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Structural and functional studies of the N-Terminal domain of the GABPα Kang, Hyun-Seo


GA-binding protein (GABP) is a heterotetrameric αβ₂α transcription factor composed of two structurally dissimilar subunits, GABPα and GABPβ. The modular DNA-binding subunit, GABPα, was known previously to consist of a DNA-binding ETS domain and a PNT domain, which presumably mediates protein-protein interactions with other components of the signalling or transcription machinery. The transactivation subunit, GABPβ consists of three domains, namely the ankyrin repeats, which bind GABPα, a leucine zipper to form β₂ dimers, and a transactivation domain. GABP is known to regulate gene expression involved in many different cellular functions, such as cell cycle control, apoptosis, and viral pathogen expression. An investigation of the N-terminal region of GABPα revealed the existence of third structured domain. Using partial proteolysis and NMR spectroscopy, this new domain was localized to residues 35-121, and was found to be flanked by unstructured residues and independent of the adjacent PNT domain. The gene encoding this domain was cloned and expressed, yielding a new truncation fragment, GABPα³⁵⁻¹²¹. As a step towards discussing its function, the structure of GABPα³⁵⁻¹²¹ was determined by NMR spectroscopy. The protein is composed of a 5-stranded β-sheet crossed by a distorted helix. Although globally resembling ubiquitin, GABPα³⁵⁻¹²¹ adopts a novel fold as evident by the arrangement of its secondary structure elements. An analysis of GABPα³⁵⁻¹²¹ for features indicative of a macromolecular binding interface revealed only a region of negative charge. Therefore, the structure of GABPα³⁵⁻¹²¹ provides few clues to its function. To determine the function of GABPα³⁵⁻¹²¹, these strategies will be pursued based on the hypothesis that this domain is a protein-protein interaction module. First, potential interactions with other domains of GABPα and GABPβ will be examined using approaches such as native gel electrophoresis, chemical crosslinking, and NMR spectroscopy. Second, interactions of GABPα³⁵⁻¹²¹ with reported partners of GABP, such as ATF, CBP/p300, or Spl, will be tested. Finally, unbiased affinity methods will be used to identify proteins from cellular extracts that bind specifically to GABPα³⁵⁻¹²¹.

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