posters 5th Asia-Pacific NMR Symposium 2013

Structure and dynamics of the human Nedd4-1 WW3* domain (#119)

Vineet Panwalkar 1 , Romel Bobby 1 , Karima Medini 1 , Justin Lecher 2 , Tet Verne Lee 1 , Margaret A. Brimble 1 , Fiona J. McDonald 3 , J. Shaun Lott 1 , Philipp Neudecker 2 4 , Andrew J. Dingley 1 2
  1. School of Chemical Sciences and School of Biological Sciences, The University of Auckland, Auckland, New Zealand
  2. ICS-6 (Strukturbiochemie), Forschungszentrum Juelich, Juelich, NRW, Germany
  3. Department of Physiology, University of Otago, Dunedin, New Zealand
  4. Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Germany

Human Nedd4-1 (Neuronal precursor cell expressed developmentally down-regulated gene 4-1) is an E3 ubiquitin ligase that negatively regulates the epithelial Na+ channel (ENaC). Nedd4 is composed of a C2 domain, four WW domains and a ubiquitin ligase Hect domain. The WW domains interact with the ENaC subunits via recognition of “PY” motifs. The third WW domain (WW3*) binds the PY motif ENaC cognitive peptide with a Kd of ~30 μM; the highest of all four WW domains. To understand the mechanism of this interaction in detail, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in the free form and in complex with a C-terminal human ENaC α-subunit peptide (PPxYxxL).1  The structure reveals that the peptide interacts in a similar manner to the other WW domain-ENaC peptide structures, and the apo-state is surprisingly well-ordered, adopting a structure that is similar to the peptide-bound state. Dynamic analysis of the WW3* domain revealed that the apo-state  and the domain in complex with the EnaC α-subunit peptide have similar order parameter (S2) values. In contrast, relaxation dispersion data and model-free analysis revealed that many residues of the apo-WW3* domain display μs-ms time scale motions. In the complex, two different conformational exchange processes on the μs-ms time scale were identified. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange plays  a role in peptide recognition. Thus, structural and dynamic features appear to define the high binding affinity of WW3*. These results should aid the interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.

  1. Bobby R, Medini K, Neudecker P, Lee TV, Brimble MA, McDonald FJ, Lott JS, Dingley AJ. (2013) Structure and dynamics of human Nedd4-1 WW3 in complex with the αENaC PY motif. Biochim. Biophys. Acta. 1834(8); 1632-1641.