AI-assisted decoding of molecular signatures essential for condensation and function of NON-PHOTOTROPIC HYPOCOTYL3

The plasma membrane associated protein NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key component of the auxin-dependent phototropic growth response in plants. Blue light induces the detachment of NPH3 from the plasma membrane into the cytosol, where it dynamically transitions into membrane-less biomolecular condensates. Despite the importance of NPH3/RPT2-Like (NRL) proteins for developmental plasticity of plants, little is known about their structure-function relationship. Here, we combine experimental data with the power of AI-based protein structure prediction to uncover molecular signatures of NPH3. Our approach unveils a bipartite C-terminal motif that enables self-interaction of NPH3 with different strengths. We further demonstrate that the C-terminal homo-oligomerization is required for both association with the plasma membrane and condensate assembly, with a different part of the bipartite motif playing the key role in each case. However, multivalency-driven transition of NPH3 to the condensed state depends on the co-operative action of an N-terminal NPH3 signature. We propose that NPH3 is a single hub that can form a polymerized network based on two distinct structural domains that self-associate. NPH3 variants that are incapable of condensation are non-functional, suggesting a fundamental role of phase separation of NPH3 for auxin-dependent phototropism. This structural snapshot may have direct implications on future analyzes of NRL family members.