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, Takeshi Uchida, Emiko Sato, Ai Sasaki, Yuko Sato, Jotaro Igarashi, Hirofumi Kurokawa, Ikuko Sagami, Teizo Kitagawa, Toru Shimizu

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

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 × 107 mol-1·s-1) was more than two orders of magnitude higher than for association with apo-PAS-A (< 105 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.

Original languageEnglish
Pages (from-to)2528-2539
Number of pages12
JournalFEBS Journal
Volume273
Issue number11
DOIs
Publication statusPublished - 2006 Jun 1

Keywords

  • Circadian rhythms
  • DNA binding
  • Heme-sensor protein
  • PAS domain
  • Resonance Raman spectroscopy

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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