RESUMO
Cytochrome P450 (CYP) 17A1 is a dual-function monooxygenase with a critical role in the synthesis of many human steroid hormones. The enzyme is an important target for treatment of breast and prostate cancers that proliferate in response to estrogens and androgens. Despite the crystallographic structures available for CYP17A1, no membrane-bound structural features of this enzyme at atomic level are available. Accumulating evidence has indicated that the interactions between bounded CYPs and membrane could contribute to the recruitment of lipophilic substrates. To this end, we have investigated the effects on structural characteristics in the presence of the membrane for CYP17A1. The MD simulation results demonstrate a spontaneous insertion process of the enzyme to the lipid. Two predominant modes of CYP17A1 in the membrane are captured, characterized by the depths of insertion and orientations of the enzyme to the membrane surface. The measured heme tilt angles show good consistence with experimental data, thereby verifying the validity of the structural models. Moreover, conformational changes induced by the membrane might have impact on the accessibility of the active site to lipophilic substrates. The dynamics of internal aromatic gate formed by Trp220 and Phe224 are suggested to regulate tunnel opening motions. The knowledge of the membrane binding characteristics could guide future experimental and computational works on membrane-bound CYPs so that various investigations of CYPs in their natural, lipid environment rather than in artificially solubilized forms may be achieved.
Assuntos
Bicamadas Lipídicas/química , Fluidez de Membrana , Modelos Químicos , Simulação de Dinâmica Molecular , Esteroide 17-alfa-Hidroxilase/química , Esteroide 17-alfa-Hidroxilase/ultraestrutura , Sítios de Ligação , Simulação por Computador , Ligação Proteica , Conformação ProteicaRESUMO
The CYP2C subfamily is of particular importance in the metabolism of drugs, food toxins, and procarcinogens. Like other P450 subfamilies, 2C enzymes share a high sequence identity, but significantly contribute in different ways to hepatic capacity to metabolize drugs. They often metabolize the same substrate to more than one product with different catalytic sites. Because it is challenging to characterize experimentally, much still remains unknown about the reason for why the substrate regioselectivity of these closely related subfamily members is different. Here, we have investigated the structural features of CYP2C8, CYP2C9, and CYP2C19 bound with their shared substrate diclofenac to elucidate the underlying molecular mechanism for the substrate regioselectivity of CYP2C subfamily enzymes. The obtained results demonstrate how a sequence divergence for the active site residues causes heterogeneous variations in the secondary structures and in major tunnel selections, and further affects the shape and chemical properties of the substrate-binding site. Structural analysis and free energy calculations showed that the most important determinants of regioselectivity among the CYP2C isoforms are the geometrical features of the active sites, as well as the hydrogen bonds and the hydrophobic interactions, mainly presenting as the various locations of Arg108 and substitutions of Phe205 for Ile205 in CYP2C8. The MM-GB/SA calculations combined with PMF results accord well with the experimental KM values, bridging the gap between the theory and the experimentally observed results of binding affinity differences. The present study provides important insights into the structure-function relationships of CYP2C subfamily enzymes, the knowledge of ligand binding characteristics and key residue contributions could guide future experimental and computational work on the synthesis of drugs with better pharmacokinetic properties so that CYP interactions could be avoided.
