Molecular origin of the binding of WWOX tumor suppressor to ErbB4 receptor tyrosine kinase

Abstract

The ability of WWOX tumor suppressor to physically associate with the intracellular domain (ICD) of ErbB4 receptor tyrosine kinase is believed to play a central role in downregulating the transcriptional function of the latter. Herein, using various biophysical methods, we show that while the WW1 domain of WWOX binds to PPXY motifs located within the ICD of ErbB4 in a physiologically relevant manner, the WW2 domain does not. Importantly, while the WW1 domain absolutely requires the integrity of the PPXY consensus sequence, nonconsensus residues within and flanking this motif do not appear to be critical for binding. This strongly suggests that the WW1 domain of WWOX is rather promiscuous toward its cellular partners. We also provide evidence that the lack of binding of the WW2 domain of WWOX to PPXY motifs is due to the replacement of a signature tryptophan, lining the hydrophobic ligand binding groove, with tyrosine (Y85). Consistent with this notion, the Y85W substitution within the WW2 domain exquisitely restores its binding to PPXY motifs in a manner akin to the binding of the WW1 domain of WWOX. Of particular significance is the observation that the WW2 domain augments the binding of the WW1 domain to ErbB4, implying that the former serves as a chaperone within the context of the WW1-WW2 tandem module of WWOX in agreement with our findings reported previously. Altogether, our study sheds new light on the molecular basis of an important WW-ligand interaction involved in mediating a plethora of cellular processes.

Figure 1

Modular organization of human ErbB4 and WWOX 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 (ECD) and a C-terminal intracellular (ICD) domains. 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 motifs relative to the first proline within the PPXY motifs, which is arbitrarily assigned zero. (b) WWOX is comprised of a tandem copy of WW domains, designated WW1 and WW2, located N-terminal to the short-chain dehydrogenase/reductase (SDR) domain. The location of Y85W mutation within the WW2 domain is indicated.

Figure 2

ITC analysis for the binding of WW1 domain of WWOX 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 (43, 46).

Figure 3

Structural models of WW1 (a) and WW2 (b) domains of WWOX in complex with ErbB4_PY3 peptide containing the PPXY motif. In each case, the β-strands within the WW domain are shown in blue with loops depicted in gray and the PPXY peptide is colored yellow. The sidechain moities of residues within the WW domain and the PPXY peptide engaged in key intermolecular contacts are shown in red and green, respectively. Note that the first two N-terminal proline residues within the PPXY motif are respectively P0 and P+1, while the C-terminal Y is indicated by Y+3.

Figure 4

ITC analysis for the binding of WW1–WW2 tandem module of WWOX 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–WW2 tandem module. 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 (43, 46).

Figure 5

ALS analysis of WW1 (top panels) and WW2 (middle panels) domains alone and in the context of WW1–WW2 tandem module (bottom panels) of WWOX. (a) Elution profiles as monitored by the differential refractive index (Δn) of each protein construct plotted as a function of elution volume (V). (b) Partial Zimm plots obtained for each protein construct from analytical SLS measurements. Note that the red solid lines through the data points represent linear fits. (c) Autocorrelation function plots obtained for each protein construct from analytical DLS measurements. Note that the red solid lines represent non-linear least squares fit of data to an autocorrelation function as embodied in Eq [9].

Figure 6

MD analysis conducted on the structural model of WW1–WW2 tandem module of WWOX. (a) Root mean square deviation (RMSD) of backbone atoms (N, Cα and C) within each simulated structure relative to the initial modeled structure as a function of simulation time. (b) Root mean square fluctuation (RMSF) of backbone atoms (N, Cα and C) averaged over the entire course of MD trajectory as a function of residue number. The vertical boxes demarcate the boundaries of WW1 and WW2 domains. (c) Radius of gyration (R_g) of each simulated structure relative to the initial modeled structure as a function of simulation time.

Figure 7

Structural snapshots taken at 0ns, 50ns, 100ns and 200ns during the course of an MD simulation conducted on the structural model of WW1–WW2 tandem module of WWOX. The constituent WW1 and WW2 domains are respectively colored green and blue, while the terminal and interdomain loops are shown in gray for clarity.