Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases

Abstract

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac I_KS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the I_KS channel, thus confirming the functional importance of E3-ligase autoinhibition.

Keywords: Nedd4; Rsp5; inactivation; oligomerization; ubiquitylation.

Figure 1. Removal of α1 alters the oligomeric state of Rsp5

1. Schematic representation of the Rsp5 domain architecture. Black strips below represent truncated proteins used in this study.

2. Normalized, mean β‐galactosidase activity in a yeast two‐hybrid assay reporting association of Rsp5 with itself or with the negative controls, ELG1 or SLX5. Mean values and bars of standard deviation from triplicates are shown.

3. Representation of a trimeric Rsp5 model in the presence (left) or absence (right) of α1 (red). The structure of Rsp5 (3OLM) was superimposed onto the E6AP trimeric structure (1C4Z), with its three HECT protomers depicted in green, pink and cyan. A clash of the α1 helices is apparent at the trimerization interface.

4. Size‐exclusion chromatography of full‐length Rsp5 or of its HECT domain, with (α1) or without (Δα1) α1. Proteins were loaded on a Superdex 200 16/60 column, and elution was monitored by measuring absorption at 280 nm (A₂₈₀).

5. Time‐dependent cross‐linking of ΔC2 or Δα1 Rsp5 with 0.5 mM disuccinimidyl suberate (DSS). Samples were resolved by SDS–PAGE followed by Coomassie blue staining.

6. Sedimentation velocity data, fits and residuals from analytical ultracentrifugation of ΔC2 or Δα1 Rsp5. Upper panels show continuous distributions analyses c(s) of the sedimenting species for ΔC2 (blue) or Δα1 (orange). Observed/calculated mass (oligomeric order), and distribution of species is presented in Table EV1.

Figure 2. Ubiquitylation on α1 induces Rsp5 oligomerization and inactivation

1. A

   Rsp5_232–809 was expressed in bacteria along with ubiquitin, E1 (UBA1) and E2 (Ubc5). The left panel shows Coomassie blue‐stained SDS–PAGE of Rsp5 purified from bacterial lysates. Identification of ubiquitylation sites was performed by mass spectrometry analysis following in‐gel trypsin digestion. A representative tandem mass spectrum of a peptide containing lysine 432 is shown (right panel).

2. B

   Normalized, mean β‐galactosidase activity reporting self‐association of wild‐type, triple or single K411R/K432R/K438R mutants of Rsp5. Mean values and standard deviation bars from triplicates are shown.

3. C–E

   (C) Representative gel of time‐dependent in vitro ubiquitylation of MBP‐Rvs167_332–482 by Rsp5^WT or Rsp5^3K>R in the presence of fluorescein‐labelled ubiquitin. Imaging of fluorescent ubiquitin conjugates was carried out with a Typhoon laser scanner at 488 nm. Gel quantification of (D) ubiquitylated Rvs167 or (E) self‐ubiquitylated Rsp5. Mean values and standard deviation from three experiment replicates are shown.

4. F

   SEC of apo HECT (α1) or Ub‐fused HECT (Ub‐α1) proteins tagged with His₆‐MBP. The proteins were loaded on a Superdex 200 16/60 column, and elution was monitored by A₂₈₀ detection.

5. G

   Time‐dependent cross‐linking of α1 or Ub‐α1 HECT domain in the presence of 0.5 mM DSS. Samples were resolved by SDS–PAGE followed by Coomassie blue staining.

6. H

   Sedimentation equilibrium (SE) data, fit and residuals of α1, Δα1 or Ub‐α1 HECT domain. A₂₈₀ data were collected at 8,000 rpm (lighter colours) and at 12,000 rpm (darker colours). The best fit for α1 HECT was obtained with a model of a single ideal solute with the size with a monomer. Alternatively, the best fits for Δα1 and Ub‐α1 HECT were obtained with a model of multiple ideal solutes with the size of monomers, trimers and hexamers (see also Table EV1).

7. I

   Time‐dependent in vitro ubiquitylation of Rpn10 by apo (α1), oligomeric or monomeric Ub‐fused HECT (Ub‐α1^mono and Ub‐α1^oligo, respectively), in the presence of fluorescein‐labelled ubiquitin. Reaction set‐up and detection were performed as described in Fig 2C.

8. J

   Histogram plot showing mean values and standard deviation bars of triplicate measurements.

