title: The Nedd4-1 WW Domain Recognizes the PY Motif Peptide through Coupled Folding and Binding Equilibria.
abstract: 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.
authors.affiliation: ICS-6 (Strukturbiochemie), Forschungszentrum Jülich , 52425 Jülich, Germany.
authors.forename: Vineet
authors.initials: V
authors.lastname: Panwalkar
authors.affiliation: ICS-6 (Strukturbiochemie), Forschungszentrum Jülich , 52425 Jülich, Germany.|Institut für Physikalische Biologie, Heinrich-Heine-Universität , 40225 Düsseldorf, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Neudecker
authors.forename: Michael
authors.initials: M
authors.lastname: Schmitz
authors.affiliation: ICS-6 (Strukturbiochemie), Forschungszentrum Jülich , 52425 Jülich, Germany.|Institut für Physikalische Biologie, Heinrich-Heine-Universität , 40225 Düsseldorf, Germany.
authors.forename: Justin
authors.initials: J
authors.lastname: Lecher
authors.affiliation: ICS-6 (Strukturbiochemie), Forschungszentrum Jülich , 52425 Jülich, Germany.|Institut für Physikalische Biologie, Heinrich-Heine-Universität , 40225 Düsseldorf, Germany.
authors.forename: Marianne
authors.initials: M
authors.lastname: Schulte
authors.forename: Karima
authors.initials: K
authors.lastname: Medini
authors.affiliation: ICS-6 (Strukturbiochemie), Forschungszentrum Jülich , 52425 Jülich, Germany.|Institut für Physikalische Biologie, Heinrich-Heine-Universität , 40225 Düsseldorf, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Stoldt
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Brimble
authors.affiliation: ICS-6 (Strukturbiochemie), Forschungszentrum Jülich , 52425 Jülich, Germany.|Institut für Physikalische Biologie, Heinrich-Heine-Universität , 40225 Düsseldorf, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Willbold
authors.affiliation: ICS-6 (Strukturbiochemie), Forschungszentrum Jülich , 52425 Jülich, Germany.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Dingley
mesh_terms: D020816:Amino Acid Motifs; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D000075702:Nedd4 Ubiquitin Protein Ligases; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases