Characterization of the structure and function of W -> FWW domain variants: Identification of a natively unfolded protein that folds upon ligand binding

The WW domain adopts a compact, three-stranded, antiparallel P-sheet struct ure that mediates protein-protein interactions by binding to xPPxY-based pr otein ligands? such as the PY-ligand (EYPPYPPPPYPSG) derived from p53 bindi ng protein-2. The conserved Trp residues, after which this domain was named , were replaced with Phe so their importance in structural integrity and fo r 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 di srupt the structure of the WW domain variant, but does abolish ligand bindi ng. In contrast. the W17F WW domain variant is largely if not completely un folded; however this variant undergoes a PY-ligand induced disorder to orde r (folding) transition. The dissociation constant for the W17F WW domain-PY -ligand interaction is 15.1 +/- 1.2 mu M, only slightly higher than that ob served for the wild-type WW domain interaction (5.9 +/- 0.33 mu M). The W17 F WW domain is a natively unfolded protein which adopts a native conformati on upon PY-ligand binding.