Figure 3. Structural model of ubiquitylation‐dependent oligomerization

1. In the apo form, α1 (red) blocks the oligomerization interface.

2. Upon ubiquitylation, the conjugated ubiquitin binds to the HECT‐UBD, and by thus pulls α1 out of the interface and enables oligomerization.

3. Model of trimeric Ub‐HECT. Three HECT molecules are depicted in various shades of blue, and ubiquitin moieties are represented in orange.

4. Enlarged view of the HECT:Ub interface including key residues chosen for experimental assessment of the model.

Figure EV1. Comparing alternative models for Δα1 self‐association in SE analysis

Interpretation of SE data from analytical ultracentrifugation of Δα1 in terms of self‐assembly with orders of 1:3:6, 1:2:4 or 1:3:9. A₂₈₀ was collected at 8,000 and 12,000 rpm. Data points, best fits, residuals and local RMSD values of each analysis are shown.

Figure 4. Oligomerization depends on binding of α1‐conjugated Ub to the HECT‐UBD. The following experiments were conducted with His₆‐MBP‐tagged proteins

1. A

   Size‐exclusion chromatography of wild‐type and mutant Ub‐HECT proteins. Ub‐α1, Ub‐α1^HECT‐I537D or Ub^I44E‐α1 HECT proteins were loaded on a Superdex 200 16/60 column, and the elution profile was monitored by A₂₈₀ detection.

2. B

   Time‐dependent cross‐linking of Ub‐α1, Ub^I44E‐α1 or Ub‐α1^HECT‐I537D Ub‐HECT proteins in the presence of 0.5 mM DSS. Samples were resolved by SDS–PAGE followed by Coomassie blue staining.

3. C

   Sedimentation equilibrium (SE) data, fits and residuals of Ub^I44E‐α1 and Ub‐α1^HECT‐I537D mutants. A₂₈₀ was collected at 8,000 rpm (lighter colours) and at 12,000 rpm (darker colours). The best fits of both mutants were obtained in a model of non‐interacting ideal solutes with mostly monomeric size and < 5% trimers (see also Table EV1).

4. D

   Microscale thermophoresis (MST) measurements of Ub‐α1 (green) or Ub^I44E‐α1 (blue). Concentration of red fluorescent NT‐647‐NHS‐labelled Rsp5 was kept constant and titrated with 0.2–1,000 nM of identical unlabelled protein. The y‐axis shows the fluorescence change ratio × 1,000.

5. E, F

   (E) Representative gel and (F) quantification of in vitro ubiquitylation of Rpn10 by α1, UbI44E‐α1 or Ub‐α1HECT I537D Rsp5, in the presence of fluorescein‐labelled ubiquitin. Mean values and standard deviation bars of three replicates are shown. Reaction set‐up and detection were performed as described in Fig 2C.

Figure 5. Ubiquitylation‐independent oligomerization of the Rsp5 G747E mutant

1. A

   Model showing stabilizing hydrogen bonds within the G747 turn‐like structure, as well as hydrogen bonds between the loop (purple) and the α1 (pink) residues in the Rsp5‐HECT structure (3OLM).

2. B

   Size‐exclusion chromatography profile of Rsp5_232–809 ^WT or Rsp5_232–809 ^G747E, run on a Superdex 200 16/60 column.

3. C

   Migration of 6.6 μM and 20 μM of Rsp5_232–809 ^WT or Rsp5_232–809 ^G747E in a native polyacrylamide gel.

4. D

   AUC‐SE analysis in terms of non‐interacting ideal solutes for Rsp5_232‐809 ^WT or Rsp5_232‐809 ^G747E fused to His₆‐MBP at 8,000 and 12,000 rpm (see also Table EV1).

5. E, F

   (E) Representative gel and (F) quantification (mean and standard deviation from three replicates) of in vitro ubiquitylation of Rvs167 by Rsp5_232–809 ^WT or Rsp5_232–809 ^G747E, in the presence of fluorescein‐labelled ubiquitin. Reaction set‐up and detection were performed as described in Fig 2C.

Figure EV2. Sequence and structural conservation of the α1 helix

1. Sequence alignment of α1 showing that K525 of human Nedd4 is fully conserved in the indicated organisms.

2. The conserved Rsp5 K432 and the Nedd4 K525 are shown as ball and sticks. The loop connecting a1 to the HECT domain is highlighted. The significantly high B‐factor values of the loop atoms and the different conformations assumed in each of the structures indicate that the loop is highly flexible.

