The Tup1-Cyc8 complex acts as a corepressor for members of multiple families of transcription factors. Although its physiological functions have been extensively studied, it remained unclear how the corepressor assembles, and how the assembly involves in the molecular functions. Here, we report the crystal structure of N-terminal domain of Tup1, which is essential for its self-assembly and interaction with Cyc8. The N-terminal domain of Tup1 tetramerizes to form a novel antiparallel four-helix bundle, and is organized as a dimer of dimers. Coiled-coil interactions stabilize each dimer together, and mutagenesis study confirmed that the hydrophobic residues responsible for the association of the protomers as dimers are required for transcriptional repression. We confirmed the functional and structural importance of the hydrophobic residues by further X-ray crystallography.
