A single WW domain is the predominant mediator of the interaction between the human ubiquitin-protein ligase Nedd4 and the human epithelial sodium channel.
The activity of the epithelial Na(+) channel (ENaC) is required for the maintenance of salt and water balance in the body. Channel activity is regulated by the ubiquitin-protein ligase Nedd4 ['neuronal precursor cell-expressed developmentally down-regulated (gene 4)'] that interacts with the channel via its WW domains. Mutations in channel subunits that disrupt this interaction cause Liddle's syndrome, a severe inherited form of hypertension. In previous studies we showed that WW domains 2, 3 and 4 of human Nedd4 bound to the human ENaC (hENaC) subunits, whereas WW domain 1 did not. Here we extend this observation to determine the binding affinities of the human Nedd4 WW domains for hENaC C-terminal peptides. We show that WW domains 2, 3 and 4 bind with differing affinities to Na(+) channel subunit peptides. WW domain 3 has the highest affinity and we predict that WW domain 3 contributes most of the binding because a construct containing the three WW domains bound no better than WW domain 3 alone. Further, a single amino acid change (Arg(165)-->Thr) in WW domain 1 enables binding to the alpha subunit of the channel to occur, with an affinity comparable with that of WW domain 4. Differential binding propensities between the various WW domains and Na(+) channel subunit peptides are explained on the basis of quantitative structural modelling of the complexes and their isolated components.
Lott
J Shaun
JS
School of Biological Sciences, University of Auckland, Private Bag 92-019, Auckland, New Zealand.
Coddington-Lawson
Sarah J
SJ
Teesdale-Spittle
Paul H
PH
McDonald
Fiona J
FJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D015536:Down-Regulation; D056827:Endosomal Sorting Complexes Required for Transport; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors; D044767:Ubiquitin-Protein Ligases
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases