Structural basis for C-degron selectivity across KLHDCX family E3 ubiquitin ligases

Abstract

Specificity of the ubiquitin-proteasome system depends on E3 ligase-substrate interactions. Many such pairings depend on E3 ligases binding to peptide-like sequences - termed N- or C-degrons - at the termini of substrates. However, our knowledge of structural features distinguishing closely related C-degron substrate-E3 pairings is limited. Here, by systematically comparing ubiquitylation activities towards a suite of common model substrates, and defining interactions by biochemistry, crystallography, and cryo-EM, we reveal principles of C-degron recognition across the KLHDCX family of Cullin-RING ligases (CRLs). First, a motif common across these E3 ligases anchors a substrate's C-terminus. However, distinct locations of this C-terminus anchor motif in different blades of the KLHDC2, KLHDC3, and KLHDC10 β-propellers establishes distinct relative positioning and molecular environments for substrate C-termini. Second, our structural data show KLHDC3 has a pre-formed pocket establishing preference for an Arg or Gln preceding a C-terminal Gly, whereas conformational malleability contributes to KLHDC10's recognition of varying features adjacent to substrate C-termini. Finally, additional non-consensus interactions, mediated by C-degron binding grooves and/or by distal propeller surfaces and substrate globular domains, can substantially impact substrate binding and ubiquitylatability. Overall, the data reveal combinatorial mechanisms determining specificity and plasticity of substrate recognition by KLDCX-family C-degron E3 ligases.

Fig. 1. KLHDCX family members mediate ubiquitylation of an assortment of Gly terminating substrates.

a C-terminal consensus degron sequences recognized by KLHDCX substrate receptor family members (top panel). *The consensus sequence of KLHDC1 has not been reported, but has been show to recognize degrons terminating in GlyGly. C-terminal sequences of UBLs used in this study (bottom panel). b Quantification of pulse-chase ubiquitylation assays monitoring monomeric CRL2^KLHDC2 dependent ubiquitylation of the indicated Gly terminating substrates by the ubiquitin-carrying enzymes UBE2D2 (gray), UBE2R2 (cyan), and UBE2L2/ARHI1 (purple). c Same as (b) but with monomeric CRL2^KLHDC3. d Same as (b) but with CRL2^KLHDC10. e Same as (b) but with CRL5^KLHDC1, and with the CRL5 specific UCE UBE2L2/ARIH2. Bar graphs are the average of n = 2 independent experiments. Source data are provided as a Source Data file.

Fig. 2. Structure of KLHDC3-EloB/C bound to a C-terminal Gly degron.

a Overall structure of UB bound to KLHDC3-EloB/C. UB (orange), KLHDC3 (light blue), EloC (cyan), and EloB (pale cyan) are shown in surface representation. b Cartoon representation of UB (orange) bound to KLHDC3 (light blue). UB’s C-terminal Gly76 (orange) is shown in sticks. Blades 1–6 of the KLHDC3 propeller are labeled. The barrel beta-strands for one blade are labeled A-D. c Cartoon representation of the “funnel-like” arrangement of KLHDC3’s substrate binding pocket. UB (orange) is shown in cartoon with its C-terminal Gly76 shown in spheres. KLHDC3 (light blue) is shown in cartoon with transparent surface. d Bottom (left) and top (right) views of KLHDC3 (light blue; surface). UB (orange,cartoon) is shown with its C-terminal Gly76 in sticks. The relative dimensions of the C-degron binding pocket and the C and N chambers of the KLHDC3 are labeled. e Electrostatic representation of KLHDC3 bound to UB (orange) with its C-terminal Gly76 in sticks. Hydrophobic surfaces are colored white, positively charged surfaces blue, and negatively charged surfaces red. f LigPlot of interactions between UB and KLHDC3, hydrogen bonds between KLHDC3 and UB or water (red, spheres) are show in black dashes. g Close up view of KLHDC3’s (light blue) C-degron binding pocket. Residues from UB (orange) and KLHDC3 forming interactions are shown in sticks with black dashes between interacting partners. h Quantification of pulse-chase ubiquitylation assays monitoring UBE2R2 mediated ubiquitylation of UB or the indicated UB mutants by WT monomericc CRL2^KLHDC3 or the indicated KLHDC3 mutants. i Structural view of interactions between the globular domain of UB (orange) and KLHDC3 (light blue). Probable interacting residues are shown in sticks. j Quantification of pulse-chase ubiquitylation assays monitoring UBE2R2 mediated ubiquitylation of WT UB or UB globular domain mutants by monomeric CRL2^KLHDC3. Bar graphs are the average of n = 2 independent experiments. Source data are provided as a Source Data file.

