Structure study of a plant dual-affinity transceptor, CHL1

Abstract

Nitrate is a key nutrient and signaling molecule for plant development and growth. It is the main source of inorganic nitrogen for plants grown in the aerobic soil condition. The growth of many species, including cultivated crops, is strictly dependent on the effective processes of nitrate uptake under frequent fluctuations of nitrate levels in soil. Nitrate is also an important signal molecule that affects plants' metabolism and development. In order to accommodate the changing nitrate concentrations in the soil, which can be varied by four orders of magnitude, plants have evolved a dual-affinity transporter system, NRT1 (low-affinity) and NRT2 (high-affinity) families. Previous studies have shown that Arabidopsis thaliana CHL1 (NRT1.1), which belongs to the low affinity family, functions as a dual-affinity nitrate transporter and sensor, whose high- and low-affinity states are switched upon phosphorylation of a key residue Thr101. However, the following two questions remain unknown: 1) What is the structural mechanism underlying the dual-affinity nature of CHL1; and 2) What is the molecular mechanism that governs the integration of nitrate transporter and sensor function in one protein, CHL1. Therefore, I solved the crystal structure of CHL1 at atomic resolution, which reveals a face-to-face inward dimer with the key residue T101 buried in the dimer interface. In chapter 2, I confirm that functional CHL1 indeed dimerizes on the cell membrane with a cell-based fluorescence resonance energy transfer (FRET) assay, and further show that decoupling of the dimer by phosphorylation mimetic mutation on Thr101 is sufficient to convert CHL1 into a monophasic high-affinity transporter. In chapter 3, I revealed a potential secondary substrate-binding site on the intracellular side of CHL1, which may be important in nitrate induced signal transduction and regulation. Together with structural analysis and mutational assays, this study provides a structure basis for the working mechanism of transceptor.