Sigurd Ramans-Harborough, Arnout P. Kalverda, Iain W. Manfield, Gary S. Thompson, Martin Kieffer, Veselina Uzunova, Mussa Quareshy, Justyna M. Prusinska, Suruchi Roychoudhry, Ken-ichiro Hayashi, Richard Napier, Charo del Genio, and Stefan Kepinski

PNAS September 27, 2023; 120 (40) e2221286120

https://doi.org/10.1073/pnas.2221286120

Significance

This paper shows the most detailed and complete view to date of a canonical Aux/IAA auxin coreceptor protein. Molecular dynamics simulation, coupled with nuclear magnetic resonance analysis shows that, although nominally disordered, the N-terminal half of the Aux/IAA AXR3 appears to show a propensity toward adoption of a small number of specific conformations. The conformational coexistence in auxin coreceptors provides an insight into a protein family that is so crucial for plant life on earth.

Abstract

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W–P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

See https://www.pnas.org/doi/10.1073/pnas.2221286120

Figure 1: Overview of the Aux/IAA degron and the intrinsic disorder of AXR3 DI/DII. (A) Structure of IAA7/AXR2 degron (cis-P87) bound to TIR1 and auxin, showing the two TIR1 cavities based on 2P1Q ((12)). The molecular surface of TIR1 is shown in mauve, the degron peptide in colored sticks by residue and auxin is green at the base of the auxin-binding pocket (B) Amino acid sequences of DII from different Aux/IAA proteins with polymorphisms highlighted in bold and underlined. Core residues are in orange, and the mutated residue in axr3-3 is shown in purple. The AXR2 sequence highlighted and in bold indicates the peptide crystallized by Tan et al. (12). Below the sequence alignment is a schematic of the AXR3 protein showing the four domains. The location of the degron is highlighted, and the dashed lines indicate the DI/DII region of the protein studied by NMR (C and D) ¹H–¹⁵N HSQC spectrum of the protein AXR3 DI/DII at 16.5 °C. The peaks associated with P87 in the cis isomer conformation are annotated light-blue. (D) An enlarged image of the signal-dense region of the HSQC spectrum in (C).