Predicting PY motif-mediated protein-protein interactions in the Nedd4 family of ubiquitin ligases.

A Katherine Hatstat,Michael D Pupi,Dewey G Mccafferty,Vikas Nanda

doi:10.1371/journal.pone.0258315

Abstract

The Nedd4 family contains several structurally related but functionally distinct HECT-type ubiquitin ligases. The members of the Nedd4 family are known to recognize substrates through their multiple WW domains, which recognize PY motifs (PPxY, LPxY) or phospho-threonine or phospho-serine residues. To better understand protein interactor recognition mechanisms across the Nedd4 family, we report the development and implementation of a python-based tool, PxYFinder, to identify PY motifs in the primary sequences of previously identified interactors of Nedd4 and related ligases. Using PxYFinder, we find that, on average, half of Nedd4 family interactions are likely PY-motif mediated. Further, we find that PPxY motifs are more prevalent than LPxY motifs and are more likely to occur in proline-rich regions and that PPxY regions are more disordered on average relative to LPxY-containing regions. Informed by consensus sequences for PY motifs across the Nedd4 interactome, we rationally designed a focused peptide library and employed a computational screen, revealing sequence- and biomolecular interaction-dependent determinants of WW-domain/PY-motif interactions. Cumulatively, our efforts provide a new bioinformatic tool and expand our understanding of sequence and structural factors that contribute to PY-motif mediated interactor recognition across the Nedd4 family.

Highlights

Neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) is the founding member of a family of HECT-type E3 ubiquitin ligases that share a common architecture but have distinct cellular functions
Identification and analysis PY motif sequences in the Nedd4 family interactome
To begin our analysis of PY motif-mediated interactions in the Nedd4 family, we first sought to determine the prevalence of PY motifs amongst interactors of the family

Summary

Introduction

Neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) is the founding member of a family of HECT-type E3 ubiquitin ligases that share a common architecture but have distinct cellular functions. The Nedd family is characterized by a multi-domain architecture comprised, from N- to C-terminus respectively, of a C2 domain for membrane localization, two to four WW domains for substrate recognition, and a catalytic HECT domain (Fig 1A) [1,2,3,4,5]. As the final enzyme in the ubiquitin signaling cascade, the Nedd family of HECTtype E3 ubiquitin ligases receives ubiquitin from a ubiquitin-E2 conjugating enzyme thioester adduct. Nedd and related HECT-type ligases are responsible for conferring substrate specificity in the ubiquitin signaling pathway.

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