi Jotaro,
Kurokawa Hirofumi,
Sagami Ikuko,
Kitagawa Teizo,
Shimizu Toru
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05259.x
Subject(s) - pas domain , heme , chemistry , dimer , biophysics , crystallography , biochemistry , transcription factor , biology , gene , organic chemistry , enzyme
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 wavelength 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 ·s −1 ) was more than two orders of magnitude higher than for association with apo‐PAS‐A (< 10 5 mol −1 ·s −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 specifically 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.