How the ends signal the end: Regulation by E3 ubiquitin ligases recognizing protein termini

Abstract

Specificity of eukaryotic protein degradation is determined by E3 ubiquitin ligases and their selective binding to protein motifs, termed "degrons," in substrates for ubiquitin-mediated proteolysis. From the discovery of the first substrate degron and the corresponding E3 to a flurry of recent studies enabled by modern systems and structural methods, it is clear that many regulatory pathways depend on E3s recognizing protein termini. Here, we review the structural basis for recognition of protein termini by E3s and how this recognition underlies biological regulation. Diverse E3s evolved to harness a substrate's N and/or C terminus (and often adjacent residues as well) in a sequence-specific manner. Regulation is achieved through selective activation of E3s and also through generation of degrons at ribosomes or by posttranslational means. Collectively, many E3 interactions with protein N and C termini enable intricate control of protein quality and responses to cellular signals.

Keywords: C-degron; E3 ligase; GID complex; N-degron; N-end rule; UBR; cullin-RING ligase; protein quality control; ubiquitin.

Figure 1

Diversity of E3 ligases targeting terminal degrons (A) Single-chain UBR-family E3 ligases. Cartoons of RING E3 UBR1 (top) and HECT E3 UBR5 (bottom), with catalytic domains in blue and substrate-binding domains, UBR-box-1 and UBR-box-2 (aka N-domain), in pink and violet. (B) Multi-subunit E3 ligases. Cartoons of GID (top) and CRL (bottom) RING E3s, with their catalytic subunits in blue, and interchangeable substrate receptors having indicated protein-protein interaction domain folds in red, slate, and purple.

Figure 2

N-degron recognition by UBR-family substrate-binding domains (A) UBR-box-1 from yeast Ubr1 bound to Arg/N-degron of SCC1 (PDB ID:3NIN). Substrate-binding cleft is highlighted in dotted lines. Specificity of Arg/N-degron binding is established by an acidic pocket and network of waters binding the N terminus and Arg side chain and a hydrophobic pocket binding the subsequent residue. (B) Overlay of UBR-box-2 from yeast Ubr1 (slate, PDB ID:7MEX) with N-degron peptide-bound bacterial ClpS (navy, PDB ID: 3DNJ). A hydrophobic pocket binds the hydrophobic N-terminal side chain. The N terminus and backbone are secured by hydrogen bonds. (C) Cryo-EM structure representing ubiquitin transfer to an Arg/N-degron substrate by yeast Ubr1 and E2 (PDB ID: 7MEX). UBR-box-1 and UBR-box-2 are pink and slate, respectively. Active site (chemically stable mimic of thioester-linkage between E2 and ubiquitin) is marked with a black star.

Figure 3

Pliable funnel-shaped β-barrel folds recognize diverse terminal degrons (A) C-degron-binding pocket (dotted lines) in FBXO31 CRL1 substrate receptor (PDB:5VZU). FBXO31 lysine at the base of funnel entrance to calycin-superfamily-type β-barrel anchors C terminus of helical degron from cyclin D1. (B) GID substrate receptors, human Gid4 (red, PDB ID:6CDC) and yeast Gid10 (pink, PDB ID:7QQY), embracing peptide N termini in deep pockets (shown in dotted lines) anchored by contacts with conserved glutamate and tyrosine at the base of substrate-binding tunnels. (C) Pliable loops of calycin-superfamily-type β-barrels from GID substrate receptors confer plasticity allowing recognition of diverse N-terminal sequences. Overlay of human Gid4 structures bound to indicated peptides initiating with Pro and Phe (PDB ID:6CDC, 7Q50, respectively). Loops at the entrance to N-degron-binding pocket are numbered. (D) Hydrogen bonds and hydrophobic interactions enabling diverse N-terminal sequences to bind human Gid4.