Figure 6. Conservation of ubiquitylation‐dependent oligomerization in human Nedd4 and implications in targeting of FGFR1

1. Elution profile from size‐exclusion chromatography of apo HECT (α1^Nedd4) or Ub‐fused HECT (α1^Nedd4‐Ub) of Nedd4. Proteins were loaded on a Superdex 200 16/60 column, and elution was monitored by A₂₈₀ measurements.

2. In vitro ubiquitylation of Rpn10 by apo HECT (α1^Nedd4) or Ub‐fused HECT (α1^Nedd4‐Ub) of Nedd4. Reaction products were resolved by SDS–PAGE and blotted against a primary rabbit α‐Rpn10 antibody, followed by secondary IR labelled mouse α‐rabbit. Odyssey infrared imaging system was used for IR detection.

3. Quantified ubiquitylated/total Rpn10 ratio (mean values and standard deviation bars from three replicates).

4. Documentation of Nedd4 knockdown by shRNA in HeLa cells.

5. Representative immunoblot of FGFR1 ubiquitylation (in the presence of serum) upon transfection of the indicated wild‐type (WT) and mutant human Nedd4 constructs. V5‐Nedd4 was used as an additional control.

6. Quantified (mean ± SEM) ubiquitylated/total FGFR1 ratio from three separate experiments. P‐values are from Student's t‐test.

Figure EV3. Size‐exclusion chromatography of His₆‐MBP‐Nedd4 fusion proteins

Full‐length protein and WT or I44E mutant of Ub‐fused HECT were separated on a Superdex 200 16/60 column, and elution was monitored by A₂₈₀ detection.

Figure 7. Ubiquitylation‐dependent oligomerization mechanism regulates the cardiac I_KS potassium current

1. A

   Representative current traces from CHO cells expressing either I_KS (KCNQ1 + KCNE1), I_KS + Nedd4^WT or I_KS + Nedd4^K523,525R. Cells were held at −90 mV. Membrane voltage was stepped for 3 s from −60 mV to +60 mV in 10 mV increments followed by repolarization to −60 mV for 1.5 s.

2. B, C

   (B) Current‐voltage (mean ± SEM) and (C) conductance‐voltage (mean ± SEM) relationships of the recorded cells (n = 10–11). Normalized conductance curves were fitted to a single Boltzmann function.

3. D

   Phase contrast (left panels), epi‐fluorescence (central panels) and TIRF fluorescence (right panels) images of CHO cells expressing either I_KS, I_KS + Nedd4^WT or I_KS + Nedd4^K523,525R. Scale bars: 10 μm.

4. E

   Quantification (mean ± SEM) of the TIRF signals was normalized by the TIRF/epi‐fluorescence intensity ratio, using Fiji NIH viewer. n = 10–17; one‐way ANOVA and Bonferroni's multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001.

5. F

   Representative immunoblot with anti‐Nedd4 of total CHO cell lysates from non‐transfected cells and cells transfected with wild‐type or mutant Nedd4. Lysates were also blotted with antibodies against endogenous β‐actin.

6. G

   Quantified (mean ± SEM) Nedd4/β‐actin ratio (n = 3).

Figure EV4. Dissecting the role of individual lysine residues in Nedd4 regulation

1. A, B

   Quantification (mean ± SEM) of (A) current‐voltage and (B) conductance‐voltage relationships of the recorded cells (n = 8–12), following co‐expression of I_KS with Nedd4^WT or mutants bearing individual lysine mutations, that is Nedd4^K523R or Nedd4^K525R. Cells were held at −90 mV. Membrane voltage was stepped for 3 s from −60 mV to +60 mV in 10 mV increments followed by repolarization to −60 mV for 1.5 s. Normalized conductance curves were fitted to a single Boltzmann function.

2. C

   Quantified (mean ± SEM) amplitude at −60 mV, for each cell type (n = 8–12). One‐way ANOVA; and Bonferroni's multiple comparison test; *P < 0.05, ***P < 0.001.

3. D

   Representative immunoblot of FGFR1 ubiquitylation (in the presence of serum) upon transfection of the indicated wild‐type and mutant human Nedd4 constructs.

4. E

   Quantified (mean ± SEM) ubiquitylated/total FGFR1 ratio from three separate experiments. P‐values are from Student's t‐test.

Figure EV5. Multiple sequence alignment of α1 sequences and post‐translation modification sites

The α1 sequences of human Nedd4, Rsp5 and other HECT ligases were aligned with ClustalW. The conserved lysine residue of the Nedd4 family and the threonine reside of E6AP that undergo ubiquitylation and phosphorylation are indicated.