Abstract
The epithelial Na+ channel (alphabetagammaENaC) regulates salt and fluid homeostasis and blood pressure. Each ENaC subunit contains a PY motif (PPXY) that binds to the WW domains of Nedd4, a Hect family ubiquitin ligase containing 3-4 WW domains and usually a C2 domain. It has been proposed that Nedd4-2, but not Nedd4-1, isoforms can bind to and suppress ENaC activity. Here we challenge this notion and show that, instead, the presence of a unique WW domain (WW3*) in either Nedd4-2 or Nedd4-1 determines high affinity interactions and the ability to suppress ENaC. WW3* from either Nedd4-2 or Nedd4-1 binds ENaC-PY motifs equally well (e.g. Kd approximately 10 microm for alpha- or betaENaC, 3-6-fold higher affinity than WW4), as determined by intrinsic tryptophan fluorescence. Moreover, dNedd4-1, which naturally contains a WW3* instead of WW2, is able to suppress ENaC function equally well as Nedd4-2. Homology models of the WW3*.betaENaC-PY complex revealed that a Pro and Ala conserved in all WW3*, but not other Nedd4-WW domains, help form the binding pocket for PY motif prolines. Extensive contacts are formed between the betaENaC-PY motif and the Pro in WW3*, and the small Ala creates a large pocket to accommodate the peptide. Indeed, mutating the conserved Pro and Ala in WW3* reduces binding affinity 2-3-fold. Additionally, we demonstrate that mutations in PY motif residues that form contacts with the WW domain based on our previously solved structure either abolish or severely reduce binding affinity to the WW domain and that the extent of binding correlates with the level of ENaC suppression. Independently, we show that a peptide encompassing the PY motif of sgk1, previously proposed to bind to Nedd4-2 and alter its ability to regulate ENaC, does not bind (or binds poorly) the WW domains of Nedd4-2. Collectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression by Nedd4 proteins.
Highlights
Lectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression by Nedd4 proteins
A polyproline type II (PPII) helix is formed by residues Pro-614Ј–Asn617Ј, which bind the XP grove of the WW domain [28], a pocket found in all WW domains and SH3 domains. (ENaC residues are indicated with a “Ј” to distinguish from WW domain residues.) Similar interactions are observed for the PY motifs of -dystroglycan and WBP1 that bind the dystrophin and YAP65 WW domains, respectively [31, 32], or the phospho-Ser-Pro sequence of RNA polymerase II that binds to the WW domain of Pin1 [30]
We have previously measured the affinity of interaction between the 3 WW domains of rNedd4-1 (WW1, WW2, WW4) with the 3 P2 regions (PY motifs) of ENaC, demonstrating that the highest affinity interactions (Kd of 20 M in low salt and ϳ50 M in 150 mM KCl) occurs between P2 and the WW4 domain of rNedd4-1
Summary
Peptides—Peptides representing wild-type or mutant sequences of rat ENaC subunits ␣, , and ␥ were synthesized by the Hospital for Sick Children/Advanced Protein Technology Centre (Toronto, Canada). Lyophilized peptides were re-suspended in 150 mM KCl, 10 mM Kϩ phosphate, pH 6.5, or where indicated, with 150 mM NaCl, 10 mM Naϩ phosphate, pH 6.5. Experiments were measured in 150 mM KCl, 10 mM Kϩ phosphate, pH 6.5, or 150 mM NaCl, 10 mM Naϩ phosphate, pH 6.5, with WW domain concentrations kept constant at 3 M (P2 mutant studies) or 1 M (Nedd isoform study). Oocytes were initially placed in a bath solution containing amiloride (10Ϫ6 M) to prevent changes in intracellular Naϩ concentration, and current was measured after washout of amiloride. A structural alignment of the ligands within Modeler was performed for the PY motif-containing peptides bound to the rNedd, hYAP65, and h-dystrophin WW domains. Macroscopic amiloride-sensitive Naϩ currents, defined as the difference between Naϩ currents obtained in the presence and
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