Understanding the structural basis of ALS mutations associated with resistance to sulfonylurea in wheat

Abstract

Developing herbicide-tolerant wheat varieties is highly desirable for effective weed management and improved crop yield. The enzyme acetolactate synthase (ALS) is the target enzyme for the sulfonylurea class of herbicides. The structural analysis of mutable sites in ALS is crucial for the generation of herbicide-resistant crops. Previous studies indicated that mutant lines of Triticum aestivum ALS (TaALS) with amino acid substitutions at P174, G631, and G632 residues provided resistance to sulfonylurea herbicide, nicosulfuron. The present study aimed to provide structural insights into mutable residues causing sulfonylurea herbicide resistance to TaALS enzyme through in-silico molecular docking and simulation approaches. The molecular docking analysis suggested a single point mutation at TaALS-P174S, its double mutant conformations (TaALS-G632S/P174S and TaALS-G631D/G632S) and associated triple mutant conformation (TaALS-G631D/G632S/P174S) to have the lowest binding affinity with nicosulfuron than the wild-type conformation of TaALS. Furthermore, the molecular dynamic simulation study confirms the weakest and more stable binding of the triple mutant conformation with nicosulfuron. Our computational study identifies a triple mutant conformation (TaALS-G631D/G632S/P174S) to be more effective in developing sulfonylurea herbicide-resistant wheat crops.

Introduction

Wheat is one of the most important cereal crops belonging to the family Poaceae. Hexaploid wheat (T. aestivum), also known as bread wheat, is grown globally (95%), followed by tetraploid durum wheat (T. turgidum) to meet the global food requirement¹. An increase in the grain yield of wheat crops is a prerequisite for fulfilling the demand of the growing population². However, its production is limited due to multiple constraints, including weed infestation, as the latter compete with the crop for water, nutrients, and light, which eventually lowers the crop productivity³. Therefore, it is important to explore effective weed control strategies for their management in the field. Among the various weed management strategies, chemical herbicides are the first choice as they are less expensive and more effective than other alternative options. However, addressing herbicide resistance in weeds through diverse management practices is crucial. Management of herbicide resistance has significant broader implications for agriculture, economic stability, and food security. Herbicide-resistant weeds can lead to increased production costs for farmers due to higher herbicide applications and more intensive weed management strategies. This can affect overall profitability and economic stability in agricultural sectors. As herbicide resistance increases, crop yields can decline due to increased weed competition. This can threaten food security, especially in regions heavily reliant on monocultures and chemical inputs⁴. Effective resistance management practices, including crop rotation, herbicide rotation, and integrated weed management, can lead to a more sustainable farming system. Presently, herbicide-resistant crop cultivation is a successful weed management strategy that can lower crop phytotoxicity from herbicide treatment, increase the herbicidal spectrum, and lower weeding costs⁵.

Previously, it has been reported that different classes of ALS-inhibiting herbicides, including Imidazolinone, Sulfonylurea, Pyrimidinyl thiobenzoates, Sulfonylamino-carbonyl-triazolinone and Triazolo pyrimidine act by blocking the branched amino acids (Valine, Leucine, and Isoleucine) biosynthesis pathway. In this pathway, Acetolactate synthase (ALS), also referred to as acetohydroxyacid synthase (AHAS), is a key enzyme and is considered an ideal target for designing novel herbicides^6,7. However, the physiological similarity of weeds and crop plants limits their uses because the herbicides also kill the wheat plants. Therefore, developing herbicide-tolerant wheat varieties is highly desirable as an effective weed management strategy. Different ALS-inhibiting herbicide families bind at distinct binding pockets of the ALS enzyme. Hence, specific point mutations in the ALS gene would provide tolerance against a specific class of ALS-inhibiting herbicides⁸. For example, P197 mutations in the Arabidopsis thaliana acetohydroxyacid synthase (AtALS) enzyme confer sulfonylurea tolerance, whereas G654 mutations provide tolerance against Imidazolinone family herbicides^8,9. The molecular docking study of Oryza sativa ALS suggests that OsALS-P171, the site homologous to AtALS-P197, is located at the catalytic pocket site and interacts with the aromatic ring of sulfonylurea¹⁰. Also, recent molecular docking study of Linum usitatissimum L. ALS (LuALS1) suggests that mutation at LuALS1-Pro197, a site homologous to AtALS-P197, led to reduction in hydrogen bonds formed between LuALS1-Pro197 and sulfonylurea (tribeneuron-methyl)¹¹. Therefore, mutation at OsALS-P171 and Triticum aestivum ALS (TaALS-P174) is expected to confer tolerance to sulfonylurea. These precise mutations in the ALS gene serve as a potential target for genome editing to produce herbicide-resistant agronomic crops⁸. Previously, transgene-free ALS-inhibiting herbicide-tolerant crops have been developed by cytosine base editing in different crops such as wheat, rice, maize, watermelon, arabidopsis, tomato, potato, etc^5,10,12–16. However, in silico structural analysis of the TaALS gene and its interactions with important herbicides are currently lacking, that can provide deep-structural insights about key residues associated with herbicide resistance in wheat.

