Multivalent AMOTL1 Engages NEDD4-1 and KIBRA Through Distinct Cooperative Binding Mechanisms.

Area of Science:

• Molecular Biology
• Cell Signaling
• Biophysics

Background:

• The Hippo signaling pathway is crucial for maintaining cellular homeostasis by balancing cell growth and apoptosis.
• Multivalent protein complexes are key regulators within this pathway, but how their assembly and stability are modulated by multivalency is unclear.
• Angiomotin-like 1 (AMOTL1) is a scaffold protein with three PPxY motifs involved in Hippo pathway regulation.

Purpose of the Study:

• To investigate the distinct binding mechanisms of AMOTL1 with its WW domain-containing partners, NEDD4-1 and KIBRA.
• To elucidate how multivalency influences the assembly and stability of protein complexes within the Hippo signaling pathway.
• To understand how differential binding by AMOTL1 leads to opposing functional outcomes.

Main Methods:

• Quantitative molecular biophysical analyses, including isothermal titration calorimetry (ITC).
• Nuclear magnetic resonance (NMR) spectroscopy to study protein-protein interactions.
• Characterization of AMOTL1 interactions with NEDD4-1 and KIBRA.

Main Results:

• AMOTL1 forms a highly stable complex with NEDD4-1 via simultaneous engagement of its three PPxY motifs with NEDD4-1 WW domains, enhancing binding affinity significantly.
• KIBRA primarily binds AMOTL1 through its C-terminal PPxY motif with high affinity, while secondary interactions are transient and do not strengthen binding.
• These distinct binding modes demonstrate that AMOTL1's multivalency allows for differential regulation of its stability and function.

Conclusions:

• Multivalency in the Hippo pathway acts as a tunable regulatory mechanism.
• Cooperative binding, as seen with NEDD4-1, can dramatically increase complex stability.
• Differential binding affinities, as observed with KIBRA, allow a single scaffold protein to mediate opposing cellular outcomes, fine-tuning cell growth and apoptosis.