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

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 ␤-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 hingelike 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.WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins (1). The domain is a small three-stranded ␤-sheet stabilized by the stacking of several conserved aromatic and proline residues (2). Differences in residue identity at the binding surface result in a variation in ligand specificity that is used as the basis to divide WW domains into groups. For example, in group I WW domains that bind PPXY sequences (3), the Tyr is a key specificity residue and is accommodated by a largely hydrophobic pocket on the concave binding surface consisting of conserved Ile (or Val/Leu), His, and Gln (or Arg/Lys) residues. The Pro residues of the ligand contribute to the binding by stacking against the Trp and Tyr residues that form a second interaction site (4). It is evident that, since a number of proteins are likely to contain WW domain recognition sites, further factors most probably contribute to increasing affinity and specificity of a WW domain for a target. For example, the Pro-rich sequence in ␤-dystroglycan targeted by the dystrophin WW domain requires a composite binding surface provided by the WW domain and an adjacent EF hand (4). The binding of murine Nedd4 to the amiloride-sensitive epithelial sodium channel requires direct involvement of two of its three WW domains (5). Indeed, WW domains often exist in multiple numbers within a protein. Multiple modules may act in concert to achieve greater specificity for a target ...

Structure and Function of the Two Tandem WW Domains of the Pre-mRNA Splicing Factor FBP21 (Formin-binding Protein 21)

“…Only two tandem WW structures have been reported (7,14), one of which represents the two tandem WW domains of yeast splicing factor Prp40 (7). The WW domains of the Prp40 have a defined interdomain orientation, whereas the tandem WW domains of the mammalian FBP21 are connected by a much more flexible linker.…”

“…The RDCs in the ␤-strands of WW1 are mostly negative, whereas they are positive for the corresponding residues in WW2. The opposite sign of RDCs suggests that there is no colinearity between the tandem WW domains, and the interdomain orientation between the two WW domains is not fixed (14,42). However, the similar R 2 /R 1 ratio of the two WW domains obtained from 15 N relaxation experiments indicates that the two domains, on average, tumble together in solution.…”

“…Many proteins contain multiple arrays of WW domains (11), which may increase the specificity and affinity for ligands (12,13) or enhance their functional diversity by their ability to bind more than one ligand (14,15). As the number and spatial arrangement of WW domains can be variable (14,16), it is likely that the length and structure of the interdomain linkers are of crucial significance.…”

“…As the number and spatial arrangement of WW domains can be variable (14,16), it is likely that the length and structure of the interdomain linkers are of crucial significance. Little is currently known about the functional importance of the interdomain linker in proteins with multiple WW domains.…”

“…Little is currently known about the functional importance of the interdomain linker in proteins with multiple WW domains. Although a number of structures of individual WW domains have been solved (17)(18)(19)(20)(21)(22), only two structures of tandem WW domains, namely those of yeast Prp40 and Drosophila Su(dx), have been determined, and their structures are markedly different (7,14). So far, the relationship between the functional diversity and the structural organization of multiple WW domains remains poorly characterized.…”