Therefore, in the present study, we analysed the effect of amino acid substitutions in the Triticum aestivum ALS (TaALS) enzyme against the sulfonylurea (i.e. nicosulfuron) herbicide. Our main aim was to analyse the binding mode of nicosulfuron, the effect of point mutations (amino acid substitutions) on the binding activity, and the identification of novel mutation using molecular docking and simulation study. Hence, a computational study was carried out to understand the effect of wheat TaALS mutations on the regulation of nicosulfuron herbicide resistance. The identified mutations from the present study could be further used for developing herbicide-tolerant crops through CRISPR/Cas mediated base editing or primer editing approach.

Materials and methods

Homology modelling of ALS enzyme

The experimental structure of the TaALS enzyme was not available at the public repository, i.e. RCSB- Protein Data Bank (PDB) database¹⁷, therefore the 3D structure of the protein was modelled using the SWISS-MODEL¹⁸ taking AtALS (PDB ID: 3E9Y), as a template structure. The quality of modelled structure was evaluated using the SAVES v6.0 server (https://saves.mbi.ucla.edu/) and PDBsum server¹⁹ to determine its suitability for further analysis. SAVES v6.0 server is an assembly of various programs such as ERRAT, VERIFY3D, PROCHECK, etc. ERRAT examines the statistics of the non-bonded interactions between atoms and gives the overall quality factor of the protein²⁰. VERIFY-3D analyses the compatibility of the atomic model with its amino acid sequence²¹. PROCHECK generates Ramachandran plots, which depict the stereochemical quality of the protein model²². Previously, it has been reported that the base modification in the P197 codon of ALS was effective in developing herbicide-resistant germplasm in various crops^5,8,9,23. Additionally, some recent studies in wheat and rice showed that single base editing in ALS gene of Triticum aestivum (TaALS-P174S, TaALS-P174A, TaALS-P174F TaALS-G631D, TaALS-G632S) and Oryza sativa (OsALS-P171S, OsALS-P171A, OsALS-P171F, OsALS-G628D, OsALS-G629F) could impart tolerance to ALS-inhibiting herbicides^10,13. Furthermore, the triple mutant conformations TaALS-G631D/G632S/P174F and OsALS-G628D/G629S/P171F showed higher resistance against nicosulfuron than the single proline mutants^10,13. Based on these studies, we incorporated different proline (P174S, P174A, P174F) and Glycine (G631D, G632S) substitutions on both chains of the homo-dimeric structure of TaALS protein. Different TaALS mutant models were generated using the mutagenesis wizard of open-source PyMol v2.5.0. All the models, including mutants, were subjected to SPDBV (swiss PDB viewer) for energy minimisation²⁴.

Ligand and receptor preparations

Nicosulfuron, a widely used herbicide, and member of the sulfonylurea herbicide family was selected to analyse the impact of different TaALS mutations on binding with nicosulfuron. The ligand (nicosulfuron) 3D structure was retrieved from the PubChem database (Compound CID: 73281) in SDF format and converted into PDB format using Open Babel²⁵. Autodock Tools v1.5.7²⁶ was used for preparing pdbqt files. Modelled TaALS and its mutant structures were considered as receptors, while herbicide nicosulfuron was considered as ligand. The receptors were prepared by adding polar hydrogen atoms followed by the addition of Kollman and Gasteiger charges.

Docking of modelled TaALS protein and its mutants with nicosulfuron

To determine the binding pocket, the modelled TaALS structure was superimposed over the crystal structure of AtALS complexed with monosulfuron (PDB ID: 3E9Y), whose active site for the sulfonylurea family has been well-studied²⁷. In the binding pocket, a grid box of size 47Å X 53Å X 47Å in the X, Y, and Z dimensions was set up with a grid spacing of 0.37 Å. Lamarckian genetic algorithm was used to search the binding conformations, and a total of 100 iterative dock runs were performed with 250 million ²⁸evaluations using Autodock v4.2.6 ²⁹. The best-docked complex was selected based on the lowest binding energy and maximum number of clusters for all docked complexes.

