Overexpression and functional characterisation of the human melanocortin 4 receptor in Sf9 cells

The human melanocortin 4 receptor (MC4r) was successfully expressed in Sf9 cells using the baculovirus infection system. N- and C-terminally His-tagged receptors generated B(max) values of 14 and 23 pmol receptor/mg membrane protein, respectively. The highest expression level obtained with the C-terminally His-tagged MC4r corresponded to 0.25mg active receptor/litre culture volume. Addition of a viral signal peptide at the N-terminus of the His-tagged MC4r did not improve the expression level. Confocal laser microscopy studies revealed that both the N- and C-terminally tagged MC4r did not accumulate intracellularly and were mainly located in the plasma membrane. The recombinant receptors showed similar affinity for the agonist NDP-MSH (Kd = 11 nM) as to MC4r expressed in mammalian cells. Functional coupling of the highest expressed C-terminal tagged receptor to endogenous Galpha protein was demonstrated through GTPgammaS binding upon agonist stimulation of the receptor. Ki values for the ligands MTII, HS014, alpha-, beta-, and gamma-MSH are comparable to the values obtained for MC4r expressed in mammalian cells.     

Probing the structure of falcipain-3, a cysteine protease from Plasmodium falciparum: comparative protein modeling and docking studies

Increasing resistance of malaria parasites to conventional antimalarial drugs is an important factor contributing to the persistence of the disease as a major health threat. The ongoing search for novel targets has resulted in identification and expression of several enzymes including cysteine proteases that are implicated in hemoglobin degradation. Falcipain-2 and falcipain-3 are considered to be the two principal cysteine proteases in this degradation, and hence, are potential drug targets. A homology model of falcipain-3 was built and validated by various structure/geometry verification tools as well as docking studies of known substrates. The correlation coefficient of 0.975 between interaction energies and K(m) values of these substrates provided additional support for the model. On comparison with the previously reported falcipain-2 homology model, the currently constructed falcipain-3 structure showed important differences between the S2 pockets that might explain the variations in the K(m) values of various substrates for these enzymes. Further, docking studies also provided insight into possible binding modes and interactions of ligands with falcipain-3. Results of the current study could be employed in de novo drug design leading to development of new antimalarial agents.     

Domain wise docking analyses of the modular chitin binding protein CBP50 from Bacillus thuringiensis serovar konkukian S4

This paper presents an in silico characterization of the chitin binding protein CBP50 from B. thuringiensis serovar konkukian S4 through homology modeling and molecular docking. The CBP50 has shown a modular structure containing an N-terminal CBM33 domain, two consecutive fibronectin-III (Fn-III) like domains and a C-terminal CBM5 domain. The protein presented a unique modular structure which could not be modeled using ordinary procedures. So, domain wise modeling using MODELLER and docking analyses using Autodock Vina were performed. The best conformation for each domain was selected using standard procedure. It was revealed that four amino acid residues Glu-71, Ser-74, Glu-76 and Gln-90 from N-terminal domain are involved in protein-substrate interaction. Similarly, amino acid residues Trp-20, Asn-21, Ser-23 and Val-30 of Fn-III like domains and Glu-15, Ala-17, Ser-18 and Leu-35 of C-terminal domain were involved in substrate binding. Site-directed mutagenesis of these proposed amino acid residues in future will elucidate the key amino acids involved in chitin binding activity of CBP50 protein.     

Experimental and theoretical studies of the three-dimensional structure of human interleukin-4

The structure of human interleukin 4 (IL-4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL-4 belonged to the all alpha-helix class of protein structures. Secondary structure prediction, site-directed mutagenesis, and CD spectroscopy suggested a predominantly alpha-helical structure, consistent with a four-helix bundle structural motif. A human/mouse IL-4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL-4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four-helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.     

Ligand-supported homology modeling of the human angiotensin II type 1 (AT(1)) receptor: insights into the molecular determinants of telmisartan binding

Angiotensin II type 1 (AT(1)) receptor belongs to the super-family of G-protein-coupled receptors, and antagonists of the AT(1) receptor are effectively used in the treatment of hypertension. To understand the molecular interactions of these antagonists, such as losartan and telmisartan, with the AT(1) receptor, a homology model of the human AT(1) (hAT(1)) receptor with all connecting loops was constructed from the 2.6 A resolution crystal structure (PDB i.d., 1L9H) of bovine rhodopsin. The initial model generated by MODELLER was subjected to a stepwise ligand-supported model refinement. This protocol involved initial docking of non-peptide AT(1) antagonists in the putative binding site, followed by several rounds of iterative energy minimizations and molecular dynamics simulations. The final model was validated based on its correlation with several structure-activity relationships and site-directed mutagenesis data. The final model was also found to be in agreement with a previously reported AT(1) antagonist pharmacophore model. Docking studies were performed for a series of non-peptide AT(1) receptor antagonists in the active site of the final hAT(1) receptor model. The docking was able to identify key molecular interactions for all the AT(1) antagonists studied. Reasonable correlation was observed between the interaction energy values and the corresponding binding affinities of these ligands, providing further validation for the model. In addition, an extensive unrestrained molecular dynamics simulation showed that the docking-derived bound pose of telmisartan is energetically stable. Knowledge gained from the present studies can be used in structure-based drug design for developing novel ligands for the AT(1) receptor.     

