Yorkie-Warts Complexes are an Ensemble of Interconverting Conformers Formed by Multivalent Interactions

Baker, Kasie; Kwok, Ethiene; Reardon, Patrick N.; Rodriguez, Diego J.; Rolland, Amber D.; Wilson, Jesse; Prell, James S.; Nyarko, Afua

doi:10.1016/j.jmb.2020.166776

Cited by 4 publications

(6 citation statements)

References 40 publications

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“…We conclude that the apo AMOTL1 PPxY domain, A 123 , has a limited folded structure, localized in two short helical segments, residues 193–197 and 245–257 as shown by CD and NMR. This first experimental demonstration that the full AMOTL1 PPxY segment is partially disordered is consistent with the structures of other multivalent PPxY proteins 24–26 . Second, the AMOTL1‐YAP PPxY‐WW complex is formed by one molecule of AMOTL1 bound to one molecule of YAP, as shown by the hydrodynamic method of SEC‐MALS and ITC.…”

Section: Discussionsupporting

confidence: 83%

“…Considered together, the novel information from these complementary methodologies indicates that the AMOTL1‐YAP PPxY‐WW complex is structurally dynamic, and the YAP WW tandems interact with all three PPxY sites in AMOTL1 as illustrated by the model in Figure 5. The dynamic structure is composed of different complexes of varying stabilities as previously noted for other WW‐PPxY complexes 24,26 . Complexes in which the YAP WW domains bind AMOTL1 sites P1‐P2 or P1‐P3 are the most stable; and the least stable complex is formed by relatively weak or transient binding of the YAP WW domains to AMOTL1 sites P2‐P3.…”

Section: Discussionmentioning

confidence: 80%

“…This first experimental demonstration that the full AMOTL1 PPxY segment is partially disordered is consistent with the structures of other multivalent PPxY proteins. [24][25][26] Second, the AMOTL1-YAP PPxY-WW complex is formed by one molecule of AMOTL1 bound to one molecule of YAP, as shown by the hydrodynamic method of SEC-MALS and ITC. Third, binding of the YAP tandem WW domains to A 123 perturbs residues at all three PPxY sites as shown by solution NMR spectroscopy.…”

Section: Discussionmentioning

confidence: 99%

“…The dynamic structure is composed of different complexes of varying stabilities as previously noted for other WW-PPxY complexes. 24,26 Complexes in which the YAP WW domains bind AMOTL1 sites P1-P2 or P1-P3 are the most stable; and the least stable complex is formed by relatively weak or transient binding of the YAP WW domains to AMOTL1 sites P2-P3. Simultaneous binding of both YAP WW domains to varying pairs of AMOTL1 PPxY sites leads to the formation of an equilibrating mixture of interconverting species transiently formed by two YAP sites bound to two conjugate AMOTL1 sites.…”

Section: Discussionmentioning

confidence: 99%

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Multivalent Angiomotin‐like 1 and Yes‐associated protein form a dynamic complex

2022

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Multivalent complexes formed between the cancer-promoting transcriptional co-activator, Yes-associated protein (YAP), and proteins containing short linear motifs of type PPxY modulate cell proliferation and are attractive therapeutic targets. However, challenges producing PPxY polypeptides containing the full binding domain has limited understanding of the assembly process. Here, we successfully produced a polypeptide containing the complete set of three PPxY binding sites of Angiomotin-like 1 (AMOTL1), a scaffolding protein that regulates the nucleo-cytoplasmic shuttling of YAP via WW-PPxY interactions. Using an array of biophysical techniques including isothermal titration calorimetry, size-exclusion chromatography coupled to multi-angle light scattering, and solution nuclear magnetic resonance spectroscopy, we show that the AMOTL1 polypeptide is partially disordered, and binds the YAP WW domains to form an ensemble of complexes of varying stabilities. The binding process is initiated by the binding of one YAP WW domain to one AMOTL1 PPxY motif and is completed by transient interactions of the second YAP WW domain with a second AMOTL1 PPxY motif to form an equilibrating mixture composed of various species having two YAP sites bound to two conjugate AMOTL1 sites. We rationalize that the transient interactions fine-tune the stability of the complex for rapid assembly and disassembly in response to changes in the local cellular environment.

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Section: Discussionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Multivalent Angiomotin‐like 1 and Yes‐associated protein form a dynamic complex

2022

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“…However, a second factor is the relative location of the two binding sequences. The connecting linker length is a well-recognized determinant of tandem binding affinities, as in polypeptides with multiple PPxY motifs binding predominantly occurs for proximal motifs (separated by ~15-20 residues), and only rarely for distal motifs (5,35). These two factors embody enthalpic and entropic contributions, respectively, to binding.…”

Section: A Molecular View Of the Parallel Tandem-ww/dual-ppxy Assemblymentioning

confidence: 99%

Tandem WW/PPxY motif interactions in WWOX: the multifaceted role of the second WW domain

Rotem‐Bamberger

Fahoum

Keinan‐Adamsky

et al. 2021

Preprint

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Class I WW domains mediate protein interactions by binding short linear PPxY motifs. They occur predominantly as tandem repeats, and their target proteins often contain multiple PPxY motifs, but the interplay of WW/peptide interactions is not always intuitive. WW domain-containing oxidoreductase (WWOX) protein harbors two WW domains: unstable WW1 capable of PPxY binding, and well-folded but mutated WW2 that cannot bind such motifs. WW2 is considered to act as a WW1 chaperone, but the underlying mechanism remains to be revealed. Here we combine NMR, ITC and structural modeling to elucidate the role of both WW domains in WWOX binding to single and double motif peptides derived from its substrate ErbB4. Using NMR we identified an interaction surface between the two domains that supports a WWOX conformation that is compatible with peptide substrate binding. ITC and NMR measurements reveal that while binding affinity to a single motif is marginally increased in the presence of WW2, affinity to a dual motif peptide increases tenfold, and that WW2 can directly bind double motif-peptides using its canonical binding site. Finally, differential binding of peptides in a mutagenesis study is consistent with a parallel orientation binding to the WW1-WW2 tandem domain, agreeing with structural models of the interaction. Our results reveal the complex nature of tandem WW domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both stability and binding. This opens the way to assess how evolution can utilize the multivariate nature of binding to fine-tune interactions for specific biological functions.

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