Molecular dynamics simulation

The wild-type and mutant TaALS-herbicide complexes obtained after docking were subjected to Molecular Dynamics Simulation (MDS) to observe the stability of complexes. GROMACS v.2022³⁰ was used to run 100 ns MDS for each complex under CHARMM36 force field parameters. Briefly, ligand topology and compatible input files were generated using the CHARMM general force field (CGenFF) server^30,31 with CHARMM36 force field parameters. The complex was solvated with TIP3P water molecules into a dodecahedron box, followed by the addition of appropriate Na⁺ and Cl⁻ ions to neutralise the system. The LINCS algorithm was used to constrain the bond lengths, and the energy minimisation was carried out on each assembled system for 50,000 steps using the steepest descent algorithm to attain a stable system with a maximum force (F_max) of < 10 KJ/mol.

Furthermore, two-step equilibrations were performed for 100 ps individually under NVT (constant Number of particles; constant Volume; and Temperature: 300 K) followed by NPT (constant Number of particles; Pressure: 1 bar; and Temperature: 300 K) conditions. Finally, the equilibrated systems were subjected to an MDS production run of 100 ns at 300 K temperature and 1 bar pressure with a timestep of 2 fs. Trajectories were recorded for every 100 ps and were visualised using the Visual Molecular Dynamic (VMD) visualisation tool³². After MDS, different statistical analyses like Root Mean Square Deviation (RMSD), Radius of Gyration (RoG), Root Mean Square Fluctuations (RMSF), interaction energy including Lennard-Jones interactions, and coulombic interaction energy, number of hydrogen bonds and Solvent Accessible Surface Area (SASA) were calculated using the inbuilt scripts of GROMACS. To calculate the free binding energy of complexes, the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) was calculated using gmx_MMPBSA³³ for the whole MDS run of 100 ns.

Results and discussion

Model preparation and validation

The three-dimensional structure of AtALS (PDB ID: 3E9Y) was selected as a template for building the 3D structure of TaALS protein using the SWISS-MODEL web server¹⁸. An overall 75.47% identity and 96% query coverage was observed between template (3E9Y) and target protein sequence. The obtained homo-dimeric model showed a QMEANDisCo local score of 0.87, reflecting the expected similarity of the modelled structure with the reported structures. A Qualitative Model Energy ANalysis (QMEAN) score of 0.84 also indicated an excellent predicted model similar to the experimental structure. A high ERRAT score with an quality factor of 93.93%, along with an evaluation of the VERIFY3D program, showed that 85.68% of the residues had an average 3D-1D score > = 0.2, suggested the good quality of the predicted model. Furthermore, Ramachandran plot analysis showed that 91.7% residues were present in the most favoured region and 7.9% residues in the additionally allowed region, confirming the reliability of the generated model (Fig. 1). The modelled tertiary structure of wildtype and mutated TaALS showed the RMSD in between 0.003 and 0.036 Å, showing minor changes in the protein backbone.

Fig. 1.

Ramachandran plot for the modelled TaALS protein.

Identification of TaALS active site

The superimposition of modelled TaALS with AtALS-monosulfuron complex (PDB ID: 3E9Y) showed that the active site residue for the sulfonylurea family, P197 of AtALS aligns very well with P174 of the TaALS (Fig. 2). The different TaALS mutant models demonstrated a structural change within and around the binding pocket of TaALS that could be used for understanding the enzyme-herbicide structural functional relationship.

Fig. 2.

Active site of monosulfuron, a Sulfonylurea herbicide, obtained from the superimposition of modelled TaALS (green colour), with the crystal structure of Acetohydroxyacid synthase (PDB ID: 3E9Y, cyan colour).

Molecular Docking study

Nicosulfuron binding with TaALS-wild-type showed binding energy of -7.32 kcal/mol, while the single mutant conformation TaALS-G632S, TaALS-P174S and TaALS-P174F showed comparative weak binding energy of -6.29 kcal/mol, -6.55 kcal/mol and − 7.18 kcal/mol, respectively, than wild-type (Table 1). The weak binding affinity of nicosulfuron with TaALS-G632S, followed by TaALS-P174S and TaALS-P174F, suggested that these point mutations played critical role in blocking nicosulfuron binding and, therefore, could be utilised to generate herbicide-resistant ALS enzyme in wheat. However, TaALS-P174A and TaALS-G631D mutant complex showed binding energy of -7.41 and − 7.49 kcal/mol that possibly favours the nicosulfuron binding. Our molecular docking results are consistent with the previous base editing study on wheat ALS, which demonstrated that the P174S mutation confers higher resistance to nicosulfuron than P174A¹³. Similarly, base editing at the P190S (equivalent to TaALS-P174S) residue of ALS in watermelon was reported to confer higher resistance to sulfonylurea family herbicide (chlorsulfuron) than wild-type¹². CRISPR/Cas mediated base editing at P180S (equivalent to TaALS-P174S) residue of ALS in soybean indicated a higher resistance to sulfonylurea family herbicides (chlorsulfuron, flucarbazone-sodium) than wild-type ALS³⁴.

