ABSTRACT
While the signaling properties of ubiquitin depend on the topology of polyubiquitin chains, little is known concerning the molecular basis of specificity in chain assembly and recognition. UEV/Ubc complexes have been implicated in the assembly of Lys63-linked polyubiquitin chains that act as a novel signal in postreplicative DNA repair and I kappa B alpha kinase activation. The crystal structure of the Mms2/Ubc13 heterodimer shows the active site of Ubc13 at the intersection of two channels that are potential binding sites for the two substrate ubiquitins. Mutations that destabilize the heterodimer interface confer a marked UV sensitivity, providing direct evidence that the intact heterodimer is necessary for DNA repair. Selective mutations in the channels suggest a molecular model for specificity in the assembly of Lys63-linked polyubiquitin signals.
Subject(s)
Biopolymers/metabolism , Fungal Proteins/chemistry , Ligases/chemistry , Protein Conformation , Saccharomyces cerevisiae Proteins , Ubiquitins/metabolism , Amino Acid Sequence , Binding Sites/genetics , Binding Sites/physiology , Biopolymers/chemistry , Biopolymers/genetics , Crystallography, X-Ray , Dimerization , Fungal Proteins/genetics , Fungal Proteins/metabolism , Ligases/genetics , Ligases/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Polyubiquitin , Protein Binding , Sequence Alignment , Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligases , Ubiquitins/chemistry , Ubiquitins/geneticsSubject(s)
Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Saccharomyces cerevisiae/enzymology , Catalytic Domain , Crystallography, X-Ray , Cysteine Endopeptidases/genetics , Models, Molecular , Multienzyme Complexes/genetics , Mutation/genetics , Proteasome Endopeptidase Complex , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Subunits , Saccharomyces cerevisiae/genetics , Structure-Activity RelationshipABSTRACT
The yeast Tup1 and Ssn6 proteins form a transcriptional repression complex that represses transcription of a broad array of genes. It has been shown that the N-terminal domain of the Tup1 protein interacts with a region of the Ssn6 protein that consists of 10 tandem copies of a tetratricopeptide motif. In this work, we use a surface plasmon resonance assay to measure the affinity of the N-terminal domain of Tup1 for a minimal 3-TPR domain of Saccharomyces cerevisiae Ssn6 that is sufficient for binding to Tup1. This domain of Ssn6 binds with comparable affinity to S. cerevisiae and Candida albicans Tup1, but with 100-fold lower affinity to Tup1 protein containing a point mutation that gives rise to a defect in repression in vivo. Results from studies using analytical ultracentrifugation, CD spectroscopy, limited proteolysis, and (1)H NMR show that this domain of Tup1 is primarily alpha-helical and forms a stable tetramer that is highly nonglobular in shape. X-ray diffraction recorded from poorly ordered crystals of the Tup1 tetramerization domain contains fiber diffraction typical of a coiled coil. Our results are used to propose a model for the structure of the N-terminal domain of Tup1 and its interaction with the Ssn6 protein.
