Search for: All records

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. 

The serotonin transporter, SERT, catalyzes serotonin reuptake at the synapse to terminate neurotransmission via an alternating access mechanism, and SERT inhibitors are the most widely prescribed antidepressants. Here, deep mutagenesis is used to determine the effects of nearly all amino acid substitutions on human SERT surface expression and transport of the fluorescent substrate analogue APP+, identifying many mutations that enhance APP+ import. Comprehensive simulations of the entire ion-coupled import process reveal that while binding of the native substrate, serotonin, reduces free energy barriers between conformational states to promote SERT dynamics, the conformational free energy landscape in the presence of APP+ instead resembles Na+ bound-SERT, with a higher free energy barrier for transitioning to an inward-facing state. The deep mutational scan for SERT-catalyzed import of APP+ finds mutations that promote the necessary conformational changes that would otherwise be facilitated by the native substrate. Indeed, hundreds of gain-of-function mutations for APP+ import are found along the permeation pathway, most notably mutations that favor opening of a solvent-exposed intracellular vestibule. The mutagenesis data support the simulated mechanism in which the neurotransmitter and a symported sodium share a common cytosolic exit pathway to achieve coupling. Furthermore, the mutational landscape for SERT surface trafficking, which likely filters out misfolded sequences, reveals that residues along the permeation pathway are mutationally tolerant, providing plausible evolutionary pathways for changes in transporter properties while maintaining folded structure.