title: Bivalent inhibitor of the N-end rule pathway.
abstract: The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Ubr1p, the recognition (E3) component of the Saccharomyces cerevisiae N-end rule pathway, contains at least two substrate-binding sites. The type 1 site is specific for N-terminal basic residues Arg, Lys, and His. The type 2 site is specific for N-terminal bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile. Previous work has shown that dipeptides bearing either type 1 or type 2 N-terminal residues act as weak but specific inhibitors of the N-end rule pathway. We took advantage of the two-site architecture of Ubr1p to explore the feasibility of bivalent N-end rule inhibitors, whose expected higher efficacy would result from higher affinity of the cooperative (bivalent) binding to Ubr1p. The inhibitor comprised mixed tetramers of beta-galactosidase that bore both N-terminal Arg (type 1 residue) and N-terminal Leu (type 2 residue) but that were resistant to proteolysis in vivo. Expression of these constructs in S. cerevisiae inhibited the N-end rule pathway much more strongly than the expression of otherwise identical beta-galactosidase tetramers whose N-terminal residues were exclusively Arg or exclusively Leu. In addition to demonstrating spatial proximity between the type 1 and type 2 substrate-binding sites of Ubr1p, these results provide a route to high affinity inhibitors of the N-end rule pathway.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Y T
authors.initials: YT
authors.lastname: Kwon
authors.forename: F
authors.initials: F
authors.lastname: Lévy
authors.forename: A
authors.initials: A
authors.lastname: Varshavsky
mesh_terms: D001665:Binding Sites; D004151:Dipeptides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D008024:Ligands; D008025:Ligases / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014451:Ubiquinone / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D004151:Dipeptides; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008024:Ligands; D029701:Saccharomyces cerevisiae Proteins; D014451:Ubiquinone; C066066:UBR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D001616:beta-Galactosidase; D008025:Ligases