Table 1.

Binding energies obtained after Docking of TaALS-wildtype and its mutant complexes with nicosulfuron.

Enzyme	Binding Energy (kcal/mol)	Inhibition constant (µM)
TaALS-wild-type	-7.32	4.32
TaALS-P174A	-7.41	3.71
TaALS-P174F	-7.18	5.47
TaALS-P174S	-6.55	15.75
TaALS-G631D	-7.49	3.23
TaALS-G632S	-6.29	24.66
TaALS-G631D/G632S	-6.85	9.55
TaALS-G631D/P174F	-7.44	3.53
TaALS-G631D/P174S	-8.47	0.62
TaALS-G632S/P174F	-7.07	6.57
TaALS-G632S/P174S	-5.77	59.35
TaALS-G631D/G632S/P174F	-7.20	5.24
TaALS-G631D/G632S/P174S	-6.58	14.92

To observe the effect of double mutant on herbicide resistance, all possible combinations of the studied point mutations (P174S, P174F, G631D, and G632S) except P174A, were used to generate double mutants of TaALS. It was observed that one double mutant (TaALS-G632S/P174S-nicosulfuron) complex showed the weakest interaction (-5.77 kcal/mol) as compared to wildtype as well as all other single and double mutation complex. However, two other double mutant complex namely, TaALS-G631D/G632S and TaALS-G632S/P174F showed a comparative weak binding with the binding energy of -6.85 kcal/mol and − 7.07 kcal/mol respectively (Table 1). As evident from Table 1, the other two double mutant (TaALS-G631D/P174F and TaALS-G631D/P174S) complex showed favourable interactions with nicosulfuron with binding energy of -7.44 kcal/mol and − 8.47 kcal/mol, respectively. Therefore, the double mutant conformations (TaALS-G632S/P174S, TaALS-G631D/G632S and TaALS-G632S/P174F) were selected to generate triple mutant conformations (TaALS-G631D/G632S/P174S and TaALS-G631D/G632S/P174F). The binding of nicosulfuron with TaALS-G631D/G632S/P174S and TaALS-G631D/G632S/P174F showed the binding energy of -6.58 kcal/mol, and − 7.20 kcal/mol, with an inhibition constant of 14.92 µM and 5.24 µM, respectively (Table 1). We observed that Arg354A formed one H-bond, Ser630 and Phe174 formed non-bonding interactions with nicosulfuron in wild as well as in all mutant complexes. Additionally, the triple mutant complex (TaALS-G631D/G632S/P174F) formed hydrogen bonds with Phe174B, Arg176B, Ser630A, and Asp631A residues with ligand, resulting in a good binding affinity, and thus found to be less suitable for herbicide resistance. While, second triple mutant (TaALS-G631D/G632S/P174S) complex formed two hydrogen bonds with Ser630A and Asp631A. The presence of Ser174 in triple mutant conformation widen the binding pocket and hence does not formed strong complex (Figure S1). The present molecular docking analysis showed single (TaALS-G632S, TaALS-P174S), double (TaALS-G632S/P174S, TaALS-G631D/G632S) and triple mutant (TaALS-G631D/G632S/P174S) conformations act as the best candidates for generating sulfonylurea family herbicide resistance in wheat. Our analysis also highlighted the critical role of P174S and G632S in generating herbicide resistance.

Further, we also checked the binding affinity of other classes of herbicides (Immidazolinone: Imazapic; Triazolopyrimidine: Pyroxsulam; Pyrmidinyl-thiobenzoates: Bispyribac‑sodium; and Sulfonyl-aminocarbonyl- triazolinone: Flucarbazone) in the wild- type as well as in triple mutant conformations. All herbicides except pyroxsulam, showed a reduced binding affinity with selected triple mutant conformations (TaALS-G631D/G632S/P174F and TaALS-G631D/G632S/P174S) than the wild-type (Table S1). All studied herbicides showed their binding within a single pocket, including amino acids: Val173B, Lys233B, Arg354A, Trp551A, Ser630A, Val73B, and Gly98B, while amino acids P174B, G631A, and G632A play significant roles in the binding affinity (Figure S1).

