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

Carlos Peña-Varas,Ariela Vergara-Jaque,Osman Mirza,Mariela González-Avendaño,Christa Kanstrup,Ingo Dreyer,David Ramírez,Christoph Crocoll,Hussam Nour-Eldin

doi:10.3390/ijms23031595

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.

Highlights

The nitrate/peptide transporter family (NPF) is one of the largest transporter families in the plant kingdom
The global ProQM score obtained for GTR1 transporter in an inward‐facing conformation (GTR1in) was 0.637, where 0.7 is typical for membrane protein structures solved by X‐ ray crystallography
The key amino acids in the transport process of these substrates are not well known, and the approaches that have been followed to identify potential molecular determinants have been focused on in multiple sequence alignments following extensive validation [14,15]. We tackle this problem focusing on a structural bioinformatics approach, as we modeled GTR1 in two conformations and generated an ensemble of con‐ formers to add flexibility to the transporter

Summary

Introduction

The nitrate/peptide transporter family (NPF) is one of the largest transporter families in the plant kingdom. Currently in the PDB database there are only three crystal structures deposited that belong to the NPF, all of which belong to the Arabidopsis thaliana NTR1.1 (NPF6.3) transporter (PDB codes 4OH3 [3], 5A2O, and 5A2N [2]). This transporter has been re‐ ported to be a dual‐affinity nitrate transporter and the crystallization studies have mainly focused on elucidating the structural basis for nitrate transport. The NPF belongs to the proton‐dependent oligopeptide transport family which is found in all kingdoms. Most POT crystals came from bacterial homologs such as YbgH [5], GkPOT [6], PepTSt [7], PepTSo [7], and PepTSo2 [8,9]

Methods

Results

Conclusion