Affinity and Specificity of Interactions between Nedd4 Isoforms and the Epithelial Na+ Channel

The epithelial Na+ channel (αβγENaC) 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 ∼10 μm for α- or βENaC, 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*·βENaC-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 βENaC-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. The epithelial Na+ channel (αβγENaC) 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 ∼10 μm for α- or βENaC, 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*·βENaC-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 βENaC-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. The epithelial sodium channel (ENaC) 1The abbreviations used are: ENaC, epithelial Na+ channel; x-, Xenopus; m-, mouse; d-, Drosophila; h-, human; WT, wild type; PDB, Protein Data Bank. is an apically located ion channel found in absorptive epithelia of organs involved in fluid and electrolyte homeostasis such as the kidney, lung, distal colon, and ducts of exocrine glands (1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1043) Google Scholar, 2Rossier B.C. Pradervand S. Schild L. Hummler E. Annu. Rev. Physiol. 2002; 64: 877-897Crossref PubMed Scopus (325) Google Scholar). In the kidney, ENaC participates in the unidirectional transport of Na+ ions into epithelial cells of the distal nephron in response to hormonal signaling, particularly aldosterone and vasopressin. ENaC is composed of three homologous subunits (α, β, and γ), each comprised of intracellular N and C termini, two transmembrane domains, and a glycosylated extracellular loop (3Canessa C.M. Merillat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar, 4Canessa C.M. Schild L. Buell Rossier B.C. 1994; PubMed Scopus Google Scholar). The of ENaC subunit to a Merillat A.M. Rossier B.C. Schild L. J. PubMed Scopus Google Scholar, S. J. J. PubMed Scopus Google Scholar). Each ENaC contains two and its C the the which to a PY motif to a for WW domains H. J. C.M. Rossier B.C. J. 1994; PubMed Scopus Google Scholar, L. E. Rossier B.C. J. PubMed Scopus Google Scholar, S. J. J. J. PubMed Scopus Google Scholar). The PY motif of or ENaC is or in with C.M. S. Rossier B.C. 1994; PubMed Scopus Google Scholar, H. Schild L. Rossier PubMed Scopus Google Scholar, Schild L. Rossier B.C. S. PubMed Scopus Google Scholar, H. Schild L. Rossier B.C. J. PubMed Scopus Google Scholar, H. H. S. J. PubMed Scopus Google Scholar, S. S. S. Am. J. PubMed Scopus Google a form of J. 1994; PubMed Scopus Google Scholar, PubMed Scopus Google from of ENaC Schild L. Merillat A.M. E. Rossier B.C. S. PubMed Scopus Google Scholar, C.M. PubMed Scopus Google Scholar). is by an in channel and the Schild L. Merillat A.M. E. Rossier B.C. S. PubMed Scopus Google Scholar). are and ions to regulate ENaC (1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1043) Google Scholar). well of ENaC, which has in is Nedd4 is a ubiquitin ligase comprised of a C2 or WW domains, and a ubiquitin ligase Hect domain S. J. J. J. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, J. PubMed Google Scholar). The C2 domain is involved in S. J. PubMed Scopus Google Scholar, H. J. 1997; PubMed Scopus Google the WW domains are involved in interactions and 1994; PubMed Scopus Google and the Hect domain the ubiquitin ligase S. S. PubMed Scopus Google for and (or of proteins. The between Nedd4 and ENaC between the WW domains of Nedd4 and the PY motifs of ENaC S. J. J. J. PubMed Scopus Google Scholar). WW domains are small of residues in containing two conserved tryptophan residues and an and bind S. PubMed Scopus Google Scholar). Nedd4 WW domains been as WW domains by of for binding the PY as J. PubMed Scopus Google Scholar). determined the structure of the WW domain of Nedd4 to of in complex with the PY of PubMed Scopus Google Scholar). WW domain of Nedd4 a with a binding to other WW domains PubMed Scopus Google Scholar, E. J. H. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. H. J. PubMed Scopus Google Scholar). is formed by residues which bind the of the WW domain PubMed Scopus Google a pocket found in all WW domains and residues are with a to from WW domain interactions are for the PY motifs of and that bind the and WW domains, PubMed Scopus Google Scholar, J. H. J. PubMed Scopus Google or the of that binds to the WW domain of PubMed Scopus Google Scholar). unique to the Nedd4 WW PY a is by residues to the which is by and interactions and PubMed Scopus Google Scholar). that an PY motif conserved in all ENaC is for binding to Nedd4 WW our structure between of and of the PY motif of and the domain of PubMed Scopus Google Scholar). of our is the of the of these contacts to the affinity of binding between ENaC and Moreover, we the of the unique of to binding to the Nedd4 WW domain for the of channel activity. Nedd4 is a of ENaC that regulates the of the H. J. Schild L. J. PubMed Scopus Google Scholar, C.M. J. PubMed Scopus Google in with the of in of ENaC Schild L. J. 1997; PubMed Scopus Google Scholar). which all or of the PY motif of or ENaC binding of the channel to the WW domains of Nedd4, to of ENaC L. E. Rossier B.C. J. PubMed Scopus Google Scholar, C.M. PubMed Scopus Google Scholar, H. J. Schild L. J. PubMed Scopus Google Scholar, C.M. J. PubMed Scopus Google Scholar). are Nedd4 isoforms and J. PubMed Google and in Nedd4-1 and Nedd4-2 isoforms been with to ENaC binding and E. J. PubMed Scopus Google Scholar, E. Am. J. Physiol. Physiol. PubMed Google Scholar). Nedd4-1 and are by has proposed that Nedd4-2 can bind to and regulate ENaC than Nedd4-1 H. J. Schild L. J. PubMed Scopus Google Scholar, E. J. PubMed Scopus Google Scholar, E. Am. J. Physiol. Physiol. PubMed Google Scholar, S. J. PubMed Scopus Google and the Nedd4-2 contains from involved in the of ENaC by aldosterone E. J. J. PubMed Scopus Google Scholar, B.C. J. 2002; PubMed Scopus Google Scholar). a that Nedd4-1 is a of ENaC its E. Am. J. Physiol. Physiol. PubMed Google Scholar, J. PubMed Scopus Google that the suppression of ENaC by the Nedd4 isoforms and family than and Nedd4-2 isoforms from and an WW WW3*, located between and which is from and Nedd4-1 isoforms but is found in and Nedd4-1 WW3* and to in ENaC E. J. PubMed Scopus Google Scholar, E. Am. J. Physiol. Physiol. PubMed Google Scholar). we PubMed Scopus Google and H. PubMed Scopus Google Scholar, J. 2002; PubMed Scopus Google binding between the Nedd4 WW domains and the ENaC PY a between the affinity of interactions with ENaC, particularly of the and WW domains from the Nedd4 and the for ENaC been is a of our results demonstrate that to Nedd4-2 can regulate is the presence of WW3* in either Nedd4-1 or that high affinity binding to the ENaC PY suppression of the channel by the Nedd4 or of ENaC subunits β, and by the for Protein by high a with an and of the by peptide β, and with the for the The peptide in or with Na+ in L. Protein PubMed Scopus Google Scholar). In the of the the and with a of of of with or ENaC to of in a and of WW domain of Nedd4-1 residues Nedd4-2 WW3* residues Nedd4-1 WW3* residues and Nedd4-1 WW3* residues into and as in in with an of for an and cells by for Each cells by and the by a by the The into and on a in (or Na+ Protein in L. Protein PubMed Scopus Google Scholar). and WW3* the and in and and in WW3* are to residues and in WW3* tryptophan of the WW domains used to peptide binding PubMed Scopus Google Scholar, L. 1994; PubMed Scopus Google Scholar). a with and of and and of in or Na+ with WW domain μm or μm ENaC from to Kd for is in and such that Kd and from the the of and the to S. Scopus Google of WW3*, or WW3* domains binding to ENaC or motif C.M. Schild L. Buell Rossier B.C. 1994; PubMed Scopus Google C.M. Schild L. Buell Rossier B.C. 1994; PubMed Scopus Google Merillat A.M. Rossier B.C. Schild L. J. PubMed Scopus Google Merillat A.M. Rossier B.C. Schild L. J. PubMed Scopus Google L. E. Rossier B.C. J. PubMed Scopus Google C.M. Schild L. Buell Rossier B.C. 1994; PubMed Scopus Google Merillat A.M. Rossier B.C. Schild L. J. PubMed Scopus Google C.M. Schild L. Buell Rossier B.C. 1994; PubMed Scopus Google C.M. Schild L. Buell Rossier B.C. 1994; PubMed Scopus Google C.M. Schild L. Buell Rossier B.C. 1994; PubMed Scopus Google C.M. Schild L. Buell Rossier B.C. 1994; PubMed Scopus Google WW3* S. J. J. PubMed Scopus Google in a of ENaC on ENaC as previously L. E. Rossier B.C. J. PubMed Scopus Google Scholar). of each subunit or and of or or or in and with of a containing of β, and ENaC a of The in sodium containing Na+ as the between Na+ in the presence and of μm in the the the to The a containing and in a containing to in intracellular Na+ and of with a Homology of to ENaC models of between the or WW3* domain and the PY motif the J. PubMed Scopus Google Scholar). that the WW3* domain in the Nedd4-2 isoforms and is between and by residues from that in WW domains from Nedd4 and other based on the previously determined of WW domains from PubMed Scopus Google J. H. J. PubMed Scopus Google PubMed Scopus Google PubMed Scopus Google and H. 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PubMed Scopus Google that or in not to in channel been Nedd4 bind to the the of to the of ENaC by Nedd4 is not and of of WW3* and the PY of ability of the Nedd4 isoforms to function as ENaC with Nedd4-2 isoforms proposed to bind to ENaC and to suppress channel than Nedd4-1 isoforms E. J. PubMed Scopus Google Scholar, E. Am. J. Physiol. Physiol. PubMed Google is an that has not been is the presence of an WW WW3*, in Nedd4-2 isoforms as and but not found in of the Nedd4-1 isoforms (e.g. and the of this WW3* we the binding affinity of WW3* from the three ENaC PY motifs and to that of the domain of found in all Nedd4 to these two domains has that WW3* and of Nedd4 the in ENaC E. J. PubMed Scopus Google Scholar, E. Am. J. Physiol. Physiol. PubMed Google Scholar). results show that WW3* binds the PY motif of each ENaC subunit in the with a 3–6-fold higher affinity than and The contains a WW3* domain. its binding affinity to that of in and the WW3* of the higher affinity interactions with the PY motifs of ENaC as that with the WW3* of the Nedd4-2 Moreover, the WW3* of this high affinity binding to and ENaC-PY and that is the presence of WW3* and not the affiliation with Nedd4-1 or that high affinity interactions between Nedd4 and ENaC. of ENaC by the the presence of a WW3* in a Nedd4-1 the ability to suppress ENaC, we of our that in dNedd4-1, which the is naturally with WW3* as a Nedd4-1 its in is to ENaC, the ENaC not PY motifs C.M. J. PubMed Scopus Google Scholar). of into with in a of ENaC to suppression in a dNedd4-1, Moreover, dNedd4-1, to of ENaC all three PY which as binding for Nedd4-WW domains dNedd4-1, a WW3*, can suppress ENaC to Nedd4-2 and in to its which Homology and of WW3* and WW3* and domains of Nedd4 in binding to and ENaC activity. The higher affinity interactions with ENaC of the Nedd4 WW3* with domains to these two domains to the for the higher affinity by models of the and WW3* domains in complex with the peptide of ENaC the J. PubMed Scopus Google Scholar). from the high of between the and WW3* domains, these models are and the to The models with J. 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In our we to binding between a PY motif peptide of and of the Nedd4-2 WW that either this does not (or is of or that a higher affinity in in the of the the complex in cells (e.g. of and Nedd4-2 to to these The Kd for binding of the ENaC PY motifs to Nedd4 WW domains in the μm and PubMed Scopus Google Scholar). of our S. J. PubMed Scopus Google the we in that WW3* of Nedd4-2 binds to the ENaC PY by and for the ENaC PY the Kd by S. J. PubMed Scopus Google are than the for the is an is that S. J. PubMed Scopus Google in the presence of the to the WW used in in other The results we intrinsic tryptophan to we from an peptide PubMed Scopus Google an the Kd In our of the of residues in the PY motif for binding to the Nedd4 WW domains and to the notion that Nedd4-2 can suppress ENaC. we that the presence of the high affinity WW3* domain in either Nedd4-1 or Nedd4-2 the ability to regulate ENaC. H. and for

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