Probing the “fingers” domain binding pocket of Hepatitis C virus NS5B RdRp and D559G resistance mutation via molecular docking,molecular dynamics simulation and binding free energy calculations

The NS5B RdRp polymerase is a prominent enzyme for the replication of Hepatitis C virus (HCV). During the HCV replication, the template RNA binding takes place in the “fingers” sub-domain of NS5B. The “fingers” domain is a new emerging allosteric site for the HCV drug development. The inhibitors of the “fingers” sub-domain adopt a new antiviral mechanism called RNA intervention. The details of essential amino acid residues, binding mode of the ligand, and the active site intermolecular interactions of RNA intervention reflect that this mechanism is ambiguous in the experimental study. To elucidate these details, we performed molecular docking analysis of the fingers domain inhibitor quercetagetin (QGN) with NS5B polymerase. The detailed analysis of QGN-NS5B intermolecular interactions was carried out and found that QGN interacts with the binding pocket amino acid residues Ala97, Ala140, Ile160, Phe162, Gly283, Gly557, and Asp559; and also forms π?π stacking interaction with Phe162 and hydrogen bonding interaction with Gly283. These are found to be the essential interactions for the RNA intervention mechanism. Among the strong hydrogen bonding interactions, the QGN?Ala140 is a newly identified important hydrogen bonding interaction by the present work and this interaction was not resolved by the previously reported crystal structure. Since D559G mutation at the fingers domain was reported for reducing the inhibition percentage of QGN to sevenfold, we carried out molecular dynamics (MD) simulation for wild and D559G mutated complexes to study the stability of protein conformation and intermolecular interactions. At the end of 50?ns MD simulation, the π?π stacking interaction of Phe162 with QGN found in the wild-type complex is altered into T-shaped π stacking interaction, which reduces the inhibition strength. The origin of the D559G resistance mutation was studied using combined MD simulation, binding free energy calculations and principal component analysis. The results were compared with the wild-type complex. The mutation D559G reduces the binding affinity of the QGN molecule to the fingers domain. The free energy decomposition analysis of each residue of wild-type and mutated complexes revealed that the loss of non-polar energy contribution is the origin of the resistance.

Communicated by Ramaswamy H. Sarma     

Impact of Ribosomal Modification on the Binding of the Antibiotic Telithromycin Using a Combined Grand Canonical Monte Carlo/Molecular Dynamics Simulation Approach

Resistance to macrolide antibiotics is conferred by mutation of A2058 to G or methylation by Erm methyltransferases of the exocyclic N6 of A2058 (E. coli numbering) that forms the macrolide binding site in the 50S subunit of the ribosome. Ketolides such as telithromycin mitigate A2058G resistance yet remain susceptible to Erm-based resistance. Molecular details associated with macrolide resistance due to the A2058G mutation and methylation at N6 of A2058 by Erm methyltransferases were investigated using empirical force field-based simulations. To address the buried nature of the macrolide binding site, the number of waters within the pocket was allowed to fluctuate via the use of a Grand Canonical Monte Carlo (GCMC) methodology. The GCMC water insertion/deletion steps were alternated with Molecular Dynamics (MD) simulations to allow for relaxation of the entire system. From this GCMC/MD approach information on the interactions between telithromycin and the 50S ribosome was obtained. In the wild-type (WT) ribosome, the 2′-OH to A2058 N1 hydrogen bond samples short distances with a higher probability, while the effectiveness of telithromycin against the A2058G mutation is explained by a rearrangement of the hydrogen bonding pattern of the 2′-OH to 2058 that maintains the overall antibiotic-ribosome interactions. In both the WT and A2058G mutation there is significant flexibility in telithromycin''s imidazole-pyridine side chain (ARM), indicating that entropic effects contribute to the binding affinity. Methylated ribosomes show lower sampling of short 2′-OH to 2058 distances and also demonstrate enhanced G2057-A2058 stacking leading to disrupted A752-U2609 Watson-Crick (WC) interactions as well as hydrogen bonding between telithromycin''s ARM and U2609. This information will be of utility in the rational design of novel macrolide analogs with improved activity against methylated A2058 ribosomes.     

Molecular dynamics simulations of lignin peroxidase in solution

The dynamical and structural properties of lignin peroxidase and its Trp171Ala mutant have been investigated in aqueous solution using molecular dynamics (MD) simulations. In both cases, the enzyme retained its overall backbone structure and all its noncovalent interactions in the course of the MD simulations. Very interestingly, the analysis of the MD trajectories showed the presence of large fluctuations in correspondence of the residues forming the heme access channel; these movements enlarge the opening and facilitate the access of substrates to the enzyme active site. Moreover, steered molecular dynamics docking simulations have shown that lignin peroxidase natural substrate (veratryl alcohol) can easily approach the heme edge through the access channel.     

