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Plant Hormone Auxin Functions as Novel Molecular Glue

Xu Tan

How do plants grow toward or away from sunlight? This intriguing property of phototropism drew Charles Darwin to a systematic study of plant movements under different environmental stimuli. He concluded that when plants "are freely exposed to unilateral light, some influence is transmitted from the upper to the lower part, causing the latter to bend" (1). It wasn’t until 50 years after Darwin’s study that the identity of the "influence" was revealed to be a growth-promoting small molecule, indole-3-acetic acid, later named auxin (meaning "to grow" in Greek). It was found that lateral light causes uneven distribution of auxin in plants, which leads to the differential growth rates in light-exposed and shadowed regions and therefore phototropism (2). Auxin was soon established as a master plant hormone, regulating almost every aspect of plant life besides phototropism, from early development to growth patterning to fruit bearing (2). The bewildering physiological versatility of this hormone was further complicated by its structural diversity. More than 200 different chemicals, natural or synthetic, have been shown to bear auxinic activity (see the figure, panel A). Despite this catalog of auxin mimics, the molecular mechanism of auxin action remained elusive until recently.

In 2005, a surprise came when the ubiquitin ligase TIR1 was identified as the long-sought auxin receptor (3). Ubiquitin ligases are enzymes that catalyze the covalent linkage of a small protein called ubiquitin to protein substrates, which usually results in the destruction of the substrates. This ubiquitination process has been recognized as a major route for regulating the abundance and function of a myriad of cellular proteins across eukaryotes, but no ligase had been shown to sense small molecules. In this surprising case, TIR1 perceives auxin and is activated for the ubiquitination of a family of transcriptional regulators named AUX/IAAs. The destruction of the AUX/IAAs leads to the reprogramming of gene expression and subsequent diverse auxin responses (3). Auxin promotes the association of TIR1 and the substrate AUX/IAAs, which is a daunting task for a small molecule given the huge size difference between small molecules and proteins. Knowledge of animal hormones suggests that auxin may achieve this by changing the shape of its receptor upon binding, thus switching the receptor to a conformation with high affinity for substrates. However, such a conformational switch mechanism usually demands a highly specific chemical structure, which is puzzling given the chemical diversity of auxin analogs.

Using x-ray crystallography, I solved six structures of the auxin receptor in different functional states to show how the hormone works (4). The receptor TIR1 forms a donut-like shape, presenting a large central pocket for sensing auxin at the bottom and binding the protein substrate on the top (see the figure, panel B). The conserved carboxyl group of auxin functions as an anchor by interacting with a key arginine residue of the receptor. This hormone-binding site is otherwise quite promiscuous, capable of accommodating a variety of auxin analogs as revealed by three TIR1 structures with different analogs. The substrate protein binds right above auxin using several conserved hydrophobic amino acids to interact with the aromatic ring of auxin, covering the space above the center of the donut. Unexpectedly, TIR1 maintains the same shape after hormone binding, thereby disproving the predicted conformational switch model. Rather, auxin functions by filling up a gap between the two proteins and directly bridging two proteins. In other words, the hormone acts like a "molecular glue" to stick the two proteins together. By doing this, auxin greatly increases the ubiquitination efficiency as demonstrated by reconstituted ubiquitination assays. This surprising molecular glue mechanism is consistent with the promiscuity of the hormone binding pocket of the receptor, since any hydrophobic moiety that can fit in the pocket without causing steric hindrance can be a molecular glue, explaining why so many chemicals function as auxins. Another surprise is that a previously unknown player, inositol hexakisphosphate (IP₆), a second small molecule, was found to bind beneath auxin and support auxin binding. We propose that IPM₆ is an important cofactor of the auxin receptor because its binding is facilitated by 11 positively charged residues of TIR1 that are highly conserved throughout the TIR1 family. Mutations of some of those residues have been shown to destabilize the receptor and produce prominent phenotypes in plants. Since IP₆ is known to be involved in plant phosphate homeostasis (5), it might have signaling functions in the cross-talk of different pathways.

Auxin brings together an ubiquitin ligase and its target. (A) Chemical structures of the natural auxin indole-3-acetic acid and some other auxin analogs. (B) Schemes of the auxin sensing mechanism: Auxin binds to a surface pocket of the receptor TIR1, which is supported by IP₆. Auxin subsequently recruits the substrate through a molecular glue mechanism.

My results established the first structural model of a plant hormone receptor and revealed a novel mechanism of hormone action and signal transduction. Given the hundreds of ubiquitin ligases like TIR1 in plants and humans, TIR1 represents the founding member of a potentially broad class of ubiquitin ligases that use small molecules to facilitate binding to substrates. Indeed, another plant hormone, jasmonate, appears to be sensed through a similar mechanism (4, 6). Understanding the structure-function relationship of auxin will help guide the design of better auxins or anti-auxins (7), some of which have already been widely used as herbicides.

Importantly, what we learned from auxin and its ubiquitin ligase receptor points us in a new direction for developing drugs that target ubiquitin ligases in humans. Human ubiquitin ligases are regarded as the next generation of drug targets because of their important roles in diverse cellular functions. Conventional drug development strategies aimed at discovering small-molecule inhibitors have had very limited success due to the major obstacle of finding small compounds that can disrupt protein-protein interactions, which underlie the functions of most ubiquitin ligases (8). The mechanism of auxin action revealed by my studies shows that nature uses the opposite approach to regulate ubiquitin ligases with a naturally occurring compound: Auxin agonizes its receptor’s ubiquitin ligase activity by promoting protein-protein interaction rather than disrupting the interaction. In fact, many human diseases such as cancer and Parkinson’s disease are associated with defective ubiquitin ligases, whose activities might be restorable by small molecules following the same principle auxin employs. Such compounds can be both small and effective, and therefore more feasible to develop. So next time someone tells you a new drug finds its origin in Darwin's plant pot, don’t be surprised.

References

1. C. Darwin, The Power of Movement in Plants (John Murray, London, 1880).
2. L. Taiz, E. Zeiger, Plant Physiology (Sinauer, Sunderland, MA, ed. 4, 2006).
3. G. Parry, M. Estelle, Curr. Opin. Cell Biol. 18, 152 (2006).
4. X. Tan et al., Nature 446, 640 (2007).
5. J. Stevenson-Paulik, R. J. Bastidas, S. T. Chiou, R. A. Frye, J. D. York, Proc. Natl. Acad. Sci. U.S.A. 102, 12612 (2005).
6. L. Katsir, H. S. Chung, A. J. Koo, G. A. Howe, Curr. Opin. Plant Biol. 11, 428 (2008).
7. K. Hayashi et al., Proc. Natl. Acad. Sci. U.S.A. 105, 5632 (2008).
8. K. Garber, J. Natl. Cancer Inst. 97, 166 (2005).

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