Since the molecular docking was performed in semi-rigid mode, keeping the protein rigid, hence, to get detailed insight into nicosulfuron binding, all the single point mutant conformations, along with wildtype TaALS were considered for the molecular dynamics simulation study. However, in case of double mutant conformations, only three conformations TaALS-G631D/G632S, TaALS-G632S/P174F, and TaALS-G632S/P174S were used for further MDS analysis. Similarly, both the triple mutant conformations (TaALS-G631D/G632S/P174F and TaALS-G631D/G632S/P174S) were also studied for their binding stability with nicosulfuron using MDS study.

Comparative analysis of complexes using MD simulation

The stability of TaALS-wild-type and selected mutant complexes with herbicide was examined upon an MDS run of 100 ns. Different statistical parameters like RMSD, RMSF, RoG, SASA, hydrogen bond (H-bond) count, and interaction energy were calculated to examine the stability of TaALS-wild-type and mutant complexes with herbicide.

Root mean square deviation (RMSD) analysis

The nicosulfuron complex with TaALS-P174A and TaALS-wildtype was observed to be most unstable throughout the 100 ns MDS run, with an average RMSD value of 0.312 nm and 0.297 nm, respectively. However, TaALS-G631D/G632S nicosulfuron complex appeared to be the most stable with lowest RMSD values followed by TaALS-G632S/P174S, TaALS-P174S, TaALS-P174F, TaALS-G631D/G632S/P174S, TaALS-G632S/P174F and TaALS-G631D/G632S/P174F complex (Fig. 3). The TaALS-G631D/G632S-nicosulfuron complex showed an initial increase in RMSD value ~ 9ns and then stabilised until the end of the simulation with an average RMSD of 0.875 nm, while TaALS-G632S/P174S nicosulfuron complex continued to rise slowly until 27ns and then stabilised at an average RMSD of 0.237 nm. The nicosulfuron complex with the triple mutant conformation TaALS-G631D/G632S/P174S also showed a similar kind of behaviour and stabilised at 21ns with an average RMSD of 0.263 nm. All the single mutant conformations of TaALS, except TaALS-P174A, were observed to gain an initial peak to stabilise the protein-nicosulfuron complex within an initial run of 20ns followed by a stable RMSD line throughout the simulation. The triple mutant conformation TaALS-G631D/G632S/P174F was observed to have lower RMSD at the beginning of the simulation, but two major conformational shifts were observed in the protein at 18ns and 55ns, resulting in an increase in the RMSD values to 0.164 nm and 0.273 nm, respectively. The RMSD of double mutant conformation TaALS-G632S/P174F was on the top of the graph along with TaALS-P174A and TaALS-wildtype, where the RMSD was observed to be stable until the initial run of 78ns, after which the RMSD drop down up to 0.5 nm due to conformational change in the protein, and remain partially unstable till the end of the simulation. Altogether, the RMSD graph represents the stable and low-RMSD values for double (TaALS-G631D/G632S, TaALS-G632S/P174S) and triple mutant (G631D/G632S/P174S) conformations upon binding with nicosulfuron, while the wild-type, TaALS-P174A, and TaALS-G632S/P174F appeared to be unstable, with the highest RMSD value till the end of the simulation.

Fig. 3.

RMSD graph of TaALS-wild-type protein and its mutants in complex with nicosulfuron.

Root mean square fluctuation (RMSF) analysis

Nicosulfuron complexes with TaALS-P174A showed maximum RMS fluctuations in both chains, followed by TaALS-P174F, TaALS-G631D, TaALS-G632S/P174F, TaALS-wildtype, while TaALS-G631D/G632S, TaALS-G632S/P174S and TaALS-G631D/G632S/P174S showed minimum RMS fluctuation. The major fluctuations were observed at amino acid positions 173–177, 243–256, 404–411, and 557–559 in both chains of all the complexes (Fig. 4). Furthermore, it was observed that nicosulfuron binds in the same binding pocket with different bonding patterns. In TaALS-wildtype, TaALS-P174A, TaALS-G631D/G632S, TaALS-G632S/P174F, TaALS-G1631D/G632S/P174S, and TaALS-G1631D/G632S/P174F amino acids Gly98, Gln184, Arg354, and Glu102 were observed to have maximum H-bond propensity with nicosulfuron. In TaALS-P174F, Gln184 showed a single H-bond with nicosulfuron (RMSF: 0.914 nm), while in TaALS-P174S and TaALS-G632S/P174S mutant conformation, Ser174 showed a single H-bond with nicosulfuron. Similarly, amino acids Arg175 and Arg176 in the vicinity showed RMSF values of 0.258 nm and 0.342 nm, respectively. The A- chain of mutant conformations TaALS-G631D/G632S/P174F, TaALS-G631D/G632S, TaALS-G631D/G632S/P174S, TaALS-G632S/P174S and B- chain of mutant conformations TaALS-P174F, TaALS-G631D/G632S/P174S, TaALS-G632S/P174S and TaALS-P174S were observed to be more stable with lower RMSD values (Fig. 4A, B). The mutant conformations TaALS-G632S/P174S and TaALS-G631D/G632S/P174S were observed with minor fluctuations, representing the stable structure conformations.