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.     

Importance of discriminator base stacking interactions: molecular dynamics analysis of A73 microhelix(Ala) variants

Transfer of alanine from Escherichia coli alanyl-tRNA synthetase (AlaRS) to RNA minihelices that mimic the amino acid acceptor stem of tRNA^Ala has been shown, by analysis of variant minihelix aminoacylation activities, to involve a transition state sensitive to changes in the ‘discriminator’ base at position 73. Solution NMR has indicated that this single-stranded nucleotide is predominantly stacked onto G1 of the first base pair of the alanine acceptor stem helix. We report the activity of a new variant with the adenine at position 73 substituted by its non-polar isostere 4-methylindole (M). Despite lacking N7, this analog is well tolerated by AlaRS. Molecular dynamics (MD) simulations show that the M substitution improves position 73 base stacking over G1, as measured by a stacking lifetime analysis. Additional MD simulations of wild-type microhelix^Ala and six variants reveal a positive correlation between N73 base stacking propensity over G1 and aminoacylation activity. For the two ΔN7 variants simulated we found that the propensity to stack over G1 was similar to the analogous variants that contain N7 and we conclude that the decrease in aminoacylation efficiency observed upon deletion of N7 is likely due to loss of a direct stabilizing interaction with the synthetase.     

Long-Timescale Molecular Dynamics Simulations Elucidate the Dynamics and Kinetics of Exposure of the Hydrophobic Patch in Troponin C

Troponin (Tn) is an important regulatory protein in the thin-filament complex of cardiomyocytes. Calcium binding to the troponin C (TnC) subunit causes a change in its dynamics that leads to the transient opening of a hydrophobic patch on TnC’s surface, to which a helix of another subunit, troponin I (TnI), binds. This process initiates contraction, making it an important target for studies investigating the detailed molecular processes that underlie contraction. Here we use microsecond-timescale Anton molecular dynamics simulations to investigate the dynamics and kinetics of the opening transition of the TnC hydrophobic patch. Free-energy differences for opening are calculated for wild-type Ca²⁺-bound TnC (∼8 kcal/mol), V44Q Ca²⁺-bound TnC (3.2 kcal/mol), E40A Ca²⁺-bound TnC (∼12 kcal/mol), and wild-type apo TnC (∼20 kcal/mol). These results suggest that the mutations have a profound impact on the frequency with which the hydrophobic patch presents to TnI. In addition, these simulations corroborate that cardiac wild-type TnC does not open on timescales relevant to contraction without calcium being bound.     

The effect of a Pro²⁸Thr point mutation on the local structure and stability of human galactokinase enzyme-a theoretical study

Galactokinase is responsible for the phosphorylation of α-d-galactose, which is an important step in the metabolism of the latter. Malfunctioning of galactokinase due to a single point mutation causes cataracts and, in serious cases, blindness. This paper reports a study of the Pro²⁸Thr point mutation using a variety of theories including molecular dynamics (MD), MM-PBSA/GBSA calculations and AIM analysis. Altered H-bonding networks were detected based on geometric and electron density criteria that resulted in local unfolding of the β-sheet secondary structure. Another consequence was the decrease in stability (5–7 kcal mol⁻¹) around this region, as confirmed by ΔG_bind calculations for the extracted part of the whole system. Local unfolding was verified by several other MD simulations performed with different duration, initial velocities and force field. Based on the results, we propose a possible mechanism for the unfolding caused by the Pro²⁸Thr point mutation.     

Understanding the Mechanism of Atovaquone Drug Resistance in Plasmodium falciparum Cytochrome b Mutation Y268S Using Computational Methods

The rapid appearance of resistant malarial parasites after introduction of atovaquone (ATQ) drug has prompted the search for new drugs as even single point mutations in the active site of Cytochrome b protein can rapidly render ATQ ineffective. The presence of Y268 mutations in the Cytochrome b (Cyt b) protein is previously suggested to be responsible for the ATQ resistance in Plasmodium falciparum (P. falciparum). In this study, we examined the resistance mechanism against ATQ in P. falciparum through computational methods. Here, we reported a reliable protein model of Cyt bc1 complex containing Cyt b and the Iron-Sulphur Protein (ISP) of P. falciparum using composite modeling method by combining threading, ab initio modeling and atomic-level structure refinement approaches. The molecular dynamics simulations suggest that Y268S mutation causes ATQ resistance by reducing hydrophobic interactions between Cyt bc1 protein complex and ATQ. Moreover, the important histidine contact of ATQ with the ISP chain is also lost due to Y268S mutation. We noticed the induced mutation alters the arrangement of active site residues in a fashion that enforces ATQ to find its new stable binding site far away from the wild-type binding pocket. The MM-PBSA calculations also shows that the binding affinity of ATQ with Cyt bc1 complex is enough to hold it at this new site that ultimately leads to the ATQ resistance.     

