Student Theses and Dissertations

Date of Award

1995

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Stephen Burley

Keywords

HLH transcription factors, DNA binding, Max protein, USF structure, protein dimerization, X-ray crystallography

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 2.9Å 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.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

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