Allosteric Activation of the Ubiquitin ligase UBR 1 by Short Peptides: Molecular Mechanisms and Physiological Functions

Abstract

The N-end rule relates the in vivo half life of a protein to the identity of its N-terminal residue. UBR1, the E3 of the N-end rule pathway in Sacchnromzyces cerevisiae, targets proteins that bear destabilizing N-terminal residues for Ub-dependent, processive degradation. UBR1 binds protein substrates or dipetides through two distinct sites: the type 1 site, specific for basic residues, and the type 2 site, specific for bulky hydrophobic residues. UBR1 also recognizes an internal degradation signal of the 35 kDa homeodomain protein CUP9, a transcriptional repressor of the di- and tripeptide transporter PTR2. Here I report that the internal degradation signal of CUP9 is recognized by UBR1 through its third, distinct substrate-binding site. Occupation of the type 1 or type 2 sites of UBR1 by dipeptides allosterically stimulates the UBR1-dependent multi-ubiquitylation of CUP9 in an in vitro system, which consists of purified components of the yeast N-end rule pathway. UBR1 is the first E3 shown to be allosterically regulated by small compounds. This regulation underlies, in vivo, the accelerated UBR1-dependent degradation of CUP9 in the presence of dipeptides with destabilizing N-terminal residues. The result is a positive feedback circuit that controls the peptide import in S. cerevisiae. Specifically, the imported dipeptides bind to UBR1 and accelerate the UBR1-dependent degradation of CUP9, thereby derepressing the transcription of PTR2 and increasing the cell's capacity to import peptides. I also describe a new, autoinhibition-based molecular mechanism underlying the activation of UBR1 by dipeptides. UBR1 is an autoinhibited protein, in that the binding of dipeptides to the type 1 and type 2 sites of UBR1 enhances the dissociation of the C-terminal autoinhibitory domain of UBR1 from its substrate-binding N-terminal region. Moreover, this dissociation, which allows the interaction between UBRl and CUP9, is strongly increased only if both type 1 and type 2 sites of UBRl are occupied by dipeptides. An autoinhibitory mechanism discovered in the S. cerevisiae UBRl is likely to recur in metazoan homologs of UBRl, and may also be involved in controlling the activity of other Db-dependent pathways.