From the
Programmes in Cell Biology and ||Structural Biology and Biochemistry, Hospital
for Sick Children, Toronto, Ontario M5G 1X8, Canada,
Institute of Medical Science and
Department of Biochemistry,
University of Toronto, Toronto, Ontario M5S 1A8, Canada, and
Institut de Pharmacologie et de
Toxicologie, Université de Lausanne, Lausanne, Switzerland
CH-1005
Received for publication, October 31, 2002 , and in revised form, March 18, 2003.
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ABSTRACT |
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INTRODUCTION |
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Nedd4 is a ubiquitin protein ligase (E3) comprised of a C2 domain, 3 or 4 WW domains, and a ubiquitin ligase Hect domain (9, 20, 21). The C2 domain is involved in membrane targeting (22, 23), the WW domains are involved in protein-protein interactions and substrate recognition (24) (see below), and the Hect domain provides the catalytic ubiquitin ligase (E3) activity (25) responsible for ubiquitination and degradation (or endocytosis) of target proteins. The interaction between Nedd4 and ENaC takes place between the WW domains of Nedd4 and the PY motifs of ENaC (9).
WW domains are small modules of 40 residues in length containing two highly conserved tryptophan residues and an invariant proline and bind proline-rich sequences (26). Nedd4 WW domains have been grouped as Class I WW domains by virtue of their specificity for binding the PY motif, most recently defined as (L/P)PXY (27). We have recently determined the solution structure of the third WW domain of rat Nedd4 (homologous to WW4 of human Nedd4s, hereafter called rNedd4-1 WW4) in complex with the PY motif-containing (P2 region) of
ENaC (28). This WW domain of Nedd4 adopts a three-stranded anti-parallel
-sheet with a hydrophobic binding surface, similar to other WW domains (28, 29, 30, 31, 32). A polyproline type II (PPII) helix is formed by residues Pro-614'Asn-617', 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 "'" 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). However, unique to the Nedd4 WW domain-
ENaC PY complex, a helical turn is adopted by residues Tyr-618'Leu-621' (C-terminal to the PPII helix), which is stabilized by intrapeptide and peptide-domain interactions involving both Tyr-618' and Leu-621' (28). This suggests that an elongated (extended) PY motif sequence (PPXYXXL), conserved in all ENaC chains, is responsible for binding to Nedd4 WW domains. Thus, our tertiary structure reveals contact points between side chains of Pro-615', Pro-616', Tyr-618', and Leu-621' of the extended PY motif of
ENaC (615PPNYDSL621) and the WW4 domain of rNedd4-1 (28). One focus of our present work is the investigation of the contribution of these contacts to the affinity of binding between ENaC and Nedd4. Moreover, we have probed the importance of the unique contribution of Leu-621' to binding to the Nedd4 WW domain for the regulation of channel activity.
Nedd4 is a suppressor of ENaC that regulates the number of channels at the plasma membrane (33, 34) in agreement with the role of ubiquitination in regulating cell surface stability of ENaC (35). Accordingly, Liddle's syndrome mutations, which eliminate all or part of the PY motif of or
ENaC and, thus (at least partially), impair binding of the channel to the WW domains of Nedd4, lead to increased cell surface stability of ENaC (8, 19, 33, 34).
There are several Nedd4 isoforms and relatives (21), and in particular Nedd4-1 and Nedd4-2 isoforms have been studied with regard to ENaC binding and regulation (36, 37). Although called "isoforms," Nedd4-1 and -2 are encoded by different genes. Recent work has proposed that Nedd4-2 can bind to and regulate ENaC activity much better than Nedd4-1 (33, 36, 37, 38), and moreover, the Nedd4-2 isoform contains sgk1 phosphorylation sites (missing from Nedd4-1), possibly involved in the regulation of ENaC by aldosterone (39, 40). However, a puzzling observation demonstrating that human Nedd4-1 (hNedd4-1) is a potent inhibitor of ENaC when lacking its N-terminal region (37, 41) suggests that the suppression of ENaC by the different Nedd4 isoforms and family members may be more complicated than originally anticipated and requires further investigations.
All Nedd4-2 isoforms (including those from Xenopus, mouse, and human, i.e. x/m/hNedd4-2) contain an "extra" WW domain, WW3*, located between WW2 and WW4, which is lacking from rat and mouse Nedd4-1 isoforms but is found in hNedd4-1 and Drosophila Nedd4-1 (dNedd4-1) (see Fig. 1). Both WW3* and WW4 appear to play key roles in regulating ENaC activity (36, 37). Although we (28) and others (42, 43) have quantified binding between the Nedd4 WW domains and the ENaC PY motifs, a comparison between the affinity of interactions with ENaC, particularly of the third and fourth WW domains from the different Nedd4 isoforms, and the consequence for ENaC suppression, have been lacking and, hence, is a second focus of our work. Our results presented here demonstrate that contrary to previous suggestions (that only Nedd4-2 proteins can regulate ENaC), it is the presence of WW3* in either Nedd4-1 or 2 that provides high affinity binding to the ENaC PY motifs, promoting suppression of the channel by the WW3*-containing Nedd4 protein.
