Structural and Biochemical Basis for Ubiquitin Ligase Recruitment by Arrestin-related Domain-containing Protein-3 ( ARRDC3)*

Background: ARRDC proteins bind ubiquitin ligases and are involved in receptor down-regulation. Results: ARRDC3 recruitment of Nedd4 is explained. Conclusion: The first PPXY motif of ARRDC3 binds to the third WW domain of Nedd4 with high affinity, whereas other domains also contribute. Significance: The structure explains aspects of high affinity recognition. After protracted stimulation, the 2-adrenergic receptor and many other G-protein-coupled receptors are ubiquitinated and down-regulated. Arrestin-related domain-containing protein-3 (ARRDC3) has been proposed to recruit the ubiquitin ligase Nedd4 to the 2-adrenergic receptor. ARRDC3 contains two PPXY motifs that could potentially interact with any of the four WW domains of Nedd4. Here we dissect the interaction determinants. ARRDC3 PPXY-Nedd4 WW dissociation constants vary from unmeasurable to K-d = 3 m for the third WW domain of Nedd4 binding to the first PPXY motif of ARRDC3. Structures of the uncomplexed and PPXY1-bound WW3 domain were determined at 1.1 and 1.7 resolution. The structures revealed conformational changes upon binding and the hydrogen bonding network in exquisite detail. Tight packing of ARRDC3 Val-352, part of a 3(10) helix at the C terminus of PPXY1, is important for high affinity binding to WW3. Although no single WW domain is strictly essential for the binding of Nedd4 and ARRDC3 expressed in HEK293 cells, high affinity binding of full-length ARRDC3 and Nedd4 is driven by the avid interaction of both PPXY motifs with either the WW2-WW3 or WW3-WW4 combinations, with K-d values as low as 300 nm.