Molecular mechanism underlying regulation of Arabidopsis CLCa transporter by nucleotides and phospholipids

Zhao Yang1,4, Xue Zhang1, Shiwei Ye2, 4, Jingtao Zheng3, Xiaowei Huang1,4, Fang Yu3, Zhenguo Chen5, Shiqing Cai2, Peng Zhang1,
1National Key Laboratory of Plant Molecular Genetics, Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China.
2 Center for Excellence in Brain Sciences and Intelligence Technology, Institute of Neuronscience, Chinese Academy of Sciences, Shanghai, 200031, China.
3Shanghai Key Laboratory of Plant molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai, 200234, China.
4 University of Chinese Academy of Sciences, Beijing, 100039, China.
5 The Fifth People's Hospital of Shanghai, Institutes of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.

Arabidopsis thaliana CLCa (AtCLCa), a tonoplast-localized NO3-/H+ antiporter, harnesses the proton gradient to facilitate the transport of NO3- and a minor amount of Cl- into the vacuole. The activity of AtCLCa is regulated by ATP and PIP2, whose physiological function was assumed to regulate stomatal movement. However, the molecular mechanism remains unclear. Here we determine the cryo-EM structures of AtCLCa bound with ATP and PI(4,5)P2. Structural and electrophysiological analyses reveal an auto-inhibiting N-terminal β-hairpin that is stabilized by ATP binding to block the anion transport pathway, thereby inhibiting the AtCLCa activity. While AMP loses the inhibition capacity due to lack of the β/γ-phosphates required for β-hairpin stabilization. This well explains how AtCLCa senses the ATP/AMP status to regulate the physiological nitrogen-carbon balance and stomatal opening. Our data further show that PI(4,5)P2 or PI(3,5)P2 binds to the AtCLCa dimer interface and occupies the proton-exit pathway, which may help to understand the inhibition of AtCLCa by phospholipids to facilitate guard cell vacuole acidification and stomatal closure. In a word, our work suggests the regulatory mechanism of AtCLCa by nucleotides and phospholipids under certain physiological scenarios.