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EXPERIMENTAL PROCEDURES |
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Expression and Purification of ProteinsThe third WW domain of rat Nedd4-1 (renamed WW4) (accession number AAB48949
[GenBank]
, residues 451498), Xenopus Nedd4-2 WW3* (accession number CAA03915
[GenBank]
, residues 489528), human Nedd4-1 WW3* (accession number BAA07655
[GenBank]
, residues 362411), and Drosophila Nedd4-1 WW3* (dNedd4-1(short), accession number CG7555(RD), residues 430469) were sub-cloned into pQE-30 and expressed as N-terminal MRGS His6-tagged proteins in Escherichia coli M15 pREP4 at 37 °C in LB (Sigma). Bacteria were induced with 1 mM isopropyl-1-thio--D-galactopyranoside (Promega) at an A600 of 0.6 for an additional 3 h at 37 °C, and cells were harvested by centrifugation at 6000 x g for 10 min. Each poly-His-tagged protein was purified after lysing cells by sonication and applying the soluble supernatant (cleared by 10,000 x g centrifugation) over a Ni2+-nitrilotriacetic acid-charged resin column (as described by the manufacturer, Qiagen). The protein was dialyzed overnight into 10 mM K+ phosphate, pH 6.5, plus 0.1 mM EDTA, 0.15 µg/ml aprotinin, 0.15 µg/ml leupeptin, 0.1 mM benzamidine at 4 °C before concentrating and purification on a Superdex 75 gel filtration column (Amersham Biosciences) in 150 mM KCl, 10 mM K+ phosphate, pH 6.5 (or Na+ replacing K+, where indicated). Protein concentrations were measured in 6.0 M guanidine HCl at A280 (44). rNedd4-1 WW4 His-Thr
Ala-Pro (H470A, T471P) and hNedd4-1 WW3* Ala-Pro
His-Thr (A381H, P382T) substitutions were generated using the QuikChange site-directed mutagenesis kit (Stratagene). His-470 and Thr-471 in rNedd4-1 WW4 and Ale-381 and Pro-382 in hNedd4-1 WW3* are equivalent to residues Ala-504 and Pro-505, respectively, in xNedd4-2 WW3* (Fig. 1B).
Equilibrium Dissociation Constant (Kd) MeasurementIntrinsic tryptophan fluorescence of the WW domains was used to monitor peptide binding (28, 45). Fluorescence measurements were obtained using a Hitachi F-2500 fluorescence spectrophotometer at 25 °C with excitation and emission wavelengths of 298 and 333 nm, respectively, and slit widths of 2.5 nm. Experiments were measured in 150 mM KCl, 10 mM K+ phosphate, pH 6.5, or 150 mM NaCl, 10 mM Na+ phosphate, pH 6.5, with WW domain concentrations kept constant at 3 µM (P2 mutant studies) or 1 µM (Nedd4 isoform study). ENaC peptides were added at concentrations ranging from 0 to 2 mM. Kd measurements for n experiments, where n is indicated in Tables I and II, were log-transformed such that residuals were normally distributed. Reported Kd means and S.E. were then calculated from the transformed data using the
method conversion. A one-way analysis of variance was performed, and pair-wise differences were examined using the Tukey adjustment to determine adjusted p values (46).
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Electrophysiological Measurement of Mutant ENaC ActivitySite-directed mutagenesis was performed on rat ENaC cDNA as described previously (8). Complementary RNAs of each subunit (WT or mutants) and of xNedd4-2 (WT or CS mutant) or dNedd4-1 (WT or CA mutant) were synthesized in vitro. Healthy stage V and VI Xenopus oocytes were pressure-injected with 100 nl of a solution containing equal amounts of ,
, and
ENaC complementary RNA at a total concentration of 100 ng/µl. The oocytes were kept in modified low sodium Barth's saline containing 10 mM NaCl, 2 mM KCl, 80 mM n-methyl-D-glucamine chloride, 0.4 mM CaCl2, 0.3 mM CaNO3, 0.8 mM MgSO4, 5 mM n-methyl-D-glucamine-Hepes, pH 7.4.
