Abstract:
Strigolactones (SLs) are a class of plant hormone that regulate the development of branching in plants. In petunia, SL is perceived by its receptor, DAD2, an α/β hydrolase fold protein. This receptor also functions as a slow enzyme that hydrolyses SL into two non-active by-products. The hydrolytic function of the receptor is essential for SL signalling and enables the receptor to interact with the downstream signalling proteins, such as the Skp-Cullin-F-box (SCF) E3 ligase complex and target protein(s) to relay the signal. It is known that the receptor undergoes a significant conformational change in the presence of SL and SCF components. At present, it is unclear how the hydrolytic activity of the receptor enables it to interact with the downstream signalling protein to transduce the signal. To increase our understanding about the structural contribution of hydrolysis in the receptor function and its interaction with downstream signalling proteins, I have used X-ray crystallography to examine the structures of two DAD2 mutants, DAD2-N242I and DAD2-D166A, that show WT-like hydrolytic activity but display altered protein-protein interactions with petunia’s F-box protein, PhMAX2A. Based on biochemical characterisations, the DAD2-N242I mutant was predicted to exist in a conformation similar to that proposed for the WT receptor in the presence of SL, whereas the DAD2-D166A mutant was predicted to display an altered conformation, specifically in the region where PhMAX2A protein interacts with the receptor. Additionally, mass spectrometry was used to confirm that DAD2 covalently binds the D-ring of SL, which has been previously shown for rice, Arabidopsis and pea in vitro and in vivo. The structural examination of the mutants showed that SL increases the flexibility of the receptor without changing the overall conformation of the protein. This flexibility is predicted to enable interactions with the downstream signalling partners.