Assuntos
Anti-Inflamatórios não Esteroides/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Diclofenaco/farmacocinética , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9 , Sistema Enzimático do Citocromo P-450/química , Simulação de Dinâmica MolecularRESUMO
Cytochrome P450 2E1 is widely known for its ability to oxidize both low molecular weight xenobiotics and endogenous fatty acids (e.g., arachidonic acid (AA)). In this study, we investigated the structural features of the AA-bound CYP2E1 complex utilizing molecular dynamics (MD) and found that the distinct binding modes for both AA and fatty acid analog are conserved. Moreover, multiple random acceleration MD simulations and steered MD simulations uncovered the most possible tunnel for fatty acids. The main attractions are derived from three key residues, His107, Ala108, and His109, whose side chains reorient to keep ligands bound via hydrogen bonds during the initial unbinding process. More importantly, based on the calculated binding free energy results, we hypothesize that the hydrogen bonds between the receptor and the ligand are the most important contributors involved in the binding affinity. Thus, it is inferred that the hydrogen bonds between these three residues and the ligand may help offer insights into the structural basis of the different ligand egress mechanisms for fatty acids and small weight compounds. Our investigation provides detailed atomistic insights into the structural features of human CYP2E1-fatty acid complex structures. Furthermore, the ligand-binding characteristics obtained in the present study are helpful for both experimental and computational studies of CYPs and may allow future researchers to achieve desirable changes in enzymatic activities.
Assuntos
Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Citocromo P-450 CYP2E1/química , Citocromo P-450 CYP2E1/metabolismo , Humanos , Simulação de Dinâmica MolecularRESUMO
Filoviruses often cause terrible infectious disease which has not been successfully dealt with pharmacologically. All filoviruses encode a unique protein termed VP35 which can mask doubled-stranded RNA to deactivate interferon. The interface of VP35-dsRNA would be a feasible target for structure-based antiviral agent design. To explore the essence of VP35-dsRNA interaction, molecular dynamics simulation combined with MM-GBSA calculations were performed on Marburg virus VP35-dsRNA complex and several mutational complexes. The energetic analysis indicates that nonpolar interactions provide the main driving force for the binding process. Although the intermolecular electrostatic interactions play important roles in VP35-dsRNA interaction, the whole polar interactions are unfavorable for binding which result in a low binding affinity. Compared with wild type VP35, the studied mutants F228A, R271A, and K298A have obviously reduced binding free energies with dsRNA reflecting in the reduction of polar or nonpolar interactions. The results also indicate that the loss of binding affinity for one dsRNA strand would abolish the total binding affinity. Three important residues Arg271, Arg294, and Lys298 which makes the largest contribution for binding in VP35 lose their binding affinity significantly in mutants. The uncovering of VP35-dsRNA recognition mechanism will provide some insights for development of antiviral drug.
Assuntos
Marburgvirus/metabolismo , Simulação de Dinâmica Molecular , RNA de Cadeia Dupla/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas Mutantes/química , Ligação Proteica , Estabilidade Proteica , RNA de Cadeia Dupla/química , Ribonucleotídeos/química , Eletricidade Estática , Termodinâmica , Proteínas Virais Reguladoras e Acessórias/químicaRESUMO
Cytochrome P450 (CYP) 7B1 is a steroid cytochrome P450 7α-hydroxylase that has been linked directly with bile salt synthesis and hereditary spastic paraplegia type 5 (SPG5). The enzyme provides the primary metabolic route for neurosteroids dehydroepiandrosterone (DHEA), cholesterol derivatives 25-hydroxycholesterol (25-HOChol), and other steroids such as 5α-androstane-3ß,17ß-diol (anediol), and 5α-androstene-3ß,17ß-diol (enediol). A series of investigations including homology modeling, molecular dynamics (MD), and automatic docking, combined with the results of previous experimental site-directed mutagenesis studies and access channels analysis, have identified the structural features relevant to the substrate selectivity of CYP7B1. The results clearly identify the dominant access channels and critical residues responsible for ligand binding. Both binding free energy analysis and total interaction energy analysis are consistent with the experimental conclusion that 25-HOChol is the best substrate. According to 20â ns MD simulations, the Phe cluster residues that lie above the active site, particularly Phe489, are proposed to merge the active site with the adjacent channel to the surface and accommodate substrate binding in a reasonable orientation. The investigation of CYP7B1-substrate binding modes provides detailed insights into the poorly understood structural features of human CYP7B1 at the atomic level, and will be valuable information for drug development and protein engineering.
