NEDD4L intramolecular interactions regulate its auto and substrate NaV1.5 ubiquitination

Abstract

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, Na_V1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For Na_V1.5, this process is critical as alterations in Na⁺ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of Na_V1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of Na_V1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na⁺ current reduction using the NEDD4L HECT domain as cargo of a Na_V1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding Na_V1.5 ubiquitination.

Keywords: E3 ligases; HECT; NEDD4-2; NEDD4L; NanoMaN; Nav1.5; PTM; SCN5A; mass spectrometry; nanobody; post translational modification; proteostasis; transthioesterification; ubiquitin; voltage-gated sodium channel.

Figure 1

C2 domain and 1,2-linker regulate the auto-ubiquitination of NEDD4L.A, schematic representation of NEDD4L variants; FL^NEDD4L (aa 1–975), CS^NEDD4L (aa 1–975, catalytically relevant residue Cys942 mutated to a Ser), ΔC2^NEDD4L (aa 155–356/377–975), Δ1,2-linker^NEDD4L (aa 1–226/383–975), Δ2,3-linker^NEDD4L (aa 1–418/496–975), W2-W3-W4-HECT^NEDD4L (aa 381–975), W3-W4-HECT^NEDD4L (aa 492–975), and HECT^NEDD4L (aa 594–975). The C2 domain is shown in blue; WW domains in green; HECT domain in shades of pink with the active site cysteine residue highlighted by a yellow pentagon. B, DSF thermal stability curves of the NEDD4L variants. Melting temperatures (Tm) were identified by calculating the average negative first derivative. Each Tm at the minimum is listed. DSF experiments were performed in triplicate with mean values plotted. C, in vitro ubiquitination assays of FL^NEDD4L, ΔC2^NEDD4L, Δ1,2-linker^NEDD4L, Δ2,3-linker^NEDD4L, W3-W4-HECT^NEDD4L, and HECT^NEDD4L. Samples were quenched with reducing loading buffer at the indicated time points. The amount of unmodified NEDD4L proteins, quantified by a densitometry analysis as a function of time, is shown as a percentage averaging all replicates. The average percentages ± SD for NEDD4L proteins are as follows (%): 100, 82 ± 10, 68 ± 23; 100, 63 ± 3, 34 ± 8; 100, 65 ± 10, 51 ± 17; 100, 82 ± 8, 64 ± 7; 100, 66 ± 7, 42 ± 9; 100, 96 ± 4, 79 ± 10. All the assays were repeated at least twice (N ≥ 2). D, densitometry analysis of the unmodified NEDD4L variants FL^NEDD4L, ΔC2^NEDD4L, Δ1,2-linker^NEDD4L, Δ2,3-linker^NEDD4L from (C) and colored as in (B). Error bars represent SD. DSF, differential scanning fluorimetry.

Figure 2

Ubiquitin exosite in the HECT domain influences NEDD4L regulation.A, in vitro ubiquitination assays of FL^NEDD4L, ΔC2^NEDD4L, Δ1,2-linker^NEDD4L, and Δ2,3-linker^NEDD4L in the presence and absence of 5 μM ubiquitin variant, UbvNL.1. Samples were quenched with reducing loading buffer at indicated time points. The amount of unmodified NEDD4L proteins, quantified by a densitometry analysis as a function of time, is shown as a percentage averaging all replicates. The average percentages ± SD for NEDD4L proteins in the absence and presence of UbvNL.1 are as follows (%): 100, 67 ± 1, 23 ± 4; 100, 64 ± 1, 7 ± 5; 100, 67 ± 19, 20 ± 1; 100, 68 ± 9, 14 ± 1; 100, 67 ± 7, 10 ± 7; 100, 58 ± 1, 12 ± 4; 100, 100, 77 ± 9, 39 ± 5; 100, 59 ± 21, 27 ± 14. All the assays were repeated at least twice (N ≥ 2). B, fluorescent Western blot analysis of the in vitro ubiquitination assays of FL^NEDD4L, ΔC2^NEDD4L, Δ1,2-linker^NEDD4L, and Δ2,3-linker^NEDD4L as carried out in (A) using anti-ubiquitin (red) and anti-NEDD4 (green) antibodies. N = 2. C–F, binding of fluorescein-labeled UbvNL.1 to the NEDD4L variants measured by fluorescence anisotropy. Each concentration is shown with ± S.D; N = 2. The K_d values of UbvNL.1 for each NEDD4L variant obtained using a quadratic fit are (C) 17 nM with FL^NEDD4L; (D) 2.1 nM with ΔC2^NEDD4L; (E) 9 nM with Δ1,2-linker^NEDD4L; (F) 2.5 nM with Δ2,3-linker^NEDD4L.

