The basic helix-loop-helix domain of the aryl hydrocarbon receptor nucleartransporter (ARNT) can oligomerize and bind E-box DNA specifically

The aryl hydrocarbon receptor nuclear transporter (ARNT) is a basic helix-l oop-helix (bHLH) protein that contains a Per-Arnt-Sim (PAS) domain. ARNT he terodimerizes in vivo with other bHLH PAS proteins to regulate a number of cellular activities, but a physiological role for ARNT homodimers has not y et been established. Moreover, no rigorous studies have been done to charac terize the biochemical properties of the bHLH domain of ARNT that would add ress 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 expe riments. The homodimeric peptide also undergoes a coil-to-helix transition upon E-box DNA binding. Peptide oligomerization and DNA affinity are strong ly influenced by ionic strength. These biochemical and biophysical studies on the ARNT-bHLH reveal its inherent ability to form homodimers at concentr ations supporting a physiological function and underscore the significant b iochemical differences among the bHLH superfamily.