Assuntos
Domínio Catalítico , Esteroide Hidroxilases/química , Esteroide Hidroxilases/metabolismo , Família 7 do Citocromo P450 , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Bibliotecas de Moléculas Pequenas/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
DEAD-box proteins are the largest family of helicase that are important in nearly all aspects of RNA metabolism. However, it is unclear how these proteins recognize and bind RNA. Here, we present a detailed analysis of the related DEAD-box protein Mss116p-RNA interaction, using molecular dynamics simulations with MM-GBSA calculations. The energetic analysis indicates that the two strands of double strands RNA (dsRNA) are recognized asymmetrically by Mss116p. The strand 1 of dsRNA provides the main binding affinity. Meanwhile, the nonpolar interaction provides the main driving force for the binding process. Although the contribution of polar interaction is small, it is vital in stabilizing the protein-RNA interaction. Compared with the wild type Mss116p, two studied mutants Q412A and D441A have obviously reduced binding free energies with dsRNA because of the decreasing of polar interaction. Three important residues Lys409, Arg415 and Arg438 lose their binding affinity significantly in mutants. In conclusion, these results complement previous experiments to advance comprehensive understanding of Mss116p-dsRNA interaction. The results also would provide support for the application of similar approaches to the understanding of other DEAD-box protein-RNA complexes.
Assuntos
RNA Helicases DEAD-box/química , Simulação de Dinâmica Molecular , RNA de Cadeia Dupla/química , RNA Fúngico/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Trifosfato de Adenosina/química , Sítios de Ligação , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/enzimologia , Eletricidade Estática , TermodinâmicaRESUMO
Cytochrome P450 (CYP) 17A1 is a dual-function monooxygenase with a critical role in the synthesis of many human steroid hormones. The enzyme is an important target for the treatment of breast and prostate cancers that proliferate in response to estrogens and androgens. Despite the ample experimental mutagenesis data, the molecular origin and the structural motifs for the enzymatic activities deficiencies have not been rationalized at the atomic resolution. To this end, we have investigated the effects on structural characteristics and tunnel geometry upon single point mutations in CYP17A1. The MD simulation results combined with PMF calculations and MM-GBSA calculations render an "access mechanism" which encapsulates the effects of mutations on the changes in both structural flexibility and tunnel dynamics, bridging the gap between the theory and the experimentally observed results of enzymatic activity decrease. The underlying molecular mechanism of the heterogeneities in open/closed conformational changes, as well as the wider opening of their respective major tunnels between wt17A1 and two mutants, may be attributed to the closer distances of hydrophobic residues or the disruption of a hydrophobic core. The knowledge of ligand binding characteristics and key residues contributions could guide future experimental and computational work on CYPs so that desirable changes in their enzymatic activities may be achieved. The present study provides important insights into the structure-function relationships of CYP17A1 protein, which could contribute to further understanding about 17-hydroxylase deficiencies and may also improve the understanding of polycystic ovary disease.
Assuntos
Androstenóis/química , Inibidores Enzimáticos/química , Simulação de Dinâmica Molecular , Esteroide 17-alfa-Hidroxilase/química , Androstenos , Domínio Catalítico , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Cytochrome P450 14α-sterol demethylase (CYP51), the key enzyme in sterol biosynthesis pathway, is an important target protein of cholesterol-lowering agents, antifungal drugs, and herbicides. CYP51B enzyme is one of the CYP51 family members. In the present study, we have chosen four representative inhibitors of CYP51B: diniconazole (Din), fluconazole (Flu), tebuconazole (Teb), and voriconazole (Vor), and launched to investigate the binding features of CYP51B-inhibitor and gating characteristics via molecular docking and molecular dynamics (MD) simulations. The results show that the ranking of binding affinities among these inhibitors is Din > Teb > Vor > Flu. Din shows the strongest binding affinity, whereas Flu shows the weakest binding affinity. More importantly, based on the calculated binding free energy results, we hypothesize that the nonpolar interactions are the most important contributors, and three key residues (Thr77, Ala258, and Lys454) play crucial role in protein-inhibitor binding. Besides, residue Phe180 may play a molecular switch role in the process of the CYP51B-Teb and CYP51B-Vor binding. Additionally, Tunnel analysis results show that the major tunnel of Din, Flu, and Teb is located between helix K, FG loop, and ß4 hairpin (Tunnel II).The top ranked possible tunnel (Tunnel II) corresponds to Vor exits through helix K, F and helix J. This study further revealed the CYP51B relevant structural characteristics at the atomic level as well as provided a basis for rational design of new and more efficacious antifungal agents.