Standard electrophysiological measurements were taken 1620h after injection. Macroscopic amiloride-sensitive Na+ currents, defined as the difference between Na+ currents obtained in the presence and absence of 5 µM amiloride (Sigma), in the bath were recorded using the two-electrode voltage-clamp technique. For current measurements the oocytes were voltage-clamped to 100 mV. The bath solution was a standard oocyte Ringer solution containing 120 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, and 10 mM Hepes. Oocytes were initially placed in a bath solution containing amiloride (106 M) to prevent changes in intracellular Na+ concentration, and current was measured after washout of amiloride. Currents were recorded with a Dagan TEV-200 amplifier (Minneapolis, MN).
Homology Modeling of Nedd4-WW3* Domain Complexed to P2 ENaC PeptideHomology models of complexes between the xNedd4-2 or hNedd4-1 WW3* domain and the
ENaC PY motif were obtained using the program Modeler (47). Note that the WW3* domain in the Nedd4-2 isoforms (xNedd4-2, mNedd4-2, and hNedd4-2) is identical between species and differs by four residues from that in hNedd4-1. WW domains from Nedd4 and other proteins were aligned based on the previously determined structures of WW domains from rNedd4-1 (PDB code 1I5H
[PDB]
) (28), hYAP65 (PDB code 1JMQ
[PDB]
) (32), h-dystrophin (PDB code 1EG4
[PDB]
) (31), hPin1 (PDB code 1F8A
[PDB]
) (30), mFBP28 (PDB code 1E0I
[PDB]
), and yeast YJQ8 (PDB code 1E0N
[PDB]
) (48). A structural alignment of the ligands within Modeler was performed for the PY motif-containing peptides bound to the rNedd4-1, hYAP65, and h-dystrophin WW domains. The Nedd4, dystrophin and YAP65 WW domain complexes, the Pin1 WW domain in the context of the complex, and the free FBP28 and YJQ8 WW domains were used as templates. A total of 100 models were generated from which the 15 lowest energy models were selected for analysis.
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RESULTS |
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Contribution of Pro-616' and Tyr-618' of the PY Motif to Binding to the WW4 DomainFig. 2 and Table I demonstrate that, consistent with our previous work (28), saturable binding was observed between rNedd4-1 WW4 and P2 WT peptide with a calculated Kd of 54 ± 8 µM (all Kd values were measured in 150 mM KCl, 10 mM K+ phosphate, pH 6.5 unless otherwise indicated). In contrast, no measurable binding was observed when either Pro-616' or Tyr-618' were replaced with Ala even at peptide concentrations greater than 1.2 mM. Pro-616' along with Pro-615' binds the XP groove formed by Trp-487 and Phe-476 in rNedd4-1 WW4. Mutation of this essential proline eliminates critical interactions with the aromatic residues in the WW domain (28). Tyr-618' binds into a hydrophobic pocket created by Ile-478, His-480, and Lys-483 in the WW domain. Mutation to a smaller Ala residue disrupts the stabilizing interactions between the aromatic ring of Tyr-618' and this pocket on the WW domain. In addition, Pro-616' and Tyr-618' were found mutated in Liddle's syndrome patients (12, 13), underscoring their physiological significance.
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Contribution of Pro-615' and Leu-621' of the PY Motif to Binding to the WW4 DomainA lesser but still significant degree of disruption of the rNedd4-1 WW4 domain-P2 peptide binding interaction was observed when either Pro-615' or Leu-621' was mutated to Ala. Substitution of Pro-615' reduced binding and increased the Kd by a factor of 6 (p < 0.001) compared with WT binding (Fig. 2 and Table I). This residue binds into the Xaa position of the XP groove, and although often a Pro residue, large aliphatic groups (Leu, Ile) are also accommodated and may even be preferred (27).2 The small Ala residue, however, cannot optimally fill this pocket (49), resulting in decreased binding affinity. Significantly, P615'S is a mutation also linked genetically to Liddle's syndrome (14).
A 6-fold increase in Kd (p < 0.001) resulted when Leu-621' was replaced by Ala (Fig. 2 and Table I). As described earlier (28), the methyl groups of Leu-621' are involved in multiple peptide-peptide and peptide-domain interactions; hence, its mutation disrupts those interactions, leading to reduced binding affinity.
