Biophysical Analysis of Binding of WW Domains of the YAP2 Transcriptional Regulator to PPXY Motifs within WBP1 and WBP2 Adaptors

Abstract

YAP2 transcriptional regulator mediates a plethora of cellular functions, including the newly discovered Hippo tumor suppressor pathway, by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using isothermal titration calorimery (ITC) and circular dichroism (CD) in combination with molecular modeling (MM) and molecular dynamics (MD), we provide evidence that the WW1 and WW2 domains of YAP2 recognize various PPXY motifs within WBP1 and WBP2 in a highly promiscuous and subtle manner. Thus, although both WW domains strictly require the integrity of the consensus PPXY sequence, non-consensus residues within and flanking this motif are not critical for high-affinity binding, implying that they most likely play a role in stabilizing the polyproline type II (PPII) helical conformation of the PPXY ligands. Of particular interest is the observation that both WW domains bind to a PPXYXG motif with highest affinity, implicating a preference for a non-bulky and flexible glycine one-residue C-terminal to the consensus tyrosine. Importantly, a large set of residues within both WW domains and the PPXY motifs appear to undergo rapid fluctuations on a nanosecond time scale, arguing that WW-ligand interactions are highly dynamic and that such conformational entropy may be an integral part of the reversible and temporal nature of cellular signaling cascades. Collectively, our study sheds light on the molecular determinants of a key WW-ligand interaction pertinent to cellular functions in health and disease.

The Biology of YAP/TAZ: Hippo Signaling and Beyond

“…The interaction with Smad2/3 entails the TAZ coiled-coil domain (207). The WW domains are essential for YAP-induced cell proliferation (261) and mediate binding of YAP/TAZ to PPxY motif-containing transcription factors such as RUNX, p73, and the cytoplasmic domain of ERBB4 or with the transcriptional cofactor WBP2 (28,40,104,140,164,197,214).…”

Recognition mechanism of p63 by the E3 ligase Itch

“…In binding with cyclic form of pep63, the exchanging regime of two of these residues, Y26 and H30, change from fast to slow, indicating the formation of a less shaky complex induced by a more stable conformation of the peptide. Similarly, as described for Yap2-WBP2 complex, 46 in the WW2-pep63 OX complex, the formation of a hydrogen bond between Hη phenolic hydrogen of Y within the PPxY motif and H-HSQC spectra, the lines underline some of the major differences observable.…”

“…The above data are in agreement with recent thermodynamic studies that suggested strongly that non-consensus residues within and flanking the PPxY peptides are important for stabilizing the peptide conformation, and that peptides with distinct conformations are optimally suited for binding to WW domains. 46 The balance among rigidity and flexibility of both WW domains and protein targets might also be an in vivo critical factor in this molecular recognition mechanism. Accordingly, the WW-ligand interactions are usually studied in vitro using short, proline-rich peptides, and the relatively low affinities observed (in 10 to 100 μM) are generally characteristic of several similar data strongly suggest that the interaction of the domain with the cyclic form of the peptide results in a more stable complex with a longer lifetime than that resulting from the interaction with the linear form of pep63.…”

“…It is notable that the residues Y26, V28, H30 and W37 seem to form a hydrophobic groove within Itch-WW2 domain. These residues are highly conserved across the family of WW domains that recognize PPxY ligands, [42][43][44][45][46] such as in the four WW domains of Itch, except for the substitution of the W with Y residue in the WW4 domain. In binding with cyclic form of pep63, the exchanging regime of two of these residues, Y26 and H30, change from fast to slow, indicating the formation of a less shaky complex induced by a more stable conformation of the peptide.…”

“…CD spectra interactions. 25,[46][47][48][49][50] The low affinity in many cases limited the possibility to obtain a highresolution structure of the complex by NMR spectroscopy. Our results show the possibility that an increase of the stability of the complex in the solution may induce a major conformation stability of the fragment, probably improving the mimicking of the local structure of the protein by using a cyclic form of the PPxY peptide.…”