X-ray crystal structure of the plant TIR1-ASK1 complex bound to auxin and a substrate degron peptide. The auxin receptor TIR1 (blue) binds to ASK1 (magenta), together forming a mushroom-shaped protein complex. The plant hormone auxin (green and red spheres) and the substrate degron peptide (orange) occupy a single pocket on top of the TIR1 mushroom cap with auxin sitting at the bottom. A previously unknown inositol hexakisphosphate (IP₆) molecule is found in the middle of TIR1 right under the auxin-binding site (the red stick model in the center of the mushroom "cap").

Auxin's receptor, TIR1, belongs to a large family of F-box proteins, which function as part of a protein destruction machinery in the ubiquitin–proteasome system. Specifically, TIR1 promotes the labeling of protein substrates with a tag called ubiquitin so that they will be recognized and degraded by the cellular protein disposal apparatus—proteasome. It was found that auxin is sensed by TIR1 and in turn helps TIR1 recruit a specific family of protein targets. But how does such a simple small molecule bring together two proteins that are thousands of times bigger?

To answer this question, the researchers first crystallized TIR1 together with a small adaptor protein called ASK1. Next, using the ALS beamlines, they collected x-ray diffraction data at high resolution and created an atomic 3D-model of the protein complex. The crystal structure of TIR1-ASK1 shows that the two-protein complex adopts a mushroom-like shape, with ASK1 and a part of TIR1 forming the stem and the rest of TIR1 forming the cap. By soaking the auxin compound and a peptide of the substrate protein with the crystals, the researchers also determined the quadruple structure of TIR1-ASK1 together with auxin and the substrate: auxin and the substrate bind to a single surface pocket near the center of the mushroom "cap" of TIR1, sandwiching auxin between the two proteins. Auxin's presence is important to the binding of the proteins because it fills up a void space in the hydrophobic protein–protein interface, which is otherwise energetically unfavorable. Unlike many known hormones, which regulate receptors by changing their shapes, auxin instead "glues" the two proteins together. This unexpected mechanism reveals a novel paradigm of hormone action.

Auxin functions as a "molecular glue" to enhance the association between two proteins.  At the bottom of the TIR1 surface pocket, auxin (green) helps nucleate a hydrophobic core together with TIR1 (blue) and its substrate polypeptide (orange).  By simultaneously interacting with both proteins, auxin extends the protein–protein interface.

Another surprising finding is a previously unknown small molecule—inositol hexakisphosphate (IP₆)—which is bound to the core of the auxin receptor. The IP₆ molecule is surrounded by a dozen positively charged amino acids, which lock IP₆ up in the center of TIR1. Most of these amino acids are strictly conserved among the TIR1 proteins from different plants, suggesting an essential role of IP₆ binding in TIR1's function. Its presence in the auxin receptor indicates a previously unrecognized role in plant hormone signaling.

An inositol hexakisphosphate molecule (IP₆) found in the center of TIR1 by crystallographic analysis.  Underneath the auxin-binding site of TIR1 (blue), an IP₆ molecule (red and yellow) closely interacts with more than a dozen positively charged TIR1 residues that are strictly conserved in plants.

Understanding how auxin works at the atomic level is not only important to agricultural economy—most herbicides are auxin analogues— but is also invaluable to biomedical sciences. The protein destruction machinery regulated by auxin is conserved from plants to humans. By revealing how auxin regulates the plant ubiquitin-proteasome system, the research team hopes to translate its findings to the development of therapeutic compounds targeting the corresponding human system. This surprising mechanism of auxin action also suggests that it is possible to use small molecules to rescue defective protein–protein interactions occurring in human diseases. Such a concept opens a new window in drug development.

Research conducted by: X. Tan, C. Zheng, and N. Zheng (University of Washington at Seattle), L.I.A. Calderon-Villalobos and M. Estelle (Indiana University at Bloomington), M. Sharon and C. Robinson (Cambridge University).

Research funding: National Institute of Medicine, Pew Scholar Program, National Science Foundation, and U.S. Department of Energy. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences (BES).

Publication about this research: X. Tan, L.I.A. Calderon-Villalobos, M. Sharon, C. Zheng, C.V. Robinson, M. Estelle, and N. Zheng,  "Mechanism of auxin perception by the TIR1 ubiquitin ligase," Nature 446, 640 (2007).