Role of ENaC-Leu-621' in Channel ActivitySo far, there are no known Liddle's mutations in Leu-621', but based on our structure (28) and binding data (Table I), we tested whether a L621'A mutation could elevate ENaC activity, as previously shown for mutations in the Pro and Tyr residues of the PY motif (8). Fig. 3A shows that a L621'A substitution in
ENaC indeed leads to a 2-fold elevation of ENaC activity when expressed in Xenopus oocytes; this increase, although significant, is much smaller than that seen with mutation of Pro-616' or Tyr-618' (Fig. 3A). In agreement, the elevation of channel activity by a catalytically inactive Nedd4(CS), acting in a dominant-negative fashion to block function of endogenous xNedd4-2 (33), is larger in the L621'A mutant than in the Pro-616' or Tyr-618' mutants (Fig 3C). As expected, WT-Nedd4 suppresses all these mutants (Fig. 3B), likely due to the presence of other, intact PY motifs in ENaC. Collectively, these results demonstrate that although the L621'A shows weaker binding to the Nedd4-WW4 domain than wild type, it still binds with a higher affinity than the Pro-616' and Tyr-618' mutants.
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Role of Asp-619' and Phosphorylation of Ser-620'In the solution structure of rNedd4-1 WW4 domain-ENaC-P2 peptide (28), no definitive role was predicted for Asp-619'. Few restraints were observed for Asp-619' in the complex, and the ensemble of NMR structures indicates that multiple orientations are possible for the side chain. Nevertheless, its negative charge could potentiate charge-charge interactions with a lysine residue (Lys-483) in the WW domain or be necessary for the overall integrity of the motif. Upon replacement of Asp-619' with Ala, however, only a very small difference in Kd could be detected (p = 0.20), which is not statistically significant (Table I). These results are consistent with a lack of effect of the D619'A mutation on ENaC activity (8).
The extended PY motif of ENaC also includes a potential phosphorylation site at Ser-620'. As demonstrated by recent studies, phosphorylation of residues near or within the PY motif of
or
ENaC has been implicated as an additional means of channel regulation (50, 51, 52). We, therefore, investigated the effect of phosphorylation of Ser-620' (Ser(P)-620') on binding to rNedd4-1 WW4 domain. As seen in Fig. 2 and Table I, phosphorylation of Ser-620' slightly increased binding affinity (by almost 2-fold, p = 0.16), an effect that did not reach statistical significance. Accordingly, the work of Schild et al. (8) demonstrates that S620'V or S620'E substitutions in
ENaC do not lead to significant changes in channel activity (as would have been expected if Nedd4 could bind more tightly to the phosphorylated Ser-620'). Thus, the significance of Ser-620' to the regulation of ENaC by Nedd4 is currently not clear and needs further investigation.
Contribution of WW3* Domain to the Nedd4-ENaC Interaction
Comparison of Binding Affinity of WW3* and WW4 toward the PY Motifs of ENaCThe differential ability of the Nedd4 isoforms to function as ENaC repressors, with Nedd4-2 isoforms proposed to bind better to ENaC and to more potently suppress channel activity than Nedd4-1 isoforms (36, 37), is an observation that has not yet been fully explained. An obvious culprit is the presence of an extra WW domain, WW3*, in Nedd4-2 isoforms (such as xNedd4-2, mNedd4-2, and hNedd4-2) but not found in most of the Nedd4-1 isoforms (e.g. rNedd4-1 and mNedd4-1) (Fig. 1). To assess the role of this divergent WW3* domain, we measured the binding affinity of WW3* from x/m/hNedd4-2 toward the three ENaC PY motifs and compared it to that of the WW4 domain of rNedd4-1, found in all Nedd4 isoforms. We chose to compare these two domains because previous work has demonstrated that WW3* and WW4 of Nedd4 play the most pivotal roles in regulating ENaC activity (36, 37). Our results show that WW3* binds the PY motif of each ENaC subunit (included in the P2 region, i.e.
P2,
P2,
P2) with a 36-fold higher affinity than WW4 (Fig. 4 and Table II). The human hNedd4-1, however, also contains a WW3* domain. We thus compared its binding affinity to that of rNedd4-1 WW4. As seen in Fig. 4 and Table II, the WW3* of hNedd4-1 shows the same higher affinity interactions with the PY motifs of ENaC as that seen with the WW3* of the Nedd4-2 isoforms. Moreover, the WW3* of dNedd4-1 (see below) also exhibited this high affinity binding to
and
ENaC-PY motifs.3 This suggests that it is the presence of WW3* and not the affiliation with Nedd4-1 or -2 isoforms, that imparts high affinity interactions between Nedd4 and ENaC.
