Atomistic Insights Into The Mechanism of Dual Affinity Switching In Plant Nitrate Transporter NRT1.1

Abstract

Improving nitrogen use efficiency is critical to enhancing agricultural productivity and to mitigate environmental pollution. To overcome the fluctuations in soil nitrate concentration, plants have evolved an elaborate nitrate transporting mechanism that switches between high and low affinity. In plants, NRT1.1, a root-associated nitrate transporter, switches its affinity upon phosphorylation at Thr101. However, the molecular basis of this unique functional behavior known as dual-affinity switching remains elusive. Crystal structures of the NRT1.1 nitrate transporter have provided evidence for the two competing hypotheses to explain the origin of dual-affinity switching. It is not known how the interplay between transporter phosphorylation and dimerization regulates the affinity switching. To reconcile the different hypotheses, we have performed extensive simulations of nitrate transporter in conjunction with Markov state models to elucidate the molecular origin for a dual-affinity switching mechanism. Simulations of monomeric transporter reveal that phosphorylation stabilizes the outward-facing state and accelerates dynamical transitions for facilitating transport. On the other hand, phosphorylation of the transporter dimer decouples dynamic motions of dimer into independent monomers and thus facilitates substrate transport. Therefore, the phosphorylation-induced enhancement of substrate transport and dimer decoupling not only reconcile the competing experimental results but also provide an atomistic view of how nitrate transport is regulated in plants.

Thirty years of molecular dynamics simulations on posttranslational modifications of proteins

“…258,259 To explore the effects of phosphorylation on enhanced transport and transporter oligomerization, the Shukla group performed unbiased MD simulations for four systems: unphosphorylated NRT1.1 (UnpNRT1.1) (B9 ms) and phosphorylated (pNRT1.1) (B5 ms) dimer, as well as UnpNRT1.1 (B142 ms) and pNRT1.1 (B63 ms) monomer. 255 Cross-correlation analyses among all Ca atoms within each system demonstrated that dynamic coupling disappeared at the dimeric interface for pNRT1.1 versus UnpNRT1.1 dimer (Fig. 10A-C).…”

“…The Shukla group did so by implementing transition path theory in their NRT1.1 phosphorylation study. 255,256 The plant nitrate transporter NRT1.1 is regulated by phosphorylation. With affinity inverse to the levels of environmental nitrate, NRT1.1 is either a low-affinity transporter during saturating nitrate conditions or a high-affinity transporter during desaturating nitrate conditions.…”

“…Overall, this study suggested that phosphorylation accelerates necessary conformational transitions, resulting in a higher transport rate. 255…”

“…Intermediate states are labeled as (1) inward-facing (IF), (2) occluded (OC), (3) outward-facing (OF), (4) partial IF-OF (inward-outward facing), and (5) hourglass-like state, respectively. Adapted with permission from B. Selvam, J. Feng, and D. Shukla, bioRxiv, 2022 255. Permission directly granted from authors for use of preprint figures.…”

Interplay between phosphorylation and oligomerization tunes the conformational ensemble of SWEET transporters

“…Functional characterization of PIN3 showed that without PID kinase-dependent phosphorylation, end-point PIN3-mediated efflux of indole-3-acetic acid was halved. For the nitrate transporter AtNRT1.1, a monomeric and phosphorylated state encodes low-affinity transport, whereas being dephosphorylated and dimerized encourages high-affinity transport (17)(18)(19).…”