E3 ligase autoinhibition by C-degron mimicry maintains C-degron substrate fidelity

Abstract

E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2^KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2^KLHDC2. Without substrate, neddylated CRL2^KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.

Keywords: Autoinhibition; C-END degron; CUL2; Cullin-RING Ligase; E3; KLHDC10; KLHDC3; Kinetic proofreading; NEDD8; Targeted protein degradation; allostery; higher-order assembly; protein-protein interaction; ubiquitin.

Figure 1.. KLHDC2 displays selectivity for the diGly C-terminus of NEDD8.

(a) Superposition of the KLHDC2 SBD (surface representation colored slate) bound to the di-Gly motif of EPHB2 (sticks colored magenta), SELK (sticks colored light blue; 6DO3.pdb), and USP1 (red, 6DO5.pdb). Inset depicts the structural conservation of C-degron binding. C-terminal sequences from known physiological substrates and for UB and NEDD8. (b) Results of TR-FRET assay between KLHDC2 and Ric8b peptide and the dose-dependent TR-FRET in the presence of five di-Gly containing proteins. (c) Pulse-chase ubiquitylation assay monitoring the transfer of fluorescently labeled Lys-less UB from UB~UBE2R2B to KLHDC2 or the indicated diGly containing substrates. (d) Quantification of assays shown in (c). Results shown in (b-d) are representative of n=3 replicates.

Figure 2.. KLHDC2’s C-terminal GlySer acts as a C-degron mimic.

(a) Crystal structure of full-length KLHDC2-EloBC. The crystallographic asymmetric unit contains subunits A (slate) and B (light pink) that interact with symmetry related subunits A’ (blue) and B’ (magenta). Insets depict the binding of KLHDC2’s C-degron mimic from protomer A (slate; left panel) to B (light pink) or protomer A’ (blue; right panel) to B (light pink) compared to USP1’s diGly C-degron bound to KLHDC2’s SBD (red, 6DO5.pdb). (b) Superposition of KLHDC2 SBD (slate) bound to a peptide derived from KLHDC2’s C-degron mimic and the SBD bound to USP1’s diGly C-degron (red; 6DO5.pdb). Inset shows the structural conservation of interactions. (c) SPR between the KLHDC2 SBD and various peptides with WT or mutant C-degron sequences from KLHDC2’s C-terminus. Asterisks denote estimated kinetic parameters as the extreme slow-off rate of the -GG peptide precluded accurate determinations.

Figure 3.. KLHDC2-EloBC forms C-degron mimic dependent tetramers in solution.

(a) SEC-MALS of KLHDC2-EloBC. Nr is peak number with the mass (μg) of injected protein sample in parenthesis; Mp is kDa molar mass of the protein complex at the peak apex (molar mass of monomer shown in parenthesis); Mw is the weight-averaged molar mass from multi-angle light scattering; and Mw/Mn is the polydispersity of mass in the peak. (b) SV-AUC with AlexaFluor488 labeled KLHDC2-EloBC. The y-axis shows the normalized continuous sedimentation coefficient c(s) as a function of the sedimentation coefficient (S) at the indicated concentrations of KLHDC2-EloBC (left panel). Species population plots as a function of KLHDC2-EloBC concentration (right panel). Model-derived dissociation constants K_D14 for tetramer to monomer dissociation, K_D12 for KLHDC2-EloBC dimerization, and K_D24 for dimer-dependent tetramerization are shown. Root-mean square deviation (Rmsd) of the fit to the model is shown. (c) Superposition of promoters A and B from the KLHDC2-EloBC tetramer. Structural elements including the SBD, BC-box, and EloBC align well, with structural differences restricted to the extreme C-terminal degron mimic sequence of KLHDC2 subunits. (d) Cartoon schematic of the KLHDC2-EloBC tetramer highlighting locations of inter-molecular interactions between protomers and their associated tetramerization elements. (e) Primary structure of KLHDC2 highlighting positional boundaries for the SBD, BC-box, and CUL2-box. The C-terminal 12 residues of the C-degron mimic are shown. Ribbon diagrams highlighting tetramerization elements and the C-degron mimic between KLHDC2 protomers A and B (left panel) or A’ and B (right panel). (f) Coomassie stained native gel of WT KLHDC2-EloBC or indicated mutants monitoring the formation of oligomeric species.

Figure 4.. The KLHDC2-EloBC tetramer does not bind protein substrates.

