Abstract
WW domains target proline-tyrosine (PY) motifs and frequently function as tandem pairs. When studied in isolation, single WW domains are notably promiscuous and regulatory mechanisms are undoubtedly required to ensure selective interactions. Here, we show that the fourth WW domain (WW4) of Suppressor of Deltex, a modular Nedd4-like protein that down-regulates the Notch receptor, is the primary mediator of a direct interaction with a Notch-PY motif. A natural Trp to Phe substitution in WW4 reduces its affinity for general PY sequences and enhances selective interaction with the Notch-PY motif via compensatory specificity-determining interactions with PY-flanking residues. When WW4 is paired with WW3, domain-domain association, impeding proper folding, competes with Notch-PY binding to WW4. This novel mode of autoinhibition is relieved by binding of another ligand to WW3. Such cooperativity may facilitate the transient regulatory interactions observed in vivo between Su(dx) and Notch in the endocytic pathway. The highly conserved tandem arrangement of WW domains in Nedd4 proteins, and similar arrangements in more diverse proteins, suggests domain-domain communication may be integral to regulation of their associated cellular activities.
Highlights
The Nedd4 protein family constitute a major class of E3 ubiquitin ligases implicated in a variety of processes including neurodegeneration [1], blood pressure regulation [2], cell-cell communication and differentiation [3,4,5], and viral budding [6]
Nedd4-like proteins are defined by a modular architecture: an N-terminal C2 domain, two to four WW domains, and a C-terminal catalytic HECT domain
The proteins exert their effects though the regulated trafficking and degradation of targets, usually located in the plasma membrane or nucleus, that are recognized by the WW domains [7]
Summary
Su(dx), Suppressor of Deltex; GST, glutathione S-transferase; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum correlation; TROSY, transverse relaxation optimized spectroscopy. Multiple WW domain modules may function by binding separate targets or act together to increase specificity as demonstrated with tandem SH2 (38 – 40) and PDZ domains [41, 42] They may work together in an autoregulatory manner. The solution structures of paired WW domains for two examples: yeast Prp40 [43] and Drosophila Su(dx) [44], reveal a rigid ␣-helical linker in the former case and a flexible linker in the latter In both examples, the nature of the linker and resultant relative domain orientation has functional implications; Prp mediating a bridging interaction and Su(dx) likely undergoing regulated, transient interactions with a number of ligands.
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