Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking

Abstract

Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4-methylthiobutyl (4MTB) and indol-3-ylmethyl (I3M) glucosinolates to study how GTR1 from Arabidopsis thaliana transports these substrates in computational simulation approaches. The designed pipeline reported here includes massive docking of 4MTB and I3M in an ensemble of GTR1 conformations (in both inward and outward conformations) extracted from molecular dynamics simulations, followed by clustered and substrate–protein interactions profiling. The identified key residues were mutated, and their role in substrate transport was tested. We were able to identify key residues that integrate a major binding site of these substrates, which is critical for transport activity. In silico approaches employed here represent a breakthrough in the plant transportomics field, as the identification of key residues usually takes a long time if performed from a purely wet-lab experimental perspective. The inclusion of structural bioinformatics in the analyses of plant transporters significantly speeds up the knowledge-gaining process and optimizes valuable time and resources.

Mechanistic insight into substrate specificity of plant glucosinolate transporters

“…The dockings indicate strong interactions between the selectivity filter and the side chains for glucosinolates being transported and show that the core glucosinolate structure interacts with two conserved residues that are assumed to be involved in general substrate recognition in the POT family. Whereas interactions with the two conserved residues have previously been observed in dockings of I3M, 4MTB, 4MSB and benzyl in NPF2.10 using inward and outward facing conformations (Chung et al 2022, Peña-Varas et al 2022), but the selectivity filter has not been described before.…”

“…In order to facilitate a structural interpretation of the eight mutations identified in NPF2.9Δ8#3, we docked I3M and pOHB in NPF2.9, NPF2.9Δ8#3 and NPF2.10 homology models. We have previously performed dockings of 4MTB and I3M in outward and inward facing conformations of NPF2.10 (Jørgensen et al 2017, Peña-Varas et al 2022). In this study, we were interested in performing the dockings in an occluded state, to better study the binding cavity.…”

“…The models were relaxed using Molecular Dynamics as previously described (Peña-Varas et al 2022) before docking. Briefly, all the models were optimized at pH 4.5 ± 2.0 with Epik (Shelley et al 2007) and PROPKA (Olsson et al 2011).…”

“…The models were created and validated using SWISS-MODEL. After generation of the models, they were relaxed through Molecular Dynamics simulations (MDs) as previously described (Peña-Varas et al 2022).…”

Specialty grand challenge in plant biophysics and modeling

“…With the expected increasing accessibility of plant protein structures, efforts can be intensified to gain detailed knowledge of molecular processes in plants. And here, computational simulations such as docking or Molecular Dynamics simulations will play an increasingly crucial role in the near future (Navarro-Retamal et al, 2016a,b, 2018Khan et al, 2019;Schott-Verdugo et al, 2019;Rasheed et al, 2020;Lehmann et al, 2021;Moe-Lange et al, 2021;Peña-Varas et al, 2022).…”