A molecular dynamics model of the Bt toxin Cyt1A and its validation by resonance energy transfer

Cyt1A is a cytolytic toxin from Bacillus thuringiensis var. israelensis. A computer model of the toxin in solution was generated and validated by resonance energy transfer (RET). The average distance between the two tryptophans (residues 158 and 161) and the fluorescently labeled cysteine 190 was 2.16 nm, which closely matched the distance predicted in computer simulations, 2.2 nm. The simulation results were able to explain two previous experimental observations: (i) amino-acid sequences of all Cyt toxins contain four blocks of highly conserved residues; and (ii) several single-point mutations drastically abrogated Cyt1A's toxicity. Selective randomization of atomic coordinates in the computer model revealed that the conserved blocks are important for proper folding and stability of the toxin molecule. Replacing lysine 225 with alanine, a mutation that renders the toxin inactive, was shown to result in breaking the hydrogen bonds between K225 and V126, L123, and Y189. Calculated Helmholtz free energy difference of the inactive mutation K225A was higher by 12 kcal/mol and 5 kcal/mol than the values for the benign mutations K118A and K198A, respectively, which indicates that the K225A mutant is significantly destabilized. The normal-mode and principal-component analyses revealed that in the wild-type Cyt1A the region around the residue K225 is quite stationary, due to the hydrogen-bond network around K225. In contrast, pronounced twisting and stretching were observed in the mutant K225A, and the region around the residue K225 becomes unstable. Our results indicate that conformational differences in this mutant spread far away from the site of the mutation, suggesting that the mutant is inactivated due to an overall change in conformation and diminished stability rather than due to a localized alteration of a “binding” or “active” site.     

Conformational flexibility and binding interactions of the G protein βγ heterodimer

Previous NMR experiments on unbound G protein βγ heterodimer suggested that particular residues in the binding interface are mobile on the nanosecond timescale. In this work we performed nanosecond‐timescale molecular dynamics simulations to investigate conformational changes and dynamics of Gβγ in the presence of several binding partners: a high‐affinity peptide (SIGK), phosducin, and the GDP‐bound α subunit. In these simulations, the high mobility of GβW99 was reduced by SIGK, and it appeared that a tyrosine might stabilize GβW99 by hydrophobic or aromatic stacking interactions in addition to hydrogen bonds. Simulations of the phosducin‐Gβγ complex showed that the mobility of GβW99 was restricted, consistent with inferences from NMR. However, large‐scale conformational changes of Gβγ due to binding, which were hypothesized in the NMR study, were not observed in the simulations, most likely due to their short (nanosecond) duration. A pocket consisting of hydrophobic amino acids on Gα appears to restrict GβW99 mobility in the crystal structure of the Gαβγ? heterotrimer. The simulation trajectories are consistent with this idea. However, local conformational changes of residues GβW63, GβW211, GβW297, GβW332, and GβW339 were detected during the MD simulations. As expected, the magnitude of atomic fluctuations observed in simulations was greater for α than for the βγ subunits, suggesting that α has greater flexibility. These observations support the notion that to maintain the high mobility of GβW99 observed by solution NMR requires that the Gβ–α interface must open up on time scale longer than can be observed in nanosecond scale simulations. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

Residues M42 and G121 of Escherichia coli dihydrofolate reductase (ecDHFR) are on opposite sides of the catalytic centre (15 and 19 A away from it, respectively). Theoretical studies have suggested that these distal residues might be part of a dynamics network coupled to the reaction catalysed at the active site. The ecDHFR mutant G121V has been extensively studied and appeared to have a significant effect on rate, but only a mild effect on the nature of H-transfer. The present work examines the effect of M42W on the physical nature of the catalysed hydride transfer step. Intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters were studied. The findings presented here are in accordance with the environmentally coupled hydrogen tunnelling. In contrast to the wild-type (WT), fluctuations of the donor-acceptor distance were required, leading to a significant temperature dependence of KIEs and deflated intercepts. A comparison of M42W and G121V to the WT enzyme revealed that the reduced rates, the inflated primary KIEs and their temperature dependences resulted from an imperfect potential surface pre-arrangement relative to the WT enzyme. Apparently, the coupling of the enzyme's dynamics to the reaction coordinate was altered by the mutation, supporting the models in which dynamics of the whole protein is coupled to its catalysed chemistry.     