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Suppression of ENaC Activity by the WW3*-containing dNedd4-1To test whether the presence of a WW3* in a Nedd4-1 protein could impart the ability to suppress ENaC, we took advantage of our observation that in dNedd4-1, which most closely resembles rNedd4-1 (its likely orthologue), the WW2 is naturally replaced with WW3* (Fig. 1). DNedd4 was chosen here as a model Nedd4-1 (full-length) protein, although its natural substrate in flies is unlikely to be ENaC, since the fly ENaC homologues (Pickpocket, Ripped pocket) do not have PY motifs (53). We thus injected complementary RNA of dNedd4-1 into Xenopus oocytes together with ENaC. As seen in Fig. 5, dNedd4-1 was a strong suppressor of ENaC (
90% suppression), similar to xNedd4-2. This suppression was prevented in a catalytically inactive mutant dNedd4-1, dNedd4-1(CA) (Fig. 5). Moreover, dNedd4-1, like xNedd4-2, was unable to inhibit activity of ENaC lacking all three PY motifs, which serve as binding sites for Nedd4-WW domains (Fig. 5). Thus, dNedd4-1, possessing a WW3*, can effectively suppress ENaC activity, similar to Nedd4-2 proteins and in sharp contrast to its orthologue, rNedd4-1, which lacks WW3*.
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Homology Modeling and Comparison of WW3* and WW4 ComplexesAs indicated above, both WW3* and WW4 domains of Nedd4 play key roles in binding to and regulating ENaC activity. The higher affinity interactions with ENaC of the Nedd4 WW3* compared with WW4 domains prompted us to compare these two domains to identify the reason for the higher affinity imparted by WW3*.
We have generated homology models of the x/m/hNedd4-2 and hNedd4-1 WW3* domains in complex with the P2 peptide of ENaC using the program Modeler (47). As expected from the high degree of sequence identity (88.6%) between the xNedd4-2 and hNedd4-1 WW3* domains, these models are very similar, and hence, only the xNedd4-2 WW3*·
ENaC model will be referred to here. The xNedd4-2 WW3*·
ENaC models were examined with PROCHECK (54) and were shown to have good geometry and sampled favorable regions of Ramachandran space with 91, 7, and 1% of residues in the favorable, allowed, and disallowed regions, respectively. These models are well defined with a pairwise root mean square deviation of 0.20 ± 0.04 Å and 0.72 ± 0.10 Å for backbone and all heavy atoms, respectively. This degree of precision in the models indicates that the same conclusions are obtained for all members of the ensemble. The generated models are very similar to our recently determined solution structure of the rNedd4-1 WW4 domain-
P2 peptide complex, with backbone root mean square deviation values of 0.33 ± 0.03 Å. This is expected given the sequence identity between rNedd4-1 WW4 and xNedd4-2 WW3* domains of 65.7% for all residues and 77.8% for residues involved in ligand binding (Fig. 1). Furthermore, because of the high degree of sequence conservation between these domains, there is a high degree of confidence in the model of the x/m/hNedd4-2 WW3* domain-ENaC
P2 peptide complex, supporting the validity of the conclusions obtained from analysis of this model.
Examination of the peptide binding site reveals an amino acid substitution (Fig. 6); His-470 in rNedd4-1 WW4, which forms the back of the XP groove and makes extensive contacts to the bound peptide through its aromatic ring, is an Ala (Ala-504) in WW3*. The result is a larger pocket to accommodate Pro-616' and Pro-615'. In addition, the presence of a Pro (Pro-505) residue in WW3* instead of a Thr (Thr-471) in WW4 provides additional interactions to Pro-616'. In contrast to Thr-471 in the rNedd4-1 WW4, the side chain of Pro-505 is oriented toward the binding interface, allowing for interactions with the P2 peptide (Fig. 6B) not seen in the rNedd4-1 WW4 complex. The combination of a larger XP groove to accommodate the ligand and additional contacts conferred by the Pro residue likely contributes to the greater binding affinity of WW3* for ENaC PY motifs.
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Mutation Analysis to Test the Role of WW3* Ala-504/Pro-505 in Conferring High Affinity Binding to PY MotifsThe calculated model depicted in Fig. 6 implicates Ala-504/Pro-505 in contributing to the high affinity binding of WW3* to the PY motifs of ENaC. To experimentally test this prediction, we mutated Ala-504/Pro-505 in WW3* to His-504/Thr-505 to mimic the equivalent residues in WW4 (and numerous other WW domains), which do not bind as tightly to the ENaC PY motifs. Table II shows that such substitutions indeed reduced binding affinity to the ENaC-PY motif 23-fold, in support of the model (Fig. 6). However, the reciprocal substitutions in WW4 (H470A/T471P) did not lead to gain of high affinity binding toward the
ENaC PY motif (Table II). Taken together, these results suggest that the Ala-504/Pro-505 in WW3* are necessary but not sufficient to impart high affinity interactions of this domain toward the PY motifs of ENaC and that another region(s) in the domain is likely involved as well.
