Structural and mechanistic insights into nitrate transport in <scp> <i>At</i> NRT1 </scp> .1: The impact of His356 protonation and Thr101 phosphorylation effect

The nitrate transporter AtNRT1.1 in Arabidopsis thaliana exhibits a unique dual-affinity transport system that optimizes nitrate uptake across variable environmental conditions. Using molecular dynamics simulations, we investigate the dynamic mechanisms governing nitrate retention and transport of AtNRT1.1. Our results reveal that protonated state of His356 stabilizes nitrate binding through interactions with Arg45, while deprotonation of His356 facilitates nitrate release toward the cytosol. We also observed that the loop between transmembrane helices TM4 and TM5, and the conserved FXXFYXXINXG motif in TM5 involved in conformational changes between inward-open and occluded states. Specifically, the Glycine in this motif serves as a pivot point, enabling TM5 bending and promoting the formation of a hydrophobic layer between TM5 and TM10, thereby sealing the intracellular gate. Additionally, phosphorylation of Thr101 further modulates AtNRT1.1 dynamic, enhancing flexibility in the TM2 and TM4 region and potentially influencing the probability of occluded state formation. These findings provide mechanistic insights into the sophisticated dual-affinity transport system of AtNRT1.1.