Theoretical and Experimental Study of the D2194G Mutation in the C2 Domain of Coagulation Factor V

Coagulation factor V (FV) is a large plasma glycoprotein with functions in both the pro- and anticoagulant pathways. In carriers of the so-called R2-FV haplotype, the FV D2194G mutation, in the C2 membrane-binding domain, is associated with low expression levels, suggesting a potential folding/stability problem. To analyze the molecular mechanisms potentially responsible for this in vitro phenotype, we used molecular dynamics (MD) and continuum electrostatic calculations. Implicit solvent simulations were performed on the x-ray structure of the wild-type C2 domain and on a model of the D2194G mutant. Because D2194 is located next to a disulfide bond (S-S bond), MD calculations were also performed on S-S bond depleted structures. D2194 is part of a salt-bridge network and investigations of the stabilizing/destabilizing role of these ionic interactions were carried out. Five mutant FV molecules were created and the expression levels measured with the aim of assessing the tolerance to amino acid changes in this region of molecule. Analysis of the MD trajectories indicated increased flexibility in some areas and energetic comparisons suggested overall destabilization of the structure due to the D2194G mutation. This substitution causes electrostatic destabilization of the domain by ~3 kcal/mol. Together these effects likely explain the lowered expression levels in R2-FV carriers.     

Stability of Transmembrane Amyloid β-Peptide and Membrane Integrity Tested by Molecular Modeling of Site-Specific Aβ 42 Mutations

Interactions of the amyloid β-protein (Aβ) with neuronal cell membranes, leading to the disruption of membrane integrity, are considered to play a key role in the development of Alzheimer’s disease. Natural mutations in Aβ ₄₂, such as the Arctic mutation (E22G) have been shown to increase Aβ ₄₂ aggregation and neurotoxicity, leading to the early-onset of Alzheimer’s disease. A correlation between the propensity of Aβ ₄₂ to form protofibrils and its effect on neuronal dysfunction and degeneration has been established. Using rational mutagenesis of the Aβ ₄₂ peptide it was further revealed that the aggregation of different Aβ ₄₂ mutants in lipid membranes results in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also have a variable ability to disrupt bilayer integrity. To further test the connection between Aβ ₄₂ mutation and peptide–membrane interactions, we perform molecular dynamics simulations of membrane-inserted Aβ ₄₂ variants (wild-type and E22G, D23G, E22G/D23G, K16M/K28M and K16M/E22G/D23G/K28M mutants) as β-sheet monomers and tetramers. The effects of charged residues on transmembrane Aβ ₄₂ stability and membrane integrity are analyzed at atomistic level. We observe an increased stability for the E22G Aβ ₄₂ peptide and a decreased stability for D23G compared to wild-type Aβ ₄₂, while D23G has the largest membrane-disruptive effect. These results support the experimental observation that the altered toxicity arising from mutations in Aβ is not only a result of the altered aggregation propensity, but also originates from modified Aβ interactions with neuronal membranes.     

Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel,hHV1

Part of the “signature sequence” that defines the voltage-gated proton channel (H_V1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H_V1 genes. Replacing Trp²⁰⁷ in human H_V1 (hH_V1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, E_a, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp²⁰⁷ establishes the major energy barrier between closed and open hH_V1. Cation–π interaction between Trp²⁰⁷ and Arg²¹¹ evidently latches the channel closed. Trp²⁰⁷ mutants lost proton selectivity at pH_o >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H_V1 that is essential to its biological functions, was compromised. In the WT hH_V1, ΔpH-dependent gating is shown to saturate above pH_i or pH_o 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp²⁰⁷ mutants, ΔpH-dependent gating saturated at lower pH_o but not at lower pH_i. That Trp²⁰⁷ mutation selectively alters pH_o sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H_V1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pH_o and pH_i in H_V1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.     