Assuntos
Antifúngicos/química , Azóis/antagonistas & inibidores , Azóis/química , Sistema Enzimático do Citocromo P-450/química , Proteínas Fúngicas/química , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
Bacterial quorum sensing is a well-characterized communication system that governs a large variety of collective behaviours. By comparison, quorum sensing regulation in eukaryotic microbes remains poorly understood, especially its functional role in eukaryote-specific behaviours, such as sexual reproduction. Cryptococcus neoformans is a prevalent fungal pathogen that has two defined sexual cycles (bisexual and unisexual) and is a model organism for studying sexual reproduction in fungi. Here, we show that the quorum sensing peptide Qsp1 serves as an important signalling molecule for both forms of sexual reproduction. Qsp1 orchestrates various differentiation and molecular processes, including meiosis, the hallmark of sexual reproduction. It activates bisexual mating, at least in part through the control of pheromone, a signal necessary for bisexual activation. Notably, Qsp1 also plays a major role in the intercellular regulation of unisexual initiation and coordination, in which pheromone is not strictly required. Through a multi-layered genetic screening approach, we identified the atypical zinc finger regulator Cqs2 as an important component of the Qsp1 signalling cascade during both bisexual and unisexual reproduction. The absence of Cqs2 eliminates the Qsp1-stimulated mating response. Together, these findings extend the range of behaviours governed by quorum sensing to sexual development and meiosis.
Assuntos
Cryptococcus neoformans/fisiologia , Peptídeos/genética , Peptídeos/metabolismo , Percepção de Quorum , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos Tipo Acasalamento , Testes Genéticos , Hifas/crescimento & desenvolvimento , Meiose , Feromônios/metabolismo , Transdução de SinaisRESUMO
Increasing sophistication in methods used to account for human polymorphisms in susceptibility to drug metabolism has been one of the success stories in the prevention of adverse drug reactions. Genetic polymorphisms in drug-metabolizing enzymes can affect enzyme activity and cause differences in treatment response or drug toxicity. CYP2C19 is one of the most highly polymorphic CYP enzymes and acts on 10-15% of drugs in current clinical use. Despite the number of experimental analyses carried out for this system, the detailed structural basis for altered catalytic properties of polymorphic CYP2C19 variants remains unexplained at the atomic level. To this end, we have investigated the mutation effects on structural characteristics and tunnel geometry upon single point mutations to elucidate the underlying molecular mechanism for the enzymatic activity deficiencies by using the fully atomistic molecular dynamics (MD) simulations in their native, membrane-bound cellular environment. The obtained results demonstrate how significant sequence divergence causes heterogeneous variations, and further affects the shape and chemical properties of the substrate binding site. Principal component analysis (PCA) results combined with free energy calculations have revealed distinct mechanisms for different peripheral variants, implying a more complicated process for the decrease/loss of enzymatic activity upon the introduction of point mutations in CYP2C19 rather than simply structural changes of the region where the mutation is located. Overall, our present study provides important insights into the current pharmacogenetic knowledge of human drug-metabolizing CYP2C19 to understand the large inter-individual variability in drug clearance. The knowledge of heterogeneous variations in structural features could guide future experimental and computational work on efficient and safe drug treatment with better pharmacokinetic properties based on the common variant alleles of CYP genes, which varies among different ethnic populations.
