Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking
Abstract
:1. Introduction
2. Results
2.1. Molecular Modeling of GTR1 in Both Inward- and Outward-Facing Conformations
2.2. Generation of GTR1 Ensemble Conformations
2.3. Testing the Ensemble Docking Pipeline
2.4. Glucosinolate Centrally Located Binding Site Revealed by Ensemble Docking
2.5. Contacts of Glucosinolates with Key Residues of the Centrally Located Binding Site and the Nature of the Chemical Interactions
2.6. Glucosinolate Transport Is Governed by Ionic Interactions
3. Discussion
4. Materials and Methods
4.1. Molecular Modeling of GTR1
4.2. Molecular Modeling of GkPOT
4.3. Molecular Dynamics Simulations (MDs)
4.4. Ensemble Docking of 4MTB and I3M into GTR1 Transporter
4.5. Clustering and Docking Postprocessing
4.6. Cloning of Mutants for Xenopus Oocyte Expression and cRNA Generation
4.7. Xenopus Oocytes Transport Assay
4.8. Glucosinolate Analysis by LC-MS/MS
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Crystallographic Structures (Inward-Facing Conformation) | |||
NTR1.1in (PDB code: 4OH3) | GkPOTin (PDB code: 4IKZ) | ||
Model in an inward-facing conformation | |||
GTR1in | |||
Models in an outward-facing conformation | |||
NTR1.1out | GkPOTout | GTR1out |
Cluster Nº | GkPOTin | GkPOTout |
---|---|---|
1 | 100 | 142 |
2 | 96 | 142 |
3 | 95 | 91 |
4 | 87 | 86 |
GTR1in-4MTB | GTR1in-I3M | GTR1out-4MTB | GTR1out-I3M | |
---|---|---|---|---|
98 | 186 | 510 | 184 | |
81 | 173 | 96 | ||
73 | 88 | |||
70 | ||||
53 | ||||
50 | ||||
49 | ||||
# Significant cluster | 7 | 2 | 1 | 3 |
Total population | 986 | 965 | 979 | 993 |
Total population Significant clusters | 474 | 359 | 510 | 368 |
% conformers in significant clusters | 48.07% | 37.20% | 52.09% | 37.06% |
GTR1 | GkPOT (PDB: 4IKZ) [6] | PepTSt (PDB: 4D2C) [20] | PepTSt (PDB: 4D2D) [20] | Function [14] |
---|---|---|---|---|
E75 | E32 | E22 | E22 | ExxE[K/R] motif [6,14,15] |
E78 * | E35 | E25 | E25 | ExxE[K/R] motif [6] |
K79 * | R36 | R26 * | R26 | ExxE[K/R] motif [6,15] |
I82 | Y39 | Y29 | Y29 | Peptide specificity [21] |
I83 | Y40 * | Y30 * | Y30 | Peptide specificity [6] |
L86 | R43 * | R33 | R33 | Peptide specificity [6] |
N116 * | Y78 * | Y68 | Y68 | Peptide binding [6] |
T119 * | G81 | G71 | G71 | ------ |
L200 | S140 | S130 | S130 | ------ |
R196 * | K136 | K126 * | K126 | ExxE[K/R]-interactor [6] |
F222 | F158 | F148 | F148 | ------ |
F226 | Y162 | V152 | V152 | ------ |
F229 | I165 | I155 | I155 | ------ |
T230 | N166 | N156 | N156 | Peptide binding [6] |
Q233 | A169 | A159 | A159 | ------ |
Y382 | W306 | W296 | W296 | ------ |
I385 | Q309 | E299 | E299 | Peptide binding/protonation [2,3] |
T386 | E310Q * | E300 | E300 | ------ |
M389 | S313 | S303 | S303 * | ------ |
L419 | N342 * | N328 * | N328 | Peptide binding [6] |
M420 | P343 | P329 | P329 | ------ |
E513 | E413 | E400 | E400 * | Proton translocation [6] |
A517 | S417 | S404 | S404 | ------ |
F540 | W440 | W427 | W427 | ------ |
Y541 | F441 | F428 | F428 | ------ |
A544 | N444 | S431 | S431 | ------ |
S547 * | A447 | G434 | G434 | ------ |
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Peña-Varas, C.; Kanstrup, C.; Vergara-Jaque, A.; González-Avendaño, M.; Crocoll, C.; Mirza, O.; Dreyer, I.; Nour-Eldin, H.; Ramírez, D. Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking. Int. J. Mol. Sci. 2022, 23, 1595. https://doi.org/10.3390/ijms23031595
Peña-Varas C, Kanstrup C, Vergara-Jaque A, González-Avendaño M, Crocoll C, Mirza O, Dreyer I, Nour-Eldin H, Ramírez D. Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking. International Journal of Molecular Sciences. 2022; 23(3):1595. https://doi.org/10.3390/ijms23031595
Chicago/Turabian StylePeña-Varas, Carlos, Christa Kanstrup, Ariela Vergara-Jaque, Mariela González-Avendaño, Christoph Crocoll, Osman Mirza, Ingo Dreyer, Hussam Nour-Eldin, and David Ramírez. 2022. "Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking" International Journal of Molecular Sciences 23, no. 3: 1595. https://doi.org/10.3390/ijms23031595