Fig. 3. Structural basis for penultimate Arg/Gln recognition in KLHDC3 degrons.

a Structural model depicting the presumed degron-mimic mediated auto-regulation of KLHDC3-EloB/C by the inactive tetrameric assembly and active monomer-substrate complexes derived from the KLHDC2-EloB/C tetrameric structure (8EBN.pdb). b Fluorescent scan of gel monitoring the ability of UB to serve as a substrate for the wild-type KLHDC3-EloB/C tetrameric assembly. Assays were performed in pulse-chase format with or without the indicated UB-E3 pre-incubation period prior to initiating ubiquitylation by addition of the UBE2R2 ~ UB thiolester conjugate. c C-terminal sequences of the KLHDC3 degron peptide TCAP, WT UB, and the indicated C-terminal modifications of UB used in isothermal titration calorimetry and biochemical studies. Equilibrium binding affinities (K_d) are shown. Values are the average +/− 1 SD from n = 2 independent experiments. d Quantification of pulse-chase ubiquitylation assays monitoring ubiquitylation of UB and the indicated C-terminal UB modifications by monomeric CRL2^KLHDC3. e Structural superposition of KLHDC3 (light blue, cartoon) bound to UB (orange, sticks), G75R UB (light orange, sticks), and G75Q UB (olive). KLHDC3’s RSR motif, and its R72 and R74 UB interacting residues (Y78 and Y32) are shown in sticks. For clarity only KLHDC3 from the WT UB bound structure is shown. f Same as (e) but only for G75R UB. Potential KLHDC3 residues (S34, D325, and T309) for interaction with the penultimate residue in consensus degron sequences are shown in sticks. g Same as (f) but for G75Q UB. h Quantification of pulse-chase ubiquitylation assays monitoring UBE2R2 mediated ubiquitylation of UB or the indicated C-terminal UB modifications, by WT monomeric CRL2^KLHDC3 or the indicated KLHDC3 mutants. Bar graphs are the average of n = 2 independent experiments. i Close up view of the penultimate Arg binding pocket in KLHDC3 (light blue, surface). Wild-type UB (orange, surface) is shown with the penultimate pocket outlined in orange dashed lines (left). The right panel shows G75R UB (orange, surface) bound to KLHDC3 (light blue, surface). Source data are provided as a Source Data file.

Fig. 4. Structure of a trapped ARIH1-diUB-neddylated-CRL2^KLHDC10 complex reveals the basis for C-degron recognition by KLHDC10.

a Surface representation of the cryo-EM structure of a trapped transfer complex from neddylated-CRL2^KLHDC10 activated ARIH1 to Lys48 on a KLHDC10 bound acceptor ubiquitin (left). Structural coordinates from EloB/C-CUL2-RBX1 (cyan, pale cyan, green, and light pink respectively;5N4W.pdb), ARIH1-UB, (gray and wheat respectively;7B5M.pdb and 7B5N.pdb), UB-KLHDC10 (orange and marine; 1UBQ.pdb and an AlphaFold model of KLHDC10) were bulk fit into the EM density in ChimeraX (right). b Structure of UB (orange, cartoon) bound to KLHDC10 (marine, cartoon). UB’s C-terminal Gly76 is shown in sticks. Blades 1–6 of the KLHDC10 propeller are labeled. The beta-strands for one blade are labeled A-D. c Bottom (left) and top (right) view of KLHDC10 (marine; surface). UB (orange) is shown in cartoon with its C-terminal Gly76 in sticks. The relative dimensions of the C-degron binding pocket and the C- and N- chambers of KLHDC10 are labeled. d Electrostatic representation of KLHDC10 bound to UB (orange) with its C-terminal Gly76 in sticks. Hydrophobic surfaces are colored white, positively charged surfaces blue, and negatively charged surfaces red. e LigPlot of interactions between UB and KLHDC10. Hydrogen bonds between KLHDC10 and UB are show in black sticks. f Close up view of KLHDC10’s (marine) C-degron binding pocket. Residues from UB (orange) and KLHDC10 forming interactions are shown in sticks with black lines. g Quantification of pulse-chase ubiquitylation assays monitoring UBE2R2 mediated ubiquitylation of UB or the indicated UB mutants by CRL2^KLHDC10. h Structural view of the funnel-like arrangement of KLHDC10’s binding pocket. UB-KLHDC10 (orange and marine) are shown in cartoon within the cryoEM density at low contour. i Same as (g). Graphs are the average of n = 2 independent experiments. Source data are provided as a Source Data file.

