Student Theses and Dissertations

Date of Award

1995

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

The Helix-Loop-Helix (HLH) family of eukaryotic transcription factors comprises a large number of proteins which play key roles in homeostasis, the regulation cell proliferation, and differentiation. These proteins share a phylogenetically conserved bipartite b/HLH domain responsible for specific DNA binding and dimerization. The HLH region dictates dimerization affinity and specificity while the basic region (b) is primarily responsible for sequence-specific DNA binding. In some family members, such as the Myc oncoproteins, the HLH motif is followed by a heptad repeat of hydrophobic amino acids, or "leucine zipper" (Z). My X-ray crystallographic structure determination at 2.9A resolution of a dimer of the b/HLH/Z domain of the mammalian oncoprotein Max bound to its target DNA revealed that this symmetric homodimer folds into a novel parallel left-handed four-helix bundle, which is globular and stabilized by a well-defined hydrophobic core. Two pairs of α-helices protrude in opposite directions from the bundle. One, the basic regions, enters the major groove of the target B-form DNA and makes numerous contacts with the bases and phosphodiester backbone. The other, the leucine zipper, forms a left-handed coiled-coil, extending the hydrophobic interface of the homodimer. I also determined the cocrystal structure of a truncated b/HLH homodimer of the human transcription factor USF bound to DNA. As expected from the sequence conservation, this protein adopts the same three-dimensional structure as Max b/HLH. Circular dichroism spectroscopic investigation of DNA binding by Max and USF demonstrated, in concert with their cocrystal structures, that these proteins undergo a dramatic folding transition upon specific, high-affinity DNA binding. More than forty residues per dimer become α-helical upon association. I also demonstrated by hydrodynamic as well as biochemical methods that these proteins can form bivalent tetramers at physiologically meaningful concentrations. This suggests that they may play a role in DNA looping, thought to be important in the transcriptional regulation of eukaryotic genes.

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