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Abstract Phosphorylation of the penultimate residue, threonine (pen-Thr), of plasma membrane (PM) H+-ATPase is essential for its activation and blue light (BL)-induced stomatal opening. However, the regulatory mechanism of action of PM H+-ATPase pen-Thr phosphorylation is not completely understood. Here, we performed screening using a protein kinase inhibitor library and found that tyrphostin AG126 inhibited phosphorylation of PM H+-ATPase pen-Thr in guard cells in response to light and fungal toxin fusicoccin (FC), in addition to inhibition of light- and FC-induced stomatal opening. Analysis of the structure–activity relationship using AG126 derivatives revealed the hydroxyl group at the C-5 position of the compound to be essential for its activity. We further characterized one AG126 derivative, AGD-1, which effectively suppressed BL-induced stomatal opening with a half-inhibitory concentration of 2.0 μM. AGD-1 inhibited PM H+-ATPase pen-Thr phosphorylation in guard cells in response to BL and FC. In addition, AGD-1 suppressed FC-induced PM H+-ATPase pen-Thr phosphorylation in mesophyll cell protoplasts, implying that the effect of AGD-1 is not specific to guard cells. Furthermore, to improve the permeability of AGD-1, we synthesized acetylated AGD-1 (AcAGD-1), which was found to suppress BL- and FC-induced stomatal opening. AcAGD-1 suppressed light-induced PM H+-ATPase pen-Thr phosphorylation, but not Thr881 phosphorylation, in leaf discs, which is important for guard cell PM H+-ATPase activation in addition to pen-Thr phosphorylation. This study identified a novel stomatal opening inhibitor capable of specifically inhibiting PM H+-ATPase pen-Thr phosphorylation.
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Structural basis of S-adenosylmethionine-dependent allosteric transition from active to inactive states in methylenetetrahydrofolate reductase
Abstract Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation andS-adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures ofChaetomium thermophilumMTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form ofcMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites inhMTHFR, three of which were previously unidentified. Based on the structural framework provided by ourcMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.
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- Award ID(s):
- 1945174
- PAR ID:
- 10515397
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 15
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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