Biochemical and biophysical characterization of the trimerization domain from the heat shock transcription factor.

Previously, we had characterized a 91 amino acid fragment of the heat shock transcription factor from the yeast Kluyveromyces lactis and had shown it to be highly alpha-helical and sufficient for formation of homotrimers [Peteranderl, R., and Nelson, H. C. M. (1992) Biochemistry 31, 12272-12276]. Based on those data, as well as the presence of hydrophobic heptad repeats, we postulated that the trimerization domain contains a three-stranded coiled-coil and that it might resemble the trimerization domain found in influenza hemagglutinin. Here, we further characterize the trimerization domain and show that the minimal domain needs 71 residues to remain trimeric and highly alpha-helical. 19F NMR spectroscopy suggests that the structure contains three parallel strands that are in register along the long axis of the coiled-coil. Electron paramagnetic resonance spectroscopy studies show that the C-termini of the subunits are in close proximity; this is in contrast to the topology of the hemaglutinin trimerization domain where the C-termini form buttressing helices. Analytical ultracentrifugation also confirms that the structure is elongated and unlikely to have buttressing helices. Additional experiments suggest that the trimerization domain has at least two subdomains. The first subdomain has the potential to form trimers independently, though not as stably as the complete domain. The second subdomain is quite helical, forms large oligomers, and appears to provide stability to the complete domain. Our current model for the heat shock transcription factor trimerization domain is a highly elongated coiled-coil structure, with a potential break in the coiled-coil region located between the two subdomains.