Assuntos
Citocromo P-450 CYP2C19/genética , Simulação de Dinâmica Molecular , Alelos , Humanos , Mutação , Polimorfismo Genético/genética , Análise de Componente PrincipalRESUMO
BioH, an enzyme of biotin synthesis, plays an important role in fatty acid synthesis which assembles the pimelate moiety. Pimeloyl-acyl carrier protein (ACP) methyl ester, which is long known to be a biotin precursor, is the physiological substrate of BioH. Azelayl methyl ester, which has a longer chain than pimeloyl methyl ester, conjugated to ACP is also indeed accepted by BioH with very low rate of hydrolysis. To date, the substrate specificity for BioH and the molecular origin for the experimentally observed rate changes of hydrolysis by the chain elongation have remained elusive. To this end, we have investigated chain elongation effects on the structures by using the fully atomistic molecular dynamics simulations combined with binding free energy calculations. The results indicate that the substrate specificity is determined by BioH together with ACP. The added two methylenes would increase the structural flexibility by protein motions at the interface of ACP and BioH, instead of making steric clashes with the side chains of the BioH hydrophobic cavity. On the other hand, the slower hydrolysis of azelayl substrate is suggested to be associated with the loose of contacts between BioH and ACP, and with the lost electrostatic interactions of two ionic/hydrogen bonding networks at the interface of the two proteins. The present study provides important insights into the structure-function relationships of the complex of BioH with pimeloyl-ACP methyl ester, which could contribute to further understanding about the mechanism of the biotin synthetic pathway, including the catalytic role of BioH.
Assuntos
Biotina/química , Enzimas/química , Simulação de Dinâmica Molecular , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Biotina/biossíntese , Catálise , Enzimas/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoRESUMO
Cytochrome P450 (CYP) 3A7 plays a crucial role in the biotransformation of the metabolized endogenous and exogenous steroids. To compare the metabolic capabilities of CYP3A7-ligands complexes, three endogenous ligands were selected, namely dehydroepiandrosterone (DHEA), estrone, and estradiol. In this study, a three-dimensional model of CYP3A7 was constructed by homology modeling using the crystal structure of CYP3A4 as the template and refined by molecular dynamics simulation (MD). The docking method was adopted, combined with MD simulation and the molecular mechanics generalized born surface area method, to probe the ligand selectivity of CYP3A7. These results demonstrate that DHEA has the highest binding affinity, and the results of the binding free energy were in accordance with the experimental conclusion that estrone is better than estradiol. Moreover, several key residues responsible for substrate specificity were identified on the enzyme. Arg372 may be the most important residue due to the low interaction energies and the existence of hydrogen bond with DHEA throughout simulation. In addition, a cluster of Phe residues provides a hydrophobic environment to stabilize ligands. This study provides insights into the structural features of CYP3A7, which could contribute to further understanding of related protein structures and dynamics.
Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Ligantes , Simulação de Dinâmica Molecular , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sítios de Ligação , Domínio Catalítico , Citocromo P-450 CYP3A , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Molecular , Simulação de Acoplamento Molecular , Ligação Proteica , Especificidade por SubstratoRESUMO
Membrane insertion peptides have been developed in recent years and serve as cargos to deliver small molecules into cells. A class of membrane insertion peptides is the so called pH-induced peptides (pHLIPs), which are able to insert into membrane when the environment pH is acidic. Despite a number of experimental studies, the insertion process as well as the penetration mechanism is still worth study with computational methods. Thus, we performed molecular dynamics simulations in this study to elucidate the detailed penetration process and mechanism. Both protonated and unprotonated peptides are employed to interact with a POPCs bilayer. By analyzing the trajectory of the simulation, the peptide travelling across membrane is expected to take milliseconds or seconds. While the peptide penetrating through the POPC bilayer boundary is much faster (several nanoseconds). More importantly, the elaborate energies between a peptide and water molecules, the energies between a peptide and POPCs have been analyzed throughout the simulation time correspondingly. A constant decrease of interaction energies have been observed for peptide-water interaction in the protonated condition. At last, we employ the statistics of hydrogen bonds to explain the penetration mechanism tentatively. For the protonated system, the decrease of hydrogen bonds of peptide-water and the increase of hydrogen bonds of peptide- POPCs have been considered as the main driven force for the peptide insertion.
Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana/química , Fosfatidilcolinas/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Simulação de Dinâmica MolecularRESUMO
Fosfomycin resistance kinases FomA, one of the key enzymes responsible for bacterial resistances to fosfomycin, has gained much attention recently due to the raising public concern for multi-drug resistant bacteria. Using molecular docking followed by molecular dynamics simulations, our group illustrated the process of fosfomycin induced conformational change of FomA. The detailed roles of the catalytic residues (Lys18, His58 and Thr210) during the formation of the enzyme-substrate complex were shown in our research. The organization functions of Gly53, Gly54, Ile61 and Leu75 were also highlighted. Furthermore, the cation-π interaction between Arg62 and Trp207 was observed and speculated to play an auxiliary role in the conformation change process of the enzyme. This detailed molecular level illustration of the formation of FomA·ATP·Mg·Fosfomycin complex could provide insight for both anti-biotic discovery and improvement of fosfomycin in the future.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Farmacorresistência Bacteriana Múltipla , Fosfomicina/química , Fosfomicina/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Quinases/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Fosfomicina/metabolismo , Ligantes , Magnésio/química , Magnésio/metabolismo , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Proteínas Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
Transthyretin (TTR) dissociation and aggregation appear to cause several amyloid diseases. TTR dimer is an important intermediate that is hard to be observed from the biological experiments. To date, the molecular origin and the structural motifs for TTR dimer dissociation, as well as the unfolding process have not been rationalized at atomic resolution. To this end, we have investigated the effect of low pH and mutation L55P on stability as well as the unfolding pathway of TTR dimer using constant pH molecular dynamics simulations. The result shows that acidic environment results in loose TTR dimer structure. Mutation L55P causes the disruption of strand D and makes the CE-loop very flexible. In acidic conditions, dimeric L55P mutant exhibits notable conformation changes and an evident trend to separate. Our work shows that the movements of strand C and the loops nearby are the beginning of the unfolding process. In addition, hydrogen bond network at the interface of the two monomers plays a part in stabilizing TTR dimer. The dynamic investigation on TTR dimer provides important insights into the structure-function relationships of TTR, and rationalizes the structural origin for the tendency of unfolding and changes of structure that occur upon introduction of mutation and pH along the TTR dimer dissociation and unfolding process.
Assuntos
Mutação , Pré-Albumina/química , Pré-Albumina/genética , Multimerização Proteica/genética , Desdobramento de Proteína , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Estabilidade ProteicaRESUMO
Cytosolic insect theta class glutathione S-transferases (GSTs) have not been studied completely and their physiological roles are unknown. A detailed understanding of Anopheles gambiae GST (Aggst1-2) requires an accurate structure, which has not yet been determined. A high quality model structure of Aggst1-2 was constructed using homology modeling and the ligand-protein complex was obtained by the docking method. Molecular dynamics (MD) simulations were carried out to study conformational changes and to calculate binding free energy. The results of MD simulation indicate that Aggst1-2 undergoes small conformational changes after ligands dock to the protein, which facilitate the catalytic reaction. An essential hydrogen bond was found between the sulfur atom of glutathione (GSH) and the hydrogen atom of hydroxyl group in Ser9, which was in good agreement with experimental data. A π-π interaction between Phe204 and CDNB ligand was also found. This interaction seems to be important in stabilization of the ligand. Further study of binding free energy decomposition revealed a van der Waals interaction between two ligands that may play a key role in nucleophilic addition reaction. This work will be a good starting point for further determination of the biological role of cytosolic insect theta class GSTs and will aid the design of structure-based inhibitors.