Figure 4

β-propeller and helical-repeat domains binding terminal degrons (A) AlphaFold-predicted WD40-type β-propeller of yeast GID substrate receptor Gid11. (B) Kelch-type β-propeller of CRL2 substrate receptor KLHDC2 bound to diGly/C-degron of SelK (PDB ID: 6DO3, degron-binding pocket in dotted lines). KLHDC2 contacts the the diGly/C-degron via direct and water-mediated hydrogen bonds. (C) Armadillo repeats of CRL2 substrate receptor ZYG11B bound to Gly/N-degron (PDB ID: 7EP2). Gly/N-degron-binding pocket (dotted lines) is a narrow cavity forming hydrogen bonds with N-terminal residues. (D) Ankyrin repeats of CRL2 substrate receptor FEM1B bound to Arg/C-degron of CDK5R1 (PDB ID:7CNG). Arg/C-degron degron-binding pocket (dotted lines) recognizes extended peptide structure: C-terminal Arg by hydrophobics flanking aliphatic portion of its side chain and acidic pocket capturing its guanidino group, and upstream hydrophobic residues by a hydrophobic pocket. (E) The same groove in FEM1B binds Arg/C-degrons and Cys/His-rich internal degron of FNIP1 internal degron (PDB IDs: 7CNG, 7ROY). Cys/His-rich surfaces of FEM1B and Cys/His-rich internal degron form intermolecular zinc-binding site. Zinc ions (gray spheres) function as molecular glue.

Figure 5

Regulation of terminal degron E3 ligases (A) Regulated incorporation of substrate receptors activates GID E3 ligases: Gid4 that is induced during glucose recovery targets Pro/N-degron gluconeogenic enzymes Fbp1, Mdh2, Icl1, and Pck1; Gid10 that is induced during heat and osmotic shock targets Pro/N-degron substrate Art2; Gid11 that is induced in distinct environmental conditions is thought to target Thr/N-degron substrates Phm8, Gpm3, Yor283w, Cpa1, and Blm10. (B) Supramolecular assembly of yeast GID E3 allows avid binding between two central-facing Gid4 substrate receptors and Pro/N-degrons from two protomers of oligomeric substrate Fbp1, much like how an organometallic chelate binds a central ligand. The so-called Chelator-GID^SR4 comprises the core consisting of the substrate receptor, and scaffolding and catalytic modules, joined by a supramolecular assembly module (SA). The Chelator assembly targets ubiquitin ligation to functionally relevant sites depending on Fbp1 quaternary structure. (C) Pseudosubstrate regulation of substrate targeting by yeast E3 Ubr1. Occupancy of UBR-box-1 and UBR-box-2 sites by dipeptides mimicking N-degrons, with N-terminal Arg (RX) and hydrophobic residues (ΦX), promotes binding of Cup9 internal degron to a distinct substrate-binding site. UBR-box-1-binding to N-degron-like terminus of Roq1 generated by stress-induced Ynm3-mediated cleavage induces Ubr1 targeting of stress-related substrates.

Figure 6

Regulation generating terminal degrons on substrates (A) Degron generation by proteolysis: exoproteases trim protein termini to generate a new degron at one protein end, while endoproteases cleave internally to potentially simultaneously generate both C- and N-degrons. (B) N-terminal acetylation of Met (M), Ala (A), Ser (S), Thr (T), and Cys (C) generates Ac/N-degrons. (C) Degron generation by N-terminal amino acid addition, either by direct coupling of Arg to an N-terminal Asp (D), Glu (E), or oxidized Cys (C^∗) or by sequential deamidation of Asn (N) or Gln (Q) followed by arginylation. (D) Degron generation by C-terminal amino acid addition during ribosomal stalling. Mistranslated polypeptides are targeted for degradation by either Listerin-dependent pathway or by CRL2^KLHDC10 and Pirh2 E3 ligases recognizing C-terminal polyAla tails generated by NEMF. (E) Premature termination of selenoprotein translation under selenium-deficient conditions leads to the formation of diGly/C-degron. (F) Defective myristoylation exposes Gly/N-degrons. (G) In eukaryotes, formyl-Met (fMet), produced by enzyme Fmt1, is incorporated at N termini of mitochondrial proteins. Fmt1 mitochondrial import is blocked under starvation conditions by phosphorylation by Gcn2, leading to its cytosolic accumulation. Cytoplasmic proteins that are misinitiated by fMet due to Fmt1 activity are eliminated by fMet/N-degron pathway.