Cryo-EM structure of the plant nitrate transporter AtCLCa reveals characteristics of the anion-binding site and the ATP-binding pocket

Jin He; Mingxing Wang; Shanshan Li; Long Chen; Kaiming Zhang; Ji She

doi:10.1016/j.jbc.2022.102833

Cryo-EM structure of the plant nitrate transporter AtCLCa reveals characteristics of the anion-binding site and the ATP-binding pocket

J Biol Chem. 2023 Feb;299(2):102833. doi: 10.1016/j.jbc.2022.102833. Epub 2022 Dec 26.

Authors

Jin He¹, Mingxing Wang¹, Shanshan Li¹, Long Chen¹, Kaiming Zhang², Ji She³

Affiliations

¹ MOE Key Laboratory for Cellular Dynamics, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China; Biomedical Sciences and Health Laboratory of Anhui Province, University of Science and Technology of China, Hefei, China.
² MOE Key Laboratory for Cellular Dynamics, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China; Biomedical Sciences and Health Laboratory of Anhui Province, University of Science and Technology of China, Hefei, China. Electronic address: kmzhang@ustc.edu.cn.
³ MOE Key Laboratory for Cellular Dynamics, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China; Biomedical Sciences and Health Laboratory of Anhui Province, University of Science and Technology of China, Hefei, China. Electronic address: jishe@ustc.edu.cn.

Abstract

Nitrate is one of the major nitrogen sources for most plants. Chloride channel (CLC) proteins mediate the transport and vacuole storage of nitrate in plants, but the structural basis of nitrate transport by plant CLC proteins remains unknown. Here, we solved the cryo-EM structure of ATP-bound Arabidopsis thaliana CLCa (AtCLCa) at 2.8 Å resolution. Structural comparison between nitrate-selective AtCLCa and chloride-selective CLC-7 reveals key differences in the central anion-binding site. We observed that the central nitrate is shifted by ∼1.4 Å from chloride, which is likely caused by a weaker interaction between the anion and Pro160; the side chains of aromatic residues around the central binding site are rearranged to accommodate the larger nitrate. Additionally, we identified the ATP-binding pocket of AtCLCa to be located between the cytosolic cystathionine β-synthase domains and the N-terminus. The N-terminus may mediate the ATP inhibition of AtCLCa by interacting with both ATP and the pore-forming transmembrane helix. Together, our studies provide insights into the nitrate selectivity and ATP regulation of plant CLCs.

Keywords: ATP; AtCLCa; CLC; antiporter; cryo-EM; nitrate; vacuole.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenosine Triphosphate / metabolism
Anions / metabolism
Arabidopsis Proteins* / metabolism
Arabidopsis* / metabolism
Binding Sites
Chloride Channels* / metabolism
Chlorides / metabolism
Cryoelectron Microscopy
Nitrate Transporters* / metabolism
Nitrates / metabolism

Substances

Adenosine Triphosphate
Anions
Chloride Channels
Chlorides
Nitrate Transporters
Nitrates
ATCLC-A protein, Arabidopsis
Arabidopsis Proteins