Three-dimensional model of the extracellular domain of the type 4a metabotropic glutamate receptor: new insights into the activation process

Metabotropic glutamate receptors (mGluRs) belong to the family 3 of G-protein-coupled receptors. On these proteins, agonist binding on the extracellular domain leads to conformational changes in the 7-transmembrane domains required for G-protein activation. To elucidate the structural features that might be responsible for such an activation mechanism, we have generated models of the amino terminal domain (ATD) of type 4 mGluR (mGlu4R). The fold recognition search allowed the identification of three hits with a low sequence identity, but with high secondary structure conservation: leucine isoleucine valine-binding protein (LIVBP) and leucine-binding protein (LBP) as already known, and acetamide-binding protein (AmiC). These proteins are characterized by a bilobate structure in an open state for LIVBP/LBP and a closed state for AmiC, with ligand binding in the cleft. Models for both open and closed forms of mGlu4R ATD have been generated. ACPT-I (1-aminocyclopentane 1,3,4-tricarboxylic acid), a selective agonist, has been docked in the two models. In the open form, ACPT-I is only bound to lobe I through interactions with Lys74, Arg78, Ser159, and Thr182. In the closed form, ACPT-I is trapped between both lobes with additional binding to Tyr230, Asp312, Ser313, and Lys317 from lobe II. These results support the hypothesis that mGluR agonists bind a closed form of the ATDs, suggesting that such a conformation of the binding domain corresponds to the active conformation.     

In pursuit of virtual lead optimization: the role of the receptor structure and ensembles in accurate docking

Accurate ranking during in silico lead optimization is critical to drive the generation of new ligands with higher affinity, yet it is especially difficult because of the subtle changes between analogs. In order to assess the role of the structure of the receptor in delivering accurate lead ranking results, we docked a set of forty related inhibitors to structures of one species of dihydrofolate reductase (DHFR) derived from crystallographic, NMR solution data, and homology models. In this study, the crystal structures yielded the superior results: the compounds were placed in the active site in the conserved orientation and the docking scores for 80% percent of the compounds clustered into the same bins as the measured affinity. Single receptor structures derived from NMR data or homology models did not serve as accurate docking receptors. To our knowledge, these are the first experiments that assess ranking of homologous lead compounds using a variety of receptor structures. We then extended the study to investigate whether ensembles, either computationally or experimentally derived, of all of the single starting structures aid, hinder or have no effect on the performance of the starting template. Impressively, when ensembles of receptor structures derived from NMR data or homology models were employed, docking accuracy improved to a level equal to that of the high resolution crystal structures. The same experiments using a second species of DHFR and set of ligands confirm the results. A comparison of the structures of the individual ensemble members to the starting structures shows that the effect of the ensembles can be ascribed to protein flexibility in addition to absorption of computational error.     

Molecular modeling of the three-dimensional structure of GLP-1R and its interactions with several agonists

Glucagon-like peptide-1 receptor (GLP-1R) is a promising molecular target for developing drugs treating type 2 diabetes. We have predicted the complete three-dimensional structure of GLP-1R and the binding modes of several GLP-1R agonists, including GLP-1, Boc5, and Cpd1, through a combination of homology modeling, molecular docking, and long-time molecular dynamics simulation on a lipid bilayer. Our model can reasonably interpret the results of a number of mutation experiments regarding GLP-1R as well as the successful modification to GLP-1 by Liraglutide. Our model is also validated by a recently revealed crystal structure of the extracellular domain of GLP-1R. An activation mechanism of GLP-1R agonists is proposed based on the principal component analysis and normal mode analysis on our predicted GLP-1R structure. Before the complete structure of GLP-1R is determined through experimental means, our model may serve as a valuable reference for characterizing the interactions between GLP-1R and its agonists. Figure Comparison of our predicted model of rGLP-1R (left) with the recently revealed crystal structure of hGLP-1R (right)     