Fig. 4.

RMSF graph of TaALS-wildtype protein and its mutants (A) chain-A; and (B) chain-B in complex with nicosulfuron herbicide.

Radius of gyration (RoG) analysis

TaALS-wildtype and different mutant conformations showed a similar range of RoG (3.13–3.35 nm) upon binding with nicosulfuron. The RoG graph pattern was similar to that of the RMSD graph, where TaALS-wildtype and TaALS-P174A showed maximum RoG due to a shift from the initial structure, followed by TaALS-G632S/P174F conformation, which showed a peak upto 3.3 nm at around 58ns. The triple mutant conformation TaALS-G631D/G632S/P174F was observed to be unstable throughout the simulation, with a continuous shift in RoG value between 3.23 and 3.27 nm. TaALS-G631D/G632S was observed to have the lowest and stable RoG value throughout the simulation, followed by TaALS-P174S, TaALS-G632S/P174S, and TaALS-P174F mutant conformations (Fig. 5). The triple mutant conformation TaALS-G631D/G632S/P174S showed intermediate but stable RoG throughout the simulation. The TaALS conformation i.e. TaALS-G631D/G632S, TaALS-P174S, TaALS-G632S/P174S, TaALS-G631D/G632S/P174S, TaALS-P174F were observed to have lower and stable RoG values throughout the simulation.

Fig. 5.

Radius of Gyration (RoG) graph of the TaALS-wildtype protein and its mutants in complex with nicosulfuron.

Solvent accessible surface area (SASA) analysis

SASA indicates the accessible surface of the protein solvent or a part of a protein that is exposed to the solvent. Overall accessibility of protein surface showed similar results as of RMSD, and RoG analysis. Nicosulfuron complex with TaALS-wildtype, TaALS-P174A and TaALS-G632S/P174F showed an increase in solvent-accessible surface area at the late stage of simulation (Fig. 6). SASA for TaALS-G631D/G632S conformation was found to be lowest, but at the end of the simulation, it was observed to be unstable. A similar pattern was observed with TaALS-G632S/P174S and TaALS-G631D/G632S/P174F, which showed an unstable peak throughout the simulations, which might be due to some continuous alteration at the protein surface. Most of the point mutant conformations showed fluctuations in the SASA plot, stating unstable protein surface (Fig. 6). Only TaALS-G631D/G632S/P174S and TaALS-P174S showed constant SASA plots, with minor fluctuations, throughout the simulation, representing a stable and compact structure throughout the simulation.

Fig. 6.

Solvent Accessible Surface Area (SASA) graph of the TaALS-wildtype protein and its mutants in complex with nicosulfuron.

Interaction energy (IE) analysis

The non-bonded interaction energy was also calculated to get an overview of the strength of interaction between nicosulfuron and TaALS mutants. We calculated short-range Lennard-Jones and coulombic interactions between TaALS and nicosulfuron during the MDS of 100 ns. Nicosulfuron complex with TaALS-wildtype showed a total interaction energy of -160.28 KJ/mol, with coulombic and Lennard-Jones interactions of -46.25 KJ/mol and − 114.02 KJ/mol, respectively (Table 2). Nicosulfuron binding with TaALS-G632S/P174S showed the highest interaction energy of -88.15 KJ/mol, with coulombic and Lennard-Jones interactions of -55.19 KJ/mol and − 32.96 KJ/mol, respectively (Table 2). However, the binding interaction energies were unstable, which was observed to be -107 KJ/mol at the beginning of the simulation, but after 32ns, no interaction was observed between TaALS-G632S/P174S and nicosulfuron (Fig. 7). Previously SASA plot (Fig. 6) also showed a shift in surface area at 32ns, which can be due to nonbonding on herbicide with the mutant TaALS. However, minor changes were observed in RMSD and RoG graphs at 32ns after the dissociation of nicosulfuron with the TaALS-G632S/P174S. Hence, it cannot be considered as favourable mutant model.

Table 2.