Role of Na+ in PY Motif-WW Domain InteractionsElevation of intracellular Na+ concentration is well known to inhibit ENaC activity (55, 56, 57, 58), and moreover, previous work suggests that the regulation of ENaC by Nedd4 in salivary glands is dependent on intracellular Na+ concentrations (59). Substituting 150 mM NaCl for 150 mM KCl in the binding solution to mimic the high intracellular concentration of Na+, however, did not alter the affinity of WW3* to P2 (Table II), suggesting that the inhibitory role of intracellular Na+ is not mediated by directly affecting the binding of Nedd4 to ENaC. These results are in agreement with those of Asher et al. (42).
Binding of sgk1-PY Motif to Nedd4-2-WW DomainsThe serum- and glucocorticoid-induced kinase, sgk1, which possesses a PY motif (PPFY), has been proposed recently to bind Nedd4 and to be involved in the regulation of ENaC by Nedd4-2 (39, 40). To test the affinity of these interactions, we generated a sgk1 peptide (LYGLPPFYSRNTAE), which includes the PPFY motif, and tested its binding affinity toward the WW1, WW2, WW3*, and WW4 domains of Nedd4-2. We were unable to detect saturable binding between the sgk1-PY motif and any of the Nedd4-2 WW domains (up to 1.5 mM peptide) (Table III), suggesting that sgk1 does not bind (or binds very poorly) to the WW domains of Nedd4-2.
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DISCUSSION |
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The solution structure of the rNedd4-1 WW4 domain complexed to the extended PY motif of ENaC (615PPNYDSL621) provided many insights into the basis of their interaction (28). First, the hydrophobic binding surface of the domain forms two important stabilizing surfaces with the PY motif ligand; an XP groove that accommodates the polyproline type II (PPII) helix containing the first 2 prolines of the PY motif, Pro-615' and Pro-616', and a hydrophobic binding pocket, which makes many important contacts with Tyr-618'. We show here that the invariant Pro-616' and Tyr-618' are absolutely required for binding, because their replacement by Ala prevents any saturable binding. In Liddle's syndrome, mutations at both positions lead to a full disease phenotype (12, 13). Substitution of Pro-616' (or Pro-615'; see below) destabilizes the PPII helix by leaving only two Pro residues instead of three, which ultimately compromises binding to the XP groove in Nedd4 WW domains. Its substitution to Ala in our mutant
ENaC peptide or to Ser/Leu in Liddle's syndrome eliminates critical hydrophobic stacking interactions with Phe-476 of the WW domain. Tyr-618' is also intolerant of substitution, because essential hydrophobic interactions between the aromatic ring with His-480, Ile-478, and the aliphatic region of Lys-483 of the WW domain are lost.
Liddle's syndrome has also been described for the P615'S mutation in the PY motif, a position that when replaced by Ala does not prevent binding to rNedd4-1 WW4 but reduces the affinity by 6-fold compared with wild type. Although other residues are tolerated in this position in ligands that bind WW domains in the minus () orientation, a polar Ser residue in this position or in place of Pro-616' is deleterious for binding into the hydrophobic XP groove (28). Although the Ala substitution in our mutant ENaC peptide is complimentary with the hydrophobic nature of the XP groove, its small side chain is likely to cause a cavity in the binding site leading to reduced binding affinity for this peptide.
A striking feature of the rNedd4-1 WW4 domain-P2 peptide solution structure (28) is the numerous contacts made between the methyl groups of Leu-621' and the WW domain, providing additional binding energy. This is facilitated by a sharp helical turn formed by a normally extended YXXL sequence, thus emphasizing the importance of residues C-terminal to the traditional PY motif for binding to the Nedd4 WW domain. Accordingly, a L621'A substitution dramatically reduces binding affinity of the extended PY motif to the WW domain, although not to the same extent as a P616'A or Y618'A substitutions. In agreement, ENaC activity is elevated 2-fold in
ENaC bearing a L621'A mutation relative to 68-fold in a Pro-616' or Tyr-618' substitutions (Fig. 4). A mild (but significant) increase in ENaC activity in the L621'A mutant was also observed by Snyder et al. (19). The small effect of the mutations in the PY motif of
ENaC on the suppressive effect of Nedd4 is to be expected (see Ref. 36) because Nedd4 can also bind the other ENaC subunits, which are intact (Table II), and a high enough concentration of WT-Nedd4 allows binding to lower affinity sites. So far, there have not been any Liddle's syndrome patients reported with a mutation in Leu-621'. This could be due to the rarity of the disease or because this mutation may result in a milder phenotype not easily diagnosed.
