The Substrate Recognition Domains of the N-end Rule Pathway

Abstract

The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with K d of ϳ3.4 M. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ϳ80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway.

Quorum sensing controls hyphal initiation in Candida albicans through Ubr1-mediated protein degradation

“…The third binding site recognizes an internal (non-N-degron) degradation signal in Cup9, denoted i (12,13,41). The i site is autoinhibited by the C-terminal domain Ubr1.…”

“…5A). Cup9 is likely a direct target of the Ubr1 E3 ubiquitin ligase, because the N-terminal residue of Cup9 is Lys, an N-end rule pathway degron for the type 1 binding site of Ubr1 (12,13,30,31). The stability of Cup9 in the ubr1 mutant in the absence of farnesol was reminiscent of its stability in WT cells in the presence of farnesol (Fig.…”

“…The first E3 identified and cloned was Ubr1, which recognizes proteins' N-terminal degradation signals (the N-end rule) (11). The RING-type E3 ligase contains types 1 and 2 substrate binding sites that recognize the unmodified basic (Arg, Lys, or His) and bulky hydrophobic (Leu, Phe, Tyr, Trp, or Ile) N-terminal residues, respectively (12,13). In addition, Ubr1 also contains binding sites that recognize internal degrons of the Cup9 transcriptional repressor (14).…”

Functional Roles of the E3 Ubiquitin Ligase UBR5 in Cancer

“…1; refs. 17,22). The human UBR5 gene has 59 exons, encoding an approximately 10 kb mRNA and >300 kDa protein with widespread expression in various cell types.…”

UBR box N-recognin-4 (UBR4), an N-recognin of the N-end rule pathway, and its role in yolk sac vascular development and autophagy

“…S1). These N-recognins share the UBR box, a ∼70-residue zinc finger domain that acts as a substrate recognition domain (14). Whereas the canonical N-recognins UBR1 and UBR2 have been characterized in various mammalian processes, functions and mechanisms of two noncanonical N-recognins, UBR4 and UBR5, in the N-end rule pathway remain poorly understood.…”