The Structure and Dynamics of Tandem WW Domains in a Negative Regulator of Notch Signaling, Suppressor of Deltex

Oleg Y Fedoroff,Sharon A Townson,Alexander P Golovanov,Martin Baron,Johanna M Avis

doi:10.1074/jbc.m404987200

Oleg Y Fedoroff, Sharon A Townson + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m404987200

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Abstract

WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded beta-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hinge-like motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.

Highlights

WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins [1]
Protein was purified by affinity binding to glutathione beads in phosphate-buffered saline followed by thrombin cleavage to release the WW domains from the glutathione S-transferase tag and a final gel filtration step (Sephacryl S-100, Amersham Biosciences) to remove higher molecular weight contaminants and to exchange the protein into the buffer used in NMR experiments
Fluorescence Analysis of Ligand Binding to the WW3– 4 Tandem Pair—The direct interaction of the second Trp residue within WW domains with ligand allows the monitoring of ligand binding by Trp fluorescence

Summary

Introduction

WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins [1]. Multiple modules may act in concert to achieve greater specificity for a target as observed for the SH21 domains in SHP-2 phosphatase [6, 7] Their presence may indicate a functional diversity borne out by an ability to bind more than one target. The recent solution structure of the WW domain pair of the yeast-splicing factor Prp40 [10] reveals a single rigid structure with the linker assuming an ␣-helical conformation This linker conformation results in a defined relative orientation of the WW domain binding surfaces in keeping with a bridging function for Prp early in the splicing process. Incomplete structural studies on the second and third tandem WW domains from the rat Nedd protein [11] suggest a disordered interdomain linker. Differences in the structure and dynamics of tandem WW domains are probable and possibly affect their protein recognition function

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