The Nedd4-1 WW Domain Recognizes the PY Motif Peptide through Coupled Folding and Binding Equilibria

Biochemistry. 2016 Feb 2;55(4):659-74. doi: 10.1021/acs.biochem.5b01028. Epub 2016 Jan 15.


The four WW domains of human Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) interact with the PPxY (PY) motifs of the human epithelial Na(+) channel (hENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We have shown previously that the α-hENaC PY motif binding interface of WW3* undergoes conformational exchange on the millisecond time scale, indicating that conformational sampling plays a role in peptide recognition. To further understand this role, the structure and dynamics of hNedd4-1 WW3* were investigated. The nuclear Overhauser effect-derived structure of apo-WW3* resembles the domain in complex with the α-hENaC peptide, although particular side chain conformations change upon peptide binding, which was further investigated by molecular dynamics simulations. Model-free analysis of the (15)N nuclear magnetic resonance spin relaxation data showed that the apo and peptide-bound states of WW3* have similar backbone picosecond to nanosecond time scale dynamics. However, apo-WW3* exhibits pronounced chemical exchange on the millisecond time scale that is quenched upon peptide binding. (1)HN and (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments at various temperatures revealed that apo-WW3* exists in an equilibrium between the natively folded peptide binding-competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium was determined by fitting a thermal denaturation profile monitored by circular dichroism spectroscopy in combination with the CPMG data, leading to the conclusion that the unfolded state is populated to ∼ 20% at 37 °C. These results show that the binding of the hNedd4-1 WW3* domain to α-hENaC is coupled to the folding equilibrium.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Endosomal Sorting Complexes Required for Transport / chemistry*
  • Endosomal Sorting Complexes Required for Transport / metabolism
  • Humans
  • Molecular Dynamics Simulation*
  • Nedd4 Ubiquitin Protein Ligases
  • Protein Folding
  • Protein Structure, Tertiary
  • Ubiquitin-Protein Ligases / chemistry*
  • Ubiquitin-Protein Ligases / metabolism


  • Endosomal Sorting Complexes Required for Transport
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4 protein, human
  • Ubiquitin-Protein Ligases