Although earlier work (34) demonstrates that rNedd4-1 can suppress ENaC activity when measured in Xenopus oocytes, more recent work has proposed that only Nedd4-2 isoforms (e.g. xNedd4-2, m-Nedd4-2), but not Nedd4-1 isoforms (e.g. rNedd4-1, mNedd4-1), can bind to and regulate ENaC activity (33, 36, 37). However, our observation (Table II) and that of Lott et al. (43) of a high affinity interaction of WW3* domain to the ENaC PY motifs as well as the observation of strong inhibition of ENaC activity by dNedd4-1, which possesses a WW3* instead of a WW2 domain (Fig. 5), renders this classification inappropriate. In support, a construct composed of only WW3*-WW4-Hect of either hNedd4-1 or hNedd4-2 (37) or with hNedd4-1 or WWP2 (a Nedd4-like protein) lacking their N terminus (37, 41, 60) is also able to suppress ENaC. Taken together, our work suggests that it is the presence of WW3* rather than the type of Nedd4 isoform or relative that provides a key determinant of high binding affinity between Nedd4 and ENaC, necessary for channel regulation by Nedd4. Although necessary, it is unlikely that WW3* alone is sufficient for ENaC regulation (41). However, it is clear that the presence of this high affinity binder alongside WW4 (which binds ENaC with moderate affinity) is sufficient to suppress ENaC function, at least in Xenopus oocytes. Obviously, other regions of Nedd4 proteins and isoforms are likely to be important for regulation of their substrates, including ENaC, in native tissues/cells.
Previous work by Harvey et al. (61) using a dominant negative approach with Nedd4 WW domains to prevent the inhibition of ENaC by elevated intracellular Na+ concentrations (Na+i feedback inhibition) in mandibular duct cells has not revealed blockade of inhibition by hNedd4-1 WW3* domain. This would suggest that at least with a dominant negative approach, hNedd4-1 WW3* domain is not sufficient to interfere with the Na+ feedback loop of ENaC. The comparison of that work with ours (Fig. 5) is not straight forward, however, because we studied the effect of Nedd4-mediated inhibition of ENaC under normal, not elevated intracellular salt concentrations (i.e. our studies did not focus on the Na+ feedback loop).
Through our comparison of the solution structure of the WW4 domain complex with our modeled structure of the WW3* domain complex, we have identified two residues in the WW domain that facilitate more stable interactions with its PY motif ligand. Consistent with in vivo data, the additional, divergent WW domain (WW3*) displays a significantly higher binding affinity (36-fold) for PY motifs. Models of the binding pocket (Fig. 6) suggest that this increased binding is due, at least in part, to the amino acid replacement of His-470 and Thr-471 in WW4 to Ala-504 and Pro-505 in WW3*, respectively. Indeed, A504H/P505T substitutions in WW3* leads to severe reduction in binding affinity toward the PY motif of ENaC (Table II). Residues found in the hydrophobic cluster in WW4 are conserved in WW3*, indicating that the stability of these domains is very similar and that difference in binding affinity is due primarily to changes in residues involved in ligand recognition. Substitutions of His-470 and Thr-471 change the nature of the XP groove, contributing to specificity and affinity. In the WW3* domain, the smaller Ala residue in WW3* (Ala-504) creates a larger XP groove (Fig. 6B) that may better accommodate the Pro and other aliphatic residues found in the X position. The WW1 and WW2 domains have an acidic Asp in this position, explaining their weak affinity for the hydrophobic XP dipeptides of the ENaC PY motifs (28). The Pro residue in WW3* in place of Thr-471 in WW4 makes additional contacts to Pro-616' in the peptide (Fig. 6A). Unlike the case for Ala-504, residues corresponding to Pro-505 are in different orientations in the solved structures of WW domain-ligand complexes. Thr-471 in WW4 and the structurally homologous residue in the YAP65 WW domain are oriented away from the binding site and have multiple conformations in the NMR ensembles of these structures. In contrast, Pin1 contains an Arg residue in this position that is oriented toward the phosphate group of the phospho-Ser residue bound in the XP groove. The dystrophin WW domain also contains a Pro residue in this position that adopts a conformation in both the free and peptide-complex states very similar to that of Pro-505 in our model. Because of the substitution of the backbone N in proline residues, Pro-505 in WW3* is restricted in its conformation and not able to adopt orientations seen for Thr-471 in the WW4 domain. Therefore, in addition to providing increased interactions with
ENaC, Pro-505 may also restrict the conformations of the
1/
2 loop in the free WW3* domain, resulting in a smaller entropic cost upon binding, which ultimately leads to a higher affinity interaction.
