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

详细信息    查看全文

• 作者：Vineet Panwalkar ; Philipp Neudecker ; Michael Schmitz ; Justin Lecher ; Marianne Schulte ; Karima Medini ; Matthias Stoldt ; Margaret A. Brimble ; Dieter Willbold ; Andrew J. Dingley
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：February 2, 2016
• 年：2016
• 卷：55
• 期：4
• 页码：659-674
• 全文大小：752K
• ISSN：1520-4995

文摘

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 ¹⁵N 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. ¹H_N and ¹⁵N 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.