The basic helix-loop-helix domain of the aryl hydrocarbon receptor nuclear transporter (ARNT) can oligomerize and bind E-box DNA specifically.
The aryl hydrocarbon receptor nuclear transporter (ARNT) is a basic helix-loop-helix (bHLH) protein that contains a Per-Arnt-Sim (PAS) domain. ARNT heterodimerizes in vivo with other bHLH PAS proteins to regulate a number of cellular activities, but a physiological role for ARNT homodimers has not yet been established. Moreover, no rigorous studies have been done to characterize the biochemical properties of the bHLH domain of ARNT that would address this issue. To begin this characterization, we chemically synthesized a 56-residue peptide encompassing the bHLH domain of ARNT (residues 90-145). In the absence of DNA, the ARNT-bHLH peptide can form homodimers in lower ionic strength, as evidenced by dynamic light scattering analysis, and can bind E-box DNA (CACGTG) with high specificity and affinity, as determined by fluorescence anisotropy. Dimers and tetramers of ARNT-bHLH are observed bound to DNA in equilibrium sedimentation and dynamic light scattering experiments. The homodimeric peptide also undergoes a coil-to-helix transition upon E-box DNA binding. Peptide oligomerization and DNA affinity are strongly influenced by ionic strength. These biochemical and biophysical studies on the ARNT-bHLH reveal its inherent ability to form homodimers at concentrations supporting a physiological function and underscore the significant biochemical differences among the bHLH superfamily.
Huffman
J L
JL
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Mokashi
A
A
Bächinger
H P
HP
Brennan
R G
RG
D000595:Amino Acid Sequence; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D001704:Biopolymers; D002942:Circular Dichroism; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D011485:Protein Binding; D018336:Receptors, Aryl Hydrocarbon; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D001704:Biopolymers; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator