Characterization of the Structure and Function of W → F WW Domain Variants: Identification of a Natively Unfolded Protein That Folds upon Ligand Binding

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Abstract
The WW domain adopts a compact, three-stranded, antiparallel β-sheet structure that mediates protein−protein interactions by binding to xPPxY-based protein ligands, such as the PY-ligand (EYPPYPPPPYPSG) derived from p53 binding protein-2. The conserved Trp residues, after which this domain was named, were replaced with Phe so their importance in structural integrity and for ligand binding could be evaluated. A biophysical approach was employed to compare the W17F, W39F, and W17F/W39F WW domains to the wild-type protein. The data demonstrate that replacement of Trp39 with Phe (W39F) does not disrupt the structure of the WW domain variant, but does abolish ligand binding. In contrast, the W17F WW domain variant is largely if not completely unfolded; however, this variant undergoes a PY-ligand induced disorder to order (folding) transition. The dissociation constant for the W17F WW domain−PY-ligand interaction is 15.1 ± 1.2 μM, only slightly higher than that observed for the wild-type WW domain interaction (5.9 ± 0.33 μM). The W17F WW domain is a natively unfolded protein which adopts a native conformation upon PY-ligand binding.