Revealing the earliest events of auxin perception

• Main Author: Ramans Harborough, Sigurd
• Format: Dissertation
• Language: English
• Published: University of Leeds 2016

Auxin signalling involves the ubiquitin-mediated degradation of Aux/IAA transcriptional repressor proteins. This is dependent on the interaction between a TIR1/AFB auxin co-receptor protein, an auxin molecule, and the Aux/IAA protein. Here we characterise the structure of an Aux/IAA protein AXR3 domains I and II, focussing in particular on the conformations adopted within the region of the Aux/IAA protein known as the degron before and during its interaction with the auxin molecule and TIR1. We use of a range of biophysical techniques including Nuclear Magnetic Resonance (NMR) Spectroscopy, Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR). Our work provides the first evidence that a large component of an Aux/IAA's structure is intrinsically disordered with tendencies to form secondary structure around the degron. We show that cis and trans isomer states of Pro 87 within the degron core are approximately equal in abundance and that binding to TIR1 is stronger for the cis state, but nevertheless apparent for both conformations highlighting the possibility of a distinct encounter complex that precedes the fully-docked complex revealed by previous crystallographic analysis. Residues flanking the degron core are shown to be important for the interaction with TIR1 and we show that by mutation of these peripheral sequences it is possible to both decrease and increase the kinetics of complex formation. We also report on apparent low affinity auxin binding to the Aux/IAA degron in the trans conformation. We discuss the possible regulatory significance of apparent conformation shifts within the degron core that may act as a molecular switch to control the Aux/IAA's intrinsic disorder and how this disorder may relate to its hypothesised function as a co-receptor for auxin.