- American Society of Plant Biologists
The plant defense hormone jasmonate (JA) functions through a multiprotein complex containing the F-box protein CORONATINE INSENSITIVE1 (COI1) (reviewed in Katsir et al., 2008; Browse, 2009). This complex forms an E3 ubiquitin ligase, which binds to downstream negative transcriptional regulators known as jasmonate ZIM-domain (JAZ) proteins and targets them for ubiquitin-meditated degradation. The interaction of COI1 and JAZ1 requires the active form of JA, which is the amino acid–conjugated form jasmonoyl-isoleucine (JA-Ile). The COI1–JAZ interaction is also promoted by the bacterial phytotoxin and hormone mimic coronatine. Interestingly, the auxin response pathway shows distinct similarity to the JA response pathway, with another F-box protein, TRANSPORT INHIBITOR RESPONSE1 (TIR1), in a role analogous to COI1 (reviewed in Katsir et al., 2008). TIR1 has been shown to bind auxin directly in a hydrophobic pocket on the protein's surface. Intriguingly, TIR1 has significant similarity to COI1, both in primary and secondary protein structure (33% sequence similarity, F-box motif, and leucine-rich repeats [LRRs]).
On the basis of previous studies and analogy to TIR1, the COI1-JAZ complex was clearly key to JA perception, and the COI1 protein was suspected to be the JA receptor (Browse, 2009). However, it remained unclear whether JA could bind to COI1 alone or whether binding involved both COI1 and its JAZ partner. To further examine JA binding, Yan et al. (pages 2220–2236) produced a high-quality molecular model of COI1 based on the crystal structure of TIR1. The model predicted that COI1 function requires a reducing environment because of unpaired Cys residues, a prediction that the authors verified experimentally. They also examined additional coi1 mutant alleles to verify the importance of the LRRs in COI1 protein stability. Most intriguingly, the model predicted a surface pocket that could serve as a JA-Ile binding site (see figure ). Molecular docking simulations showed that JA-Ile and coronatine fit into the surface pocket better than other jasmonate forms, such as jasmonic acid and methyl jasmonate.
Proposed model of JA-Ile binding to COI1. P1-P4 and the Bottleneck represent different regions of the COI1 protein, flanking the surface binding pocket.
In support of the model for JA-Ile binding, the authors found that COI1 could be recovered from total Arabidopsis protein extracts by binding to JA linked to sepharose, which forms a JA configuration similar to JA-Ile. Pull-down assays showed that mutations affecting the binding pocket also impaired the COI1–JAZ1 physical interaction. To further examine the COI1–JAZ1 interaction, the authors used surface plasmon resonance, wherein one protein is immobilized on a surface and a response is produced by addition of an interacting protein in solution. They found that addition of COI1 preincubated with JA-Ile or coronatine produced a positive signal with immobilized JAZ1, but no signal was produced by COI1 alone nor by JA-Ile or coronatine alone. Finally, the authors synthesized a biotin-tagged photoaffinity probe derived from coronatine; this probe comprised a coronatine moiety and a photoreactive group that covalently attaches to the binding protein upon irradiation, thereby allowing subsequent detection with antibiotin antibody. This probe, which retained coronatine biological functions, specifically labeled purified COI1, thereby demonstrating direct binding of COI1 to coronatine. Now that this interesting work has identified the protein that binds directly to JA-Ile, future work can examine the mechanisms of specificity in the JA response, which involves interactions with many JAZ proteins and affects many environmental and developmental pathways.