These properties likely contribute to the increased affinity of WW3* for ENaC PY motifs. However, although possessing a Ala-504/Pro-505 in WW3* is necessary to achieve high affinity interactions with the ENaC PY motifs, it is not sufficient because substituting the His-Thr with Ala-Pro at the equivalent position in WW4 does not restore high affinity binding. Thus, another site(s) in the WW3*, not yet identified, contributes to the high affinity binding as well.
Other models were recently published (43) to explain the interactions between the WW domains of hNedd4-1 and ENaC PY motifs. Models for - and
ENaC complexes used the
-dystroglycan ligand bound to the dystrophin WW domain as a docking template, which is not the mode in which the PY motif of
ENaC binds the WW domains of Nedd4 (28). In contrast, using the ligand from the solution structure of the rNedd4-1 WW4 domain-
P2 peptide complex to model the
ENaC complexes results in a helical turn structure for the YXXL residues. In the models of
ENaC complexes reported by Lott et al. (43), Leu-676' does not contact the WW domains, whereas the helical turn adopted by the YXXL residues in our models results in interactions between Leu-621' in
ENaC and the aliphatic portion of Arg-502 in the WW3* domain. This interaction, also observed in our solution structure of the WW4 domain-
ENaC peptide complex, is consistent with our binding data (Table I) and functional data (Fig. 3), which show that a L621'A mutation decreases binding to the WW domain and results in increased ENaC activity compared with wild type. Since the YXXL sequence is conserved in all ENaC subunits, a difference in mode of binding for ENaC PY motifs is surprising. Furthermore, the corresponding residue for Arg-502 in the WW3* domain is either an Arg or Lys in other Nedd4 WW domains, suggesting that the interaction with the C-terminal Leu of the YXXL sequence is conserved in all Nedd4 WW domain-ENaC PY motif complexes. Thus, our model in which the ENaC ligands adopt a helical conformation, permitting this interaction, is consistent with structural, biochemical, and functional data.
Recent work demonstrates that sgk1, which possesses a PY motif (PPFY), binds to Nedd4 and phosphorylates Nedd4-2 isoforms, leading to reduced ability of Nedd4-2 to down-regulate ENaC and, hence, to increased ENaC activity (39, 40). In our current work, however, we were unable to detect saturable binding between a PY motif peptide of sgk1 and any of the Nedd4-2 WW domains. This suggests that either this direct interaction does not exist (or is of very low affinity) or that a higher affinity interaction may be achieved in vivo in the context of the full-length sgk1 protein. Alternatively, the interaction may be indirect. Clearly, complex formation in native cells (e.g. kidney cortical collecting ducts) of endogenous sgk1 and Nedd4-2 needs to be demonstrated to sort out these issues.
The Kd values for binding of the wild-type ENaC PY motifs to Nedd4 WW domains range in the low µM range (present work and Ref. 28). A recent report, published after submission of our manuscript (62), supports the trend we find here, in that WW3* of Nedd4-2 binds most strongly to the ENaC PY motifs, followed by WW4. WW1 and WW2 have low affinities for the ENaC PY motifs. However, the actual Kd values provided by Fotia et al. (62) are 10-fold lower than ours. Although the reason for the difference is unknown, an obvious explanation is that Fotia et al. (62) carry out their measurements (using surface plasmon resonance) in the presence of the glutathione S-transferase still fused to the WW domains. Because glutathione S-transferase dimerizes, it cannot be used in surface plasmon resonance (nor in other methods). The results we obtained using intrinsic tryptophan fluorescence were very similar to those we obtained from an NMR peptide titration (28), an entirely independent method, thus validating the Kd values presented here.
In summary, our work presented here provides quantitative analysis of the importance of residues in the PY motif for binding to the Nedd4 WW domains and provides new data to dispel the recent notion that only Nedd4-2 proteins can suppress ENaC. Rather, we propose that the presence of the high affinity WW3* domain in either Nedd4-1 or Nedd4-2 proteins dictates the ability to regulate ENaC.
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FOOTNOTES |
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¶ Supported by a Canadian Institute of Health Research Studentship.
** Supported by a National Science and Engineering Research Council of Canada postdoctoral fellowship.
¶¶ Supported by Canadian Institute of Health Research Investigator awards.
|||| To whom correspondence should be addressed: Program in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5098; Fax: 416-813-5771; E-mail: drotin{at}sickkids.ca.
1 The abbreviations used are: ENaC, epithelial Na+ channel; x-, Xenopus; m-, mouse; d-, Drosophila; h-, human; WT, wild type; PDB, Protein Data Bank.
2 P. C. Henry and D. Rotin, unpublished results.
3 M. C. O'Brien and D. Rotin, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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