Interaction energy (KJ/mol) between TaALS-wildtype, mutant enzymes, and nicosulfuron.

Enzyme	Coulombic Interaction	Lennard-Jones Interactions	Total Interaction Energy (KJ/mol)
TaALS-wildtype	-46.25	-114.02	-160.28
TaALS-P174A	-50.87	-122.91	-173.78
TaALS-P174F	-54.56	-139.62	-194.19
TaALS-P174S	-59.68	-58.40	-118.08
TaALS-G631D	-79.95	-102.46	-182.40
TaALS-G632S	-127.73	-88.84	-216.57
TaALS-G631D/G632S	-55.72	-61.30	-117.02
TaALS-G632S/P174F	-66.05	-52.02	-118.07
TaALS-G632S/P174S	-55.19	-32.96	-88.15
TaALS-G631D/G632S/P174F	-139.30	-127.50	-266.79
TaALS-G631D/G632S/P174S	-39.29	-55.40	-94.70

Fig. 7.

(A) Coulombic interaction, (B) Lennard-Jones interactions, and (C) Total interaction energy graph of the TaALS-wildtype protein and its mutants with nicosulfuron.

Nicosulfuron binding with TaALS-G631D/G632S/P174S remained stable throughout the simulation, with coulombic interaction of -39.29 KJ/mol, Lennard-Jones interactions of -55.40 KJ/mol, and combined interaction energy of -94.70 KJ/mol (Table 2). Mutant conformations (TaALS-G631D/G632S and TaALS-P174S) binding with nicosulfuron showed a comparative weak interaction energy of -117.02 KJ/mol and − 118.08 KJ/mol, respectively, but were observed to be highly unstable and high peaks (0 to -450 KJ/mol) were observed throughout the simulation. The TaALS-G632S/P174F binding with nicosulfuron was also weak, with an interaction energy of -118.07 KJ/mol and was observed to be stable throughout the simulation. In comparison, the triple mutant conformation, TaALS-G631D/G632S/P174F, showed the lowest interaction energy (maximum binding affinity) of -266.79 KJ/mol with coulombic and Lennard-Jones interactions of -139.30 KJ/mol and − 127.50 KJ/mol, respectively. The graphical analysis throughout the simulation revealed a shift in the binding pattern of the nicosulfuron, which forms an H-bond with Glu102 of TaALS-G631D/G632S/P174F at around 47ns, resulting in an increase in the binding affinity from − 150 KJ/mol to -360 KJ/mol (Fig. 7). On the other side, single mutant docked conformations TaALS-P174A, TaALS-G631D, TaALS-P174F, and TaALS-G632S showed interaction energy of -173.78 KJ/mol, -182.40 KJ/mol, -194.19 KJ/mol, and − 216.57 KJ/mol, respectively, that showed strong binding with nicosulfuron as compared to the wildtype. The triple mutant (TaALS-G631D-G632S-P174S) conformation with the lowest and most stable interaction energy (-94.70 KJ/mol) could be considered a favourable target site for generating herbicide resistance in wheat.

Hydrogen bond analysis

The number of hydrogen bonds (H-bonds) per frame were calculated throughout the trajectories using the ‘gmx hbond’ command, taking the default values (donor-acceptor distance: 3Å and angle cut-off: 20°). Nicosulfuron binding with TaALS-wildtype showed 0.270 H-bonds per frame (Fig. 8). Herbicide binding with TaALS-G632S/P174S and TaALS-G631D/G632S/P174S showed the lowest 0.025 and 0.122 H-bonds per frame, respectively, which indicated very weak binding with nicosulfuron (Fig. 8). Initially, two H-bonds were observed in TaALS-G632S/P174S nicosulfuron complex, followed by no bonding interactions after a run of 37 ns (due to non-binding) (Figure-7). However, not more than one H-bond was observed in triple mutant TaALS-G631D/G632S/P174S-nicosulfuron complex throughout the simulation that also correlate with the stable interaction energy graph (Figure 7). The second triple mutant conformation (TaALS-G631D/G632S/P174F)-complex showed a maximum number of 1.286 H-bonds per frame, while the other double mutant conformations, TaALS-G631D/G632S and TaALS-G632S/P174F, showed 0.437 and 0.573 H-bonds per frame, respectively. In comparison, the single mutant conformations showed around half H-bonds per frame (Fig. 8). Nicosulfuron formed a maximum of six H-bonds with TaALS-G631D and TaALS-G632S in the intermediate and beginning stages of simulation, respectively which drop in the later stages of simulations (Fig. 8). In conclusion, the mutant conformations TaALS-G632S/P174S and TaALS-G631D/G632S/P174S were observed to have the lowest H-bond count per frame and were considered to be the best mutant conformation for developing herbicide resistance in wheat.

