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

Abstract

The WW domain adopts a compact, three-stranded, antiparallel beta-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 microM, only slightly higher than that observed for the wild-type WW domain interaction (5.9 +/- 0.33 microM). The W17F WW domain is a natively unfolded protein which adopts a native conformation upon PY-ligand binding.

A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

“…We used deep mutational scanning to measure the ability of 47,000 unique variants of the hYAP65 WW domain to bind to their polyproline peptide ligand (2,4). WW domains mediate protein-protein interactions, have a well-defined structure, and fold through a two-state mechanism, simplifying subsequent measurements of thermodynamic stability (11)(12)(13). We displayed a library of variants of the hYAP65 WW domain on the surface of T7 bacteriophage.…”

An Expanded WW Domain Recognition Motif Revealed by the Interaction between Smad7 and the E3 Ubiquitin Ligase Smurf2

“…Indeed, analysis of the 201 WW domains identified by the Simple Modular Architecture Research Tool reveals that the Smurf2 WW2 domain and the analogous Smurf1 WW domain are the only ones with a residue other than Trp at position 17 (numbering according to YAP65) (38). This Tyr has the potential to destabilize the WW2 domain (15). In contrast, the WW3 domain of Smurf2 has a Trp at position 17, but in place of the canonical binding site Trp at position Smurf2 WW3-Smad7 PY Structure 39 there is a Phe (supplemental Fig.…”

“…WW domains are named for two conserved Trp residues (14). The first conserved Trp is on one side of the sheet in a hydrophobic cluster that is important for domain stability, with mutation of this residue destabilizing the structure (15). The second Trp is found on the opposite face of the sheet in a hydrophobic pocket and is integrally involved in binding to polyproline sequences of ligands (15)(16)(17).…”

“…The first conserved Trp is on one side of the sheet in a hydrophobic cluster that is important for domain stability, with mutation of this residue destabilizing the structure (15). The second Trp is found on the opposite face of the sheet in a hydrophobic pocket and is integrally involved in binding to polyproline sequences of ligands (15)(16)(17). Mutation of either the binding site Trp or ligand Pro residues can disrupt ligand binding, demonstrating the importance of the Trp in the recog-nition of target ligands (15,18,19).…”

“…The second Trp is found on the opposite face of the sheet in a hydrophobic pocket and is integrally involved in binding to polyproline sequences of ligands (15)(16)(17). Mutation of either the binding site Trp or ligand Pro residues can disrupt ligand binding, demonstrating the importance of the Trp in the recog-nition of target ligands (15,18,19). WW domains can be divided into four groups based on their ligand binding specificity (20).…”