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

The Hippo signaling pathway regulates the homeostatic balance between cell growth and apoptosis through intricate networks of multivalent protein complexes. How multivalency modulates the assembly and stability of these protein complexes remains poorly understood. Here, we show that Angiomotin-like 1 (AMOTL1), a scaffold protein containing three PPxY motifs, employs distinct cooperative binding mechanisms to engage two WW domain-containing partners: NEDD4-1, which promotes AMOTL1 degradation, and KIBRA, which protects AMOTL1 from degradation. Using quantitative molecular biophysical analyses, including isothermal titration calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate that AMOTL1 forms a cooperatively stabilized complex with NEDD4-1 through simultaneous engagement of all three PPxY motifs with three of the four NEDD4-1 WW domains. This cooperative binding mode produces approximately ten-fold enhancement in affinity compared to the primary anchor interaction alone. In contrast, KIBRA engages AMOTL1 primarily through high-affinity binding at the C-terminal PPxY motif, with transient secondary interactions at the other PPxY sites that do not enhance overall binding strength. These contrasting mechanisms demonstrate that multivalency within the Hippo pathway serves as a tunable regulatory feature, where cooperative interactions can either enhance or minimally contribute to binding strength, explaining how a single scaffold protein can be differentially regulated to achieve opposing functional outcomes.