(a) Fluorescence-scan of native gel electrophoresis monitoring time dependence for binding of fluorescent SELK (^FAMSELK) to WT or the indicated C-degron mimic mutants. (b) same as in (a) but monitoring concentration dependence of ^FAMSELK or ^FAMUSP1 after 4-hour pre-incubation. (c) same as in (a) but monitoring complex formation between TAMRA-labeled CUL2 (left panel) or TAMRA-labeled NEDD8~CUL2 (right panel) and ^FAMSELK. Co-migration of CUL2 and ^FAMSELK results in yellow bands. (d) Surface representations of the cryo-EM structure of KLHDC2-EloBC bound to CUL2-RBX1. (e) Models for ubiquitylation complexes with tetrameric KLHDC2-EloBC and substrate bound monomer KLHDC2. Note positioning of protomer A or A’ correlates with the predicted positioning of a diGly substrate. (f) Pulse-chase ubiquitylation reactions monitoring the transfer of fluorescently labeled WT UB from UB~UBCH5B to WT KLHDC2-EloBC or the indicated C-degron mutants with or without the addition of SELK.

Figure 5.. KLHDC2-EloBC substrate mimic-induced tetramers select for bona fida substrates through a proofreading mechanism.

(a) Pulse-chase ubiquitylation reactions monitoring how pre-incubation of a USP1-NEDD8~CUL2^KLHDC2 complex affects transfer of UB from UB~UBE2R2 to substrate with WT or -KK monomeric KLHDC2-EloBC. (b) same as in (a) but with NEDD8. Note that pre-incubation results in partitioning of UB from KLHDC2 to USP1 but not NEDD8 in a tetramer-dependent fashion. (c) SPR traces monitoring the binding of SELK^Fragment, USP1^Fragment, or NEDD8 proteins to the KLHDC2 SBD. Association rates (k_a), dissociation rates (k_d), and the equilibrium binding constants (K_D) obtained from fits are shown. (d), same as in (c), but with the indicated GlySer insertion mutants of NEDD8. (e) same as in (a) but with -KK KLHDC2 and NEDD8 or the indicated insertion mutant substrates. (f) same as in (e) but with WT KLHDC2.

Figure 6.. KLHDC2 C-degron mimic-induced tetramerization affects myriad CRL2^KLHDC2 biochemical activities.

(a) Normalized rates of CUL2 neddylation by NEDD8~UBE2M in the absence (left panel) or presence (right panel) of CAND1 and with WT or the indicated KLHDC2-EloBC C-degron mutants and with or without SELK peptide. (b) Western blot monitoring NEDD8~CUL2 deneddylation by CSN in the absence or presence of WT or the indicated KLHDC2-EloBC C-degron mutants and with or without the addition of SELK peptide or a MBP-SELK fusion protein. (c) Model of monomeric CUL2^KLHDC2 neddylation complex. The relative position of tetramerization elements 1 and 2 are highlighted. (d) Velocity of CUL2 neddylation versus NEDD8~UBE2M concentration. Rates of NEDD8~CUL2 formation were measured in the presence of CUL2 alone (circles) or CUL2^KLHDC2(-KK). Data were fit to the Michaelis-Menten model using Graphpad Prism software. (e) same as in (a) but monitoring the stimulation of CUL2 neddylation in the presence of WT or the indicated KLHDC2-EloBC mutants with or without SELK. All results represent replicates of n=3.

Figure 7.. KLHDC2-EloBC appears to form C-degron mimic-dependent tetramers in cells that control CUL2^KLHDC2 function.

(a) HEK-293T cells were transiently transfected with constructs for FLAG-tagged WT or C-degron mutant KLHDC2. Immunoprecipitations were performed in the absence or presence of the indicated supplements followed by Western blotting. (b) Steady-state levels of proteins from WT U2OS cells, KLHDC2 knockout (KO), or KLHDC2 KO cells with ectopic expression of the indicated KLHDC2 proteins. (c) same as (b), monitoring the steady-state levels of the diGly C-terminal fragment of USP1. (d) Quantification of steady-state levels of UPS1 fragment from (c); (technical replicates are shown of n=3). (e) same as (b) but monitoring the steady-state levels of NEDD8. (f) same as (d) but for NEDD8. (g) Cartoon model for substrate selectivity amplification by the C-degron mimic KLHDC2-EloBC tetramer. Typical CRL SRs (top panel) poorly discriminate between bona fide substrates with fast association rates and “non-substrates” with slow association rates. In contrast, the C-degron mimic KLHDC2-EloBC tetramer (bottom panel) is unable to directly engage substrates. Substrates must “capture” the KLHDC2-EloBC monomer upon dissociation from tetramer. Thus, substrate selectivity is achieved by enabling SR-substrate complex formation for only substrates that have association rates that are sufficiently fast to capture monomer prior to tetramer reformation.