Abstract
The yeast Cyc8p-Tup1p protein complex is a general transcriptional corepressor of genes involved in many different physiological processes. Herein, we present the crystal structure of the Tup1p N-terminal domain (residues 1-92), essential for Tup1p self-assembly and interaction with Cyc8p. This domain tetramerizes to form a novel antiparallel four-helix bundle. Coiled coil interactions near the helical ends hold each dimer together, whereas interdimeric association involves only two sets of two residues located toward the chain centers. A mutagenesis study confirmed that the nonpolar residues responsible for the association of the protomers as dimers are also required for transcriptional repression. An additional structural study demonstrated that the domain containing an Leu(62) → Arg mutation that had been shown not to bind Cyc8p exhibits an altered structure, distinct from the wild type. This altered structure explains why the mutant cannot bind Cyc8p. The data presented herein highlight the importance of the architecture of the Tup1p N-terminal domain for self-association.
Highlights
The yeast Cyc8p-Tup1p protein complex is a general transcriptional corepressor
We solved the Tup1p NTD, which is required for tetramerization of Tup1p and association with Cyc8p in the Cyc8pTup1p corepressor complex
NTD tetramerizes as a dimer of dimers with a novel arrangement, which is stabilized by nonpolar knob residues in coiled coils formed by intra- and interdimers
Summary
The yeast Cyc8p-Tup1p protein complex is a general transcriptional corepressor. Results: The crystal structures of Tup1p N-terminal domain and its mutant were determined. The N-terminal 72 residues in Tup1p interact with Cyc8p and are required for self-tetramerization, but this region cannot independently repress transcription [29]. The C-terminal region of Tup1p (residues 333–706) directly interacts with DNA-binding repressor proteins, e.g. Mat␣2p [32]. This domain contains WD40 motifs [33, 34] that are defined by highly conserved tryptophans and aspartates. For the study reported we determined the crystal structure of the Tup1p N-terminal domain (NTD, residues 1–92), which is required for Tup1p tetramerization and association with Cyc8p. Nonpolar residues in the coiled coil regions are required for self-association and transcriptional repression of genes targeted by Tup1p. A mutant NTD Tup1p-L62R forms a dimer of dimers, it contains interactions between protomers that are markedly unlike those of the wild-type NTD and that may be related to the ability of Cyc8p-Tup1p complexes to oligomerize when associated with chromatin targets
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