A fuzzy encounter complex precedes formation of the fully-engaged TIR1-Aux/IAA auxin co-receptor system

The plant hormone auxin regulates almost every aspect of plant development via the TIR1/AFB-auxin-Aux/IAA auxin co-receptor complex. Within this ternary complex, auxin acts as a molecular glue to promote the binding of Aux/IAA transcriptional repressor proteins to SCF TIR1/AFB ubiquitin-ligase complexes, thereby catalysing their ubiquitin-mediated proteolysis. A conspicuous feature of the crystal structure of the complex is a rare cis W-P bond within the Aux/IAA degron motif. To study receptor complex assembly, we have used NMR to determine the solution structure of the amino-terminal half of the Aux/IAA protein AXR3/IAA17, including the degron, both in isolation and in complex with TIR1 and auxin. We show that this region of AXR3 is intrinsically-disordered with only limited elements of structure and yet the critical degron W-P bond occurs with an unusually high (1:1) ratio of cis to trans isomers. We show that assembly of the co-receptor complex involves both auxin-dependent and -independent interaction events in which the disorder of the Aux/IAA is retained. Further, using the synthetic auxin molecule cvxIAA and by analysing specific Aux/IAA conformers, we show that a subset of auxin-dependent binding events occur away from the base of the canonical auxin binding pocket in TIR1. Our results reveal the existence of a fuzzy, topologically-distinct ternary encounter complex and thus that auxin perception is not limited to sequential, independent binding of auxin and then Aux/IAA to TIR1.