Fig. 5. Structural basis for penultimate residue tolerance amongst KLHDC10 degrons.

a Close up view of UB (orange) bound to the KLHDC10 (marine) C-degron binding pocket. Modeled conformations of consensus penultimate residues Pro (light orange, sticks) and Trp (yellow orange, sticks) are shown and their structural clash with KLHDC10 F176 is labeled. KLHDC10 residues (I174, L127, A155, and Y110) that form a cryptic hydrophobic pocket behind F176 are shown in sticks. b Surface representation of the KLHDC10 (marine) binding pocket, highlighting the structural clash of KLHDC10 with modeled penultimate residues Pro (light orange, sticks) and Trp (yellow orange, sticks). Hydrophobic surfaces are colored white, positively charged surfaces blue, and negatively charged surfaces red. c Same as (b), but with a F176 flipped rotamer of KLHDC10 revealing a cryptic binding pocket that can accommodate penultimate Pro or Trp from KLHDC10 consensus degrons. d C-terminal sequences of the KLHDC10 degron variants used in surface plasmon resonance and biochemical studies. Equilibrium binding affinities obtained from SPR experiments (K_d) are shown. Values are the average +/− 1 SD from n = 2 independent experiments. e Quantification of pulse-chase ubiquitylation assays monitoring UBE2R2-mediated ubiquitylation of UB or the indicated C-terminal mutations by CRL2^KLHDC10. f Same as (e). Bar graphs are the average of n = 2 independent experiments. Source data are provided as a Source Data file.

Fig. 6. Millisecond ubiquitylation of UB by CRL2^KLHDC3 and CRL2^KLHDC10.

a Structural superposition of UB (orange) bound to KLHDC3 (light blue) to a UBE2R2 (cyan) acceptor UB (light orange) trapped polyubiquitylation structure (8PQL.pdb). Superpositions were generated by aligning UB from the KLHDC3 bound structure to the acceptor UB in the UBE2R2 trapped comples. UBE2R2 and acceptor UB residues making contacts are shown in spheres. b Quantification of pulse-chase ubiquitylation assays monitoring ubiquitylation of UB (orange) or SELK (gray) by WT or the indicated UBE2R2 mutants with monomeric neddylated-CRL2^KLHDC3. c Same as (b) but with neddylated-CRL2^KLHDC10. d Quantification of pulse-chase ubiquitylation assays monitoring ubiquitylation of WT UB or Arg54Asp  UB by monomeric neddylated-CRL2^KLHDC3 or neddylated-CRL2^KLHDC10. Bar graphs are the average from n = 2 independent experiments. e Graphs from pre-steady state kinetic studies showing the fraction of UB substrate remaining as a function of time with monomeric neddylated-CRL2^KLHDC3. Data were fit to an analytical closed-form solutions model in Mathematica. Datapoints from triplicate technical replicates are shown in shaded circles. f Same as (e), but with neddylated-CRL2^KLHDC10. g Estimates of K_m and K_obs for UBE2R2 or UBE2L2/ARIH1 mediated ubiquitylation of UB by monomeric neddylated-CRL2^KLHDC3 and neddylated-CRL2^KLHDC10. Shown are the average +/− 1 SEM from n = 3 independent experiments. Source data are provided as a Source Data file.

Fig. 7. KLHDCX family C-terminal anchors mediating C-degron placement and recognition.

a Cartoon representation of the arrangement of blades 1–6 from the β-propeller domain of KLHDC2. The relative positioning of the degron C-terminal carboxylate (orange, surface, colored by element) and its RSR recognition motif are shown. b Same as (a), but for KLHDC3s RSR motif. c Same as (a), but for KLHDC10s FSR motif. d Cartoon representation of the structure of KLHDC2 (rainbow, 8EBL.pdb) with the degron C-terminal carboxylate (orange, spheres, colored by element) and its RSR (pale green, surface, colored by element) recognition motif are shown. e Same as (d), but for KLHDC3 RSR motif (pale yellow, surface, colored by element). f Same as (d), but for KLHDC10 FSR motif RSR (red, surface, colored by element).