NMR studies of tandem WW domains of Nedd4 in complex with a PY motif-containing region of the epithelial sodium channel

Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) is a ubiquitin-protein ligase containing multiple WW domains. We have previ ously demonstrated the association between the WW domains of Nedd4 and PPxY (PY) motifs of the epithelial sodium channel (ENaC). In this paper, we rep ort the assignment of backbone H-1 alpha, (HN)-H-1, N-15, C-13', C-13 alpha , and aliphatic C-13 resonances of a fragment of rat Nedd4 (rNedd4) contain ing the two C-terminal WW domains, WW(II+III), complexed to a PY motif-cont aining peptide derived from the beta subunit of rat ENaC, the beta P2 pepti de. The secondary structures of these two WW domains, determined from chemi cal shifts of C-13 alpha and C-13 beta resonances, are virtually identical to those of the WW domains of the Yes-associated protein YAP65 and the pept idyl-prolyl isomerase Pin1. Triple resonance experiments that detect the H- 1 alpha chemical shift were necessary to complete the chemical shift assign ment, owing to the large number of proline residues in this fragment of rNe dd4. A new experiment, which correlates sequential residues via their N-15 nuclei and also detects H-1 alpha chemical shifts, is introduced and its ut ility for the chemical shift assignment of sequential proline residues is d iscussed. Data collected on the WW(II+III)-beta P2 complex indicate that th ese WW domains have different affinities for the beta P2 peptide.