Second-contact shell mutation diminishes streptavidin-biotin binding affinity through transmitted effects on equilibrium dynamics

We report a point mutation in the second contact shell of the high-affinity streptavidin-biotin complex that appears to reduce binding affinity through transmitted effects on equilibrium dynamics. The Y54F streptavidin mutation causes a 75-fold loss of binding affinity with 73-fold faster dissociation, a large loss of binding enthalpy (ΔΔH = 3.4 kcal/mol at 37 °C), and a small gain in binding entropy (TΔΔS = 0.7 kcal/mol). The removed Y54 hydroxyl is replaced by a water molecule in the bound structure, but there are no observable changes in structure in the first contact shell and no additional changes surrounding the mutation. Molecular dynamics simulations reveal a large increase in the atomic fluctuation amplitudes for W79, a key biotin contact residue, compared to the fluctuation amplitudes in the wild-type. The increased W79 atomic fluctuation amplitudes are caused by loss of water-mediated hydrogen bonds between the Y54 hydroxyl group and peptide backbone atoms in and near W79. We propose that the increased atomic fluctuation amplitudes diminish the integrity of the W79-biotin interaction and represents a loosening of the "tryptophan collar" that is critical to the slow dissociation and high affinity of streptavidin-biotin binding. These results illustrate how changes in protein dynamics distal to the ligand binding pocket can have a profound impact on ligand binding, even when equilibrium structure is unperturbed.     

Separating instability from aggregation propensity in γS-crystallin variants

Molecular dynamics (MD) simulations, circular dichroism (CD), and dynamic light scattering (DLS) measurements were used to investigate the aggregation propensity of the eye-lens protein γS-crystallin. The wild-type protein was investigated along with the cataract-related G18V variant and the symmetry-related G106V variant. The MD simulations suggest that local sequence differences result in dramatic differences in dynamics and hydration between these two apparently similar point mutations. This finding is supported by the experimental measurements, which show that although both variants appear to be mostly folded at room temperature, both display increased aggregation propensity. Although the disease-related G18V variant is not the most strongly destabilized, it aggregates more readily than either the wild-type or the G106V variant. These results indicate that γS-crystallin provides an excellent model system for investigating the role of dynamics and hydration in aggregation by locally unfolded proteins.     

Functional significance of some particular amino acid residues in Bombyx mori pyridoxal kinase

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme for vitamin B₆ metabolism in animals. It catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal 5′-phosphate, an important cofactor for many enzymatic reactions. Bombyx mori PLK (BmPLK) is 10 or more residues shorter than mammalian PLKs, and some amino acid residues conserved in the PLKs from mammals are not maintained in the protein. Multiple sequence alignment suggested that amino acid residues Thr⁴⁷, Ile⁵⁴, Arg⁸⁸, Asn¹²¹ and Glu²³⁰ might play important roles in BmPLK. In this study, we used a site-directed specific mutagenesis approach to determine the functional significance of these particular amino acid residues in BmPLK. Our results demonstrated that the mutation of Asn¹²¹ to Glu did not affect the catalytic function of BmPLK. The corresponding site-directed mutants of Thr⁴⁷ to Asn, Ile⁵⁴ to Phe, and Arg⁸⁸ to Ile displayed a decreased catalytic efficiency and an elevated Km value for substrate relative to the wild-type value, and no enzyme activity could be detected in mutant of Trp²³⁰ to Glu. Circular dichroism analysis revealed that the mutation of Trp²³⁰ to Glu resulted in mis-folding of the protein. Our results provided direct evidence that residue Trp²³⁰ is crucial to maintain the structural and functional integrity of BmPLK. This study will add to the existing understanding of the characteristic of structure and function of BmPLK.     

Computational simulations of structural role of the active-site W374C mutation of acetyl-coenzyme-A carboxylase: Multi-drug resistance mechanism

Herbicides targeting grass plastidic acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) are selectively effective against graminicides. The intensive worldwide use of this herbicide family has selected for resistance genes in a number of grass weed species. Recently, the active-site W374C mutation was found to confer multi-drug resistance toward haloxyfop (HF), fenoxaprop (FR), Diclofop (DF), and clodinafop (CF) in A. myosuroides. In order to uncover the resistance mechanism due to W374C mutation, the binding of above-mentioned four herbicides to both wild-type and the mutant-type ACCase was investigated in the current work by molecular docking and molecular dynamics (MD) simulations. The binding free energies were calculated by molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) method. The calculated binding free energy values for four herbicides were qualitatively consistent with the experimental order of IC₅₀ values. All the computational model and energetic results indicated that the W374C mutation has great effects on the conformational change of the binding pocket and the ligand-protein interactions. The most significant conformational change was found to be associated with the aromatic amino acid residues, such as Phe377, Tyr161′ and Trp346. As a result, the π-π interaction between the ligand and the residue of Phe377 and Tyr161′, which make important contributions to the binding affinity, was decreased after mutation and the binding affinity for the inhibitors to the mutant-type ACCase was less than that to the wild-type enzyme, which accounts for the molecular basis of herbicidal resistance. The structural role and mechanistic insights obtained from computational simulations will provide a new starting point for the rational design of novel inhibitors to overcome drug resistance associated with W374C mutation.