Figure 3

Site-specificity of NEDD4L.A, FL^NEDD4L sequence coverage after in vitro band excision. Identified peptides are highlighted in shaded regions and colored based on the domain scheme in Figure 1A. Bold, red letters with a yellow square above them are identified with Lys sites with a Gly-Gly modification. B, domain scheme of FL^NEDD4L with the Lys sites of ubiquitination labeled based on location. The peptide spectrum matches of each Lys residue is in parentheses. C, representative MS/MS spectrum and sequence coverage of the peptide containing a Gly-Gly modification on Lys471 and (D) Lys489. Lower case k indicates a Gly-Gly modification.

Figure 4

Na_V1.5 is ubiquitinated by FL^NEDD4Lon Na_V1.5 DI-DII linker.A, HEK293 cells transfected with Na_V1.5 alone or cotransfected with CS^NEDD4L, W3-4HECT^NEDD4L, or FL^NEDD4L and cultured for 24 or (B) 72 h. Cells were stained with anti-Na_V1.5 antibody (white), phalloidin dye to mark actin filaments (magenta), and Hoescht3342 to mark nuclei (blue). C, Na_V1.5 chimeric construct spans DI-DII linker (purple; residues 411–717) and CTerm extended PY motif (blue; residues 1960–1996). Composite of the existing cryo-EM structure of Na_VPas (Na_V of American cockroach Periplaneta americana; PDB ID 5X0M; green) aligned with experimental structure of Na_V1.5^CTerm (PDB ID 4OVN; blue). Dashed lines indicate regions lacking published structural data (each dash ∼10 amino acids). D, time course of the in vitro ubiquitination of FL^NEDD4L in the absence and presence of Na_V1.5^{DI-DII linker+PPSY} or Na_V1.5^{DI-DII linker+AASA} substrate. Equal amounts of samples were taken at the indicated time points and quenched with reducing loading buffer. The SDS-PAGE was stained with colloidal Coomassie blue stain. The amount of unmodified Na_V1.5^{DI-DII linker}, quantified by a densitometry analysis as a function of time, is shown as a percentage averaging all replicates. The average ± SD for Na_V1.5^{DI-DII linker+PPSY/AASA} protein in the presence of NEDD4L enzyme are as follows (%): 100, 57 ± 6, 36 ± 6, 19 ± 5, 100, 96 ± 4, 93 ± 7, 92 ± 9. All the assays were repeated at least twice (N ≥ 2). E, scheme of Na_V1.5^DI-DII+PPSY with the Lys sites of ubiquitination labeled based on location. Mono- and di-Ub Na_V1.5^DI-DII+PPSY bands were excised from 3 h in vitro gel lane. The LC/MS/MS peptide spectrum matches of each Lys residue are in parentheses. F, representative MS/MS spectrum and sequence coverage of the peptide containing a Gly-Gly modification on Na_V1.5^DI-DII+PPSY Lys430. Lower case k indicates a Gly-Gly modification and lower case m indicates oxidized methionine. G, Na_V1.5^DI-DII+PPSY sequence coverage after band excision from the in vitro assay. Identified peptides are highlighted in shaded regions and colored based on the domain scheme in (D).