Fig. 8.

The number of hydrogen bonds formed among the TaALS-wildtype protein and its mutants with nicosulfuron.

Free binding energy analysis

The free binding energy was calculated using gmx_MMPBSA to evaluate the binding between different TaALS mutants and nicosulfuron. Nicosulfuron showed the lowest binding energy of -8.66 kcal/mol with TaALS-wildtype, while the mutant conformations were observed to show weak binding affinity. TaALS-P174S showed the weakest free binding of -2.51 kcal/mol, followed by TaALS-G631D/G632S/P174S (-4.86 kcal/mol), TaALS-G631D/G632S (-5.17 kcal/mol), TaALS-P174A (-5.18 kcal/mol), TaALS-P174F (-5.35 kcal/mol), and TaALS-G632S/P174S (-5.47 kcal/mol) (Table 3). Moreover, nicosulfuron showed moderate binding with TaALS-G632S/P174F, TaALS-G631D, TaALS-G631D/G632S/P174F, and TaALS-G632S with a free binding energy of -6.78 kcal/mol, -7.55 kcal/mol, -7.75 kcal/mol and − 7.88 kcal/mol, respectively (Table 3). We observed that total interaction energy calculation is appropriately supported by free binding energy calculation where TaALS-P174S, TaALS-G631D/G632S/P174S and TaALS-G631D/G632S showed relatively higher free binding energy, suggesting the weaker efficacy of nicosulfuron binding with these mutants in comparison to the TaALS-wildtype. Therefore, these mutant conformations (TaALS-P174S, TaALS-G631D/G632S and TaALS-G631D/G632S/P174S) with the highest free binding energy (weak binding with the herbicide) are the best conformations for generating ALS-inhibiting herbicide (nicosulfuron) tolerant wheat.

Table 3.

The free binding energy (kcal/mol) between different TaALS mutants and nicosulfuron.

Enzyme	VDW	EEL	Free Binding Energy (kcal/mol)
TaALS-wildtype	-31.98	-567.06	-8.66
TaALS-P174A	-33.95	-563.81	-5.18
TaALS-P174F	-38.70	-565.93	-5.35
TaALS-P174S	-16.28	-425.23	-2.51
TaALS-G631D	-24.87	-46.48	-7.55
TaALS-G632S	-9.12	-16.38	-7.88
TaALS-G631D/G632S	-30.61	-68.51	-5.17
TaALS-G632S/P174F	-25.91	-15.40	-6.78
TaALS-G632S/P174S	-35.75	-49.96	-5.47
TaALS-G631D/G632S/P174F	-35.03	-552.08	-7.75
TaALS-G631D/G632S/P174S	-34.63	-561.70	-4.86

The analysis of molecular dynamic simulation studies revealed the stable and weak binding in the case of mutant TaALS conformations than wildtype TaALS enzyme. In comparison to single mutants, double and triple TaALS mutant conformations showed weak binding for Nicosulfuron. Among all the double mutant conformations, TaALS-G632S/P174S and TaALS-G631D/G632S were observed to show stable and weak binding, while in case of the triple mutant conformation, TaALS-G631D/G632S/P174S found in this study showed the best result in terms of binding and stability. An overall comparison highlighted that the proposed triple mutant (TaALS-G631D/G632S/P174S) found to be best among the wild type, single, double and triple mutant models due to its poor herbicide affinity, minimum H-bond, weakest interaction energy and maximum stability throughout the simulation.

Conclusion

The present study was aimed to understand ALS-herbicide structure-function relationship and the effect of mutation of key residue on herbicide binding. Our in-silico investigation suggested that the triple mutant conformation (TaALS-G631D/G632S/P174S) has the poor binding affinity for nicosulfuron and could be used for developing natural resistance against sulfonylurea family herbicides in wheat. Additionally, it should be considered that any mutation in ALS might result in minor or major secondary effects on plants, that require further in-vivo validation. This study could also be extended for other class of herbicides as well as in other crops. In conclusion, the results of our study could be utilized in targeted genome editing for boosting herbicide resistance in wheat and other crops.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

DS and PC designed and supervised the experiments. PK and PP performed protein modeling, molecular docking experiments, and molecular dynamics simulations. PK and RB analyzed the data, drafted, and revised the manuscript. Both PK and RB contributed equally to the manuscript. The manuscript is seen and approved by all the authors.

Data availability

The data generated or analysed in this study is included in the manuscript.

Declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

The Authors declare that there is no conflict of interest.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-91379-0.