Fig. 3: Structural basis for penultimate Arg/Gln recognition in KLHDC3 degrons.
From: Structural basis for C-degron selectivity across KLHDCX family E3 ubiquitin ligases
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 (Kd) 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 CRL2KLHDC3. 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 CRL2KLHDC3 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.