Figure 5

Cotransfection with NEDD4L variants reduces Na_V1.5-mediated sodium current.A, average peak current density (J_peak) – voltage relationship from wild-type Na_V1.5 channels elicited in response to a family of 10 ms steps from −60 to +50 mV from a holding potential of −120 mV. Each dot, mean ± SD with n denoted in parenthesis. B–F, J_peak for Na_V1.5 (black) and upon cotransfection with NEDD4L variants (B) FL^NEDD4L, (C) CS^NEDD4L, (D) W3-4-HECT^Nedd4L, (E) Δ1,2-linker^NEDD4L, (F) Δ2,3-linker^NEDD4L, (G) J_peak at −10 mV. Mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. H, representative Western blot showing immunodetection of the NEDD4L variant protein levels (green) used in (B–F) and GAPDH (red; loading control) in HEK293. NEDD4L antibody epitope is the HECT domain present in all NEDD4L variants. Four such experiments yielded comparable results.

Figure 6

NanoMaN targets Na_V1.5 to diminish I_Na.A, schematic of the NanoMaN construct composed of an N-terminal nanobody (either Nb17 or Nb82) and the catalytic HECT domain of NEDD4L comprising residues 640 to 975. B, top, exemplar current recordings for WT Na_V1.5 channels elicited in response to a family of voltage steps to −60 to +50 mV from a holding potential of −120 mV. Bottom, population data shows average peak current density (J_peak) – voltage relationship. Each dot, mean ± SD with n denoted in parenthesis. C, co-expression of Nb17 alone yielded minimal change in peak Na current density. D, overexpression of both NanoMaN17 diminished peak current densities. Format as in panel (B). E, Nb82 alone also minimally perturbed peak current density. F, NanoMaN82 also diminished peak current density. G, average peak current density (J_peak) – voltage relationship for Na_V1.5 PPSY/AASA mutant channels. Each dot, mean ± SD with n denoted in parenthesis. H–J, J_peak for Na_V1.5 PPSY/AASA mutant channels upon co-expression of NEDD4L variants (H) FL^NEDD4L, (I) W3-4-HECT^Nedd4L, and (J) NanoMan17. K, average peak current density (J_peak) – voltage relationship for Na_V1.5 K442/443/496–R mutant channels. Each dot, mean ± SD with n denoted in parenthesis. L–N, J_peak for NaV1.5 PPSY/AASA mutant channels upon co-expression of NEDD4L variants (L) FL^NEDD4L, (M) W3-4-HECT^NEDD4L, and (N) NanoMan17. O, bar graph summarizes changes in J_peak for Na_V1.5 PPSY/AASA mutant in the presence of NEDD4L variants. Mean ± SD, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. P, bar graph summarizes changes in J_peak for Na_V1.5 K442/443/496–R mutant in the presence of NEDD4L variants. Mean ± SD, ∗p < 0.05, ∗∗∗p < 0.001 by one-way ANOVA with Dunnett’s multiple comparisons test. NanoMaN, nanobody modulators of Na+ channels.

Figure 7

Proposed NEDD4L and Na_V1.5 mechanism of regulation.A, the intramolecular interactions of the NEDD4L C2 domain and 1,2-linker lock the HECT domain in the inactive T-shape. Upon ubiquitin exosite binding, the regulatory domains are released and the HECT domain transitions into the active L-shape. Subsequent auto-ubiquitination can occur on the preferred lysine residues within the 2,3-linker. B, the NEDD4L recognition motif located in the CTerm of the voltage-gated sodium channel Na_V1.5 binds to the WW3 or WW4 domains. Substrate binding near the 1,2-linker can initiate regulation release and robust ubiquitination. Specifically, for Na_V1.5, the ubiquitination occurs in the DI-DII-linker; not the region that has the recognition motif. C, NanoMans reduced the peak sodium current of Na_V1.5 suggesting less Na_V1.5 channels at the plasma membrane. NanoMaN, nanobody modulators of Na+ channels.