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, 101, 192-202

Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

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Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

Brett J Schuchardt et al. Biochimie.

Abstract

The newly discovered transactivation function of ErbB4 receptor tyrosine kinase is believed to be mediated by virtue of the ability of its proteolytically-cleaved intracellular domain (ICD) to physically associate with YAP2 transcriptional regulator. In an effort to unearth the molecular basis of YAP2-ErbB4 interaction, we have conducted a detailed biophysical analysis of the binding of WW domains of YAP2 to PPXY motifs located within the ICD of ErbB4. Our data show that the WW1 domain of YAP2 binds to PPXY motifs within the ICD in a differential manner and that this behavior is by and large replicated by the WW2 domain. Remarkably, while both WW domains absolutely require the integrity of the PPXY consensus sequence, non-consensus residues within and flanking this motif do not appear to be critical for binding. In spite of this shared mode of binding, the WW domains of YAP2 display distinct conformational dynamics in complex with PPXY motifs derived from ErbB4. Collectively, our study lends new insights into the molecular basis of a key protein-protein interaction involved in a diverse array of cellular processes.

Keywords: Conformational dynamics; PPXY motifs; Structural analysis; WW-ligand thermodynamics.

Figures

Figure 1
Figure 1
Modular organization of human ErbB4 and YAP2 proteins. (a) ErbB4 contains the canonical ECD-TM-ICD receptor tyrosine kinase modular cassette, where the central single-helical transmembrane (TM) domain is flanked between an N-terminal extracellular domain (ECD) and a C-terminal intracellular domain (ICD). The three PPXY motifs (designated PY1, PY2 and PY3) within the ICD are located at the extreme C-terminus. Note that the amino acid sequence of 12-mer peptides containing the PPXY motifs and flanking residues are provided. The numerals indicate the nomenclature used in this study to distinguish residues within and flanking the PPXY motifs relative to the first consensus proline, which is arbitrarily assigned zero. (b) YAP2 is comprised of a tandem copy of WW domains, designated WW1 and WW2, located N-terminal to the trans-activation (TA) domain.
Figure 2
Figure 2
Representative ITC isotherms for the binding of WW1 domain of YAP2 to ErbB4_PY1 (a), ErbB4_PY2 (b) and ErbB4_PY3 (c) peptides. The upper panels show the raw ITC data expressed as change in thermal power with respect to time over the period of titration. In the lower panels, change in molar heat is expressed as a function of molar ratio of corresponding peptide to WW1 domain. The red solid lines in the lower panels show the fit of data to a one-site binding model using the integrated ORIGIN software as described earlier [22, 20].
Figure 3
Figure 3
Far-UV CD spectra of ErbB4_PY1 (red), ErbB4_PY2 (green) and ErbB4_PY3 (blue) peptides. Note that the mean ellipticity, [θ], was calculated using Eq [3].
Figure 4
Figure 4
Structural models of WW1 (a) and WW2 (b) domains of YAP2 in complex with ErbB4_PY3 peptide containing the PPXY motif. The β-strands in the WW domains are shown in blue with loops depicted in gray and the peptide is colored yellow. Note that two orientations related by a 90°-rotation about the horizontal axis are depicted for the inquisitive eye. The sidechain moieties of all residues, including the PPXY motif (which corresponds to P0, P+1 and Y+3), within the bound peptide are shown in green. For the WW domains, the sidechain moieties colored in red denote all residues pointing toward the peptide on the concave side.
Figure 5
Figure 5
Conformational dynamics as probed through MD simulations conducted on WW1 and WW2 domains of YAP2 in complex with ErbB4_PY3 peptide containing the PPXY motif. (a) RMSD of backbone atoms (N, Cα and C) within each simulated structure relative to the initial modeled structure of WW1 (top panel) and WW2 (bottom panel) domains in complex with ErbB4_PY3 peptide as a function of simulation time. Note that the overall RMSD for each WW-peptide complex (black) is deconvoluted into the WW domain alone (red) and the peptide alone (green). (b) RMSF of backbone atoms (N, Cα and C) averaged over the entire course of corresponding MD trajectory of WW1 (top panel) and WW2 (bottom panel) domains in complex with ErbB4_PY3 peptide as a function of residue number within each WW domain. The shaded vertical rectangular boxes denote residues located within the β1-β2 and β2-β3 loops. (c) RMSF of backbone atoms (N, Cα and C) averaged over the entire course of corresponding MD trajectory of WW1 (top panel) and WW2 (bottom panel) domains in complex with ErbB4_PY3 peptide as a function of residue number within the peptide (see Figure 1a for nomenclature). The PPXY motif and the flanking residues are overlayed for reference.
Figure 6
Figure 6
Intermolecular distances, as probed through MD simulations, between consensus residues within the PPXY motif of ErbB4_PY3 peptide and residues lining the binding groove within WW1 and WW2 domains of YAP2. (a) Distance between Cγ pyrrolidine carbon of P0 within the PPXY motif and Nε1 indole nitrogens of W199 and W258 located respectively within WW1 (top panel) and WW2 (bottom panel) domains. (b) Distance between Cγ pyrrolidine carbon of P+1 within the PPXY motif and Cζ phenolic carbons of Y188 and Y247 located respectively within WW1 (top panel) and WW2 (bottom panel) domains. (c) Distance between Oη phenolic oxygen of Y+3 within the PPXY motif and Nδ1 imidazole nitrogens of H192 and H251 located respectively within WW1 (top panel) and WW2 (bottom panel) domains.
Figure 7
Figure 7
Intermolecular distances, as probed through MD simulations, between residues flanking the PPXY motif of ErbB4_PY3 peptide and residues lining the binding groove within WW1 and WW2 domains of YAP2. (a) Distance between Cγ1/Cγ2 methyl carbons of V-3 within the PPXY motif and Cδ1/Cδ2 indole carbons of W199 and W258 located respectively within WW1 (top panel) and WW2 (bottom panel) domains. (b) Distance between Cδ1/Cδ2 methyl carbons of L-2 within the PPXY motif and Cδ carbons of Q186 and E245 located respectively within WW1 (top panel) and WW2 (bottom panel) domains. (c) Distance between Nη1/Nη2 guanidine nitrogens of R+6 within the PPXY motif and Oε1/Oε2 carbonyl oxygens of E178 and E237 located respectively within WW1 (top panel) and WW2 (bottom panel) domains.

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