Multiple WW domains of Nedd4-1 undergo conformational exchange that is quenched upon peptide binding.

The third WW domain (WW3*) of the ubiquitin ligase human neuronal precursor cell expressed developmentally downregulated gene 4-1 (hNedd4-1) was reported to bind its PY motif peptide by a coupled folding-binding equilibrium. However, it is unknown whether these thermodynamic properties are retained in the context of neighboring hNedd4-1 domains. In this report, NMR data show that the WW3* displays a fold-unfold equilibrium in the presence of neighboring WW domains, and that similar fold-unfold equilibria also likely exist for neighboring WW domains. These equilibria are quenched upon interaction with peptide. Thus, the binding mechanism of hNedd4-1 WW domains to proteins involves coupled folding and binding equilibria, and this mechanism may be a general feature that modulates peptide affinities of WW domains.