米曲霉木糖醇脱氢酶的分子模建与对接

[目的]研究米曲霉木糖醇脱氢酶基因的结构与功能.[方法]克隆测序来源于米曲霉的木糖醇脱氢酶(XDH)基因,利用Swiss-MODEL和Modeller对XDH进行三级结构模建,通过PROCHECK和Prosa2003对得到的4个目标模型进行评价,从中得到一个最佳模型.在同源建模的基础上,通过分子对接软件MolsoftICM-Pro,对辅因子进行对接,预测了XDH与NAD+、Zn2+作用的相关残基.寻找底物木糖醇与XDH结合的可能活性口袋,用Molsoft模拟XDH与木糖醇的对接,预测了酶与底物作用的关键氨基酸残基.[结果]结构分析显示,米曲霉XDH含有醇脱氢酶家族锌指纹结构和典型醇脱氢酶Rossmann折叠的辅酶结合域,属于Medium-chain脱氢酶(MDR)家族.通过对接研究,预测了XDH与NAD+之间形成氢键的氨基酸有Asp206、Arg211、Ser255、Ser301和Arg303,这些氨基酸位于结合域,与Zn2+形成氢键的氨基酸有His72和Glu73,位于催化域,与天然底物木糖醇形成氢键的氨基酸有Ile46、Ile349、Lys350和Thr351,位于催化域.[结论]所得信息对XDH分子定向改造、拓展米曲霉工业应用范围有重要意义.     

Cyclic analogs of alpha-melanocyte-stimulating hormone (alphaMSH) with high agonist potency and selectivity at human melanocortin receptor 1b

Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.     

Modeled structure of the 75-kDa neurotrophin receptor.

Motifs in ligand-binding domains of the neurotrophin (NTR) and lymphotoxin (TNFR-I) receptors define a family of receptors that mediates programmed cell death. We have explored relationships of architecture and function in this family through a molecular model of NTR, also called p75NGFR or LANR. Modeling by homology took advantage of four modular subdomains in the crystal structure of TNFR-I that also occur in NTR. Hypothetical complexes between the model and a ligand structure (for nerve growth factor, NGF) were then examined using docking software. NTR appears to bind in the dimer interface of NGF, making two sets of contacts. NTR subdomains III and IV provide the ligand-contact surfaces, in contrast to TNFR, in which subdomains II and III contact TNF-beta. NTR subdomain II appears to have been evolutionarily modified, potentially contributing to an interface between receptor subunits. These and other specific predictions of the model will require experimental confirmation.     

Three dimensional modeling of N-terminal region of galanin and its interaction with the galanin receptor

The neuropeptide galanin comes under the powerful and versatile modulators of classical neurotransmitters and is present in brain tissues, which are intimately involved in epileptogenesis. It acts as appealing targets for studying basic mechanisms of seizure initiation and arrest, and for the development of novel approaches for various neurodegenerative diseases. Galanin is widely distributed in the mammalian brain which controls various processes such as sensation of pain, learning, feeding, sexual behaviour, carcinogenesis, pathophysiology of neuroendocrine tumors and others. The function of galanin can be exploited through its interaction with three G-protein coupled receptors subtypes such as GalR1, GalR2 and GalR3. The N-terminal region of galanin comprises about highly conserved 15 amino acid residues, which act as the crucial region for agonist-receptor binding. We have constructed a theoretical structural model for the N-terminal region of galanin from Homo sapiens by homology modeling. The stereochemistry of the model was checked using PROCHECK. The functionally conserved regions were identified by surface mapping of phylogenetic information generated by online web algorithm ConSurf. The docking studies on the pharmacologically important galanin receptors with the theoretical model of N-terminal region of galanin predicted crucial residues for binding which would be useful in the development of novel leads for neurodegenerative disorders.     

Modeling of mammalian olfactory receptors and docking of odorants

Olfactory receptors (ORs) belong to the superfamily of G protein-coupled receptors (GPCRs), the second largest class of genes after those related to immunity, and account for about 3 % of mammalian genomes. ORs are present in all multicellular organisms and represent more than half the GPCRs in mammalian species (e.g., the mouse OR repertoire contains >1,000 functional genes). ORs are mainly expressed in the olfactory epithelium where they detect odorant molecules, but they are also expressed in a number of other cells, such as sperm cells, although their functions in these cells remain mostly unknown. It has recently been reported that ORs are present in tumoral tissues where they are expressed at different levels than in healthy tissues. A specific OR is over-expressed in prostate cancer cells, and activation of this OR has been shown to inhibit the proliferation of these cells. Odorant stimulation of some of these receptors results in inhibition of cell proliferation. Even though their biological role has not yet been elucidated, these receptors might constitute new targets for diagnosis and therapeutics. It is important to understand the activation mechanism of these receptors at the molecular level, in particular to be able to predict which ligands are likely to activate a particular receptor (‘deorphanization’) or to design antagonists for a given receptor. In this review, we describe the in silico methodologies used to model the three-dimensional (3D) structure of ORs (in the more general framework of GPCR modeling) and to dock ligands into these 3D structures.     

Probing the active site of cellodextrin phosphorylase from Clostridium stercorarium: kinetic characterization, ligand docking, and site-directed mutagenesis

Cellodextrin phosphorylase from Clostridium stercorarium has been recombinantly expressed in Escherichia coli for the first time. Kinetic characterization of the purified enzyme has revealed that aryl and alkyl β-glucosides can be efficiently glycosylated, an activity that has not yet been described for this enzyme class. To obtain a better understanding of the factors that determine the enzyme's specificity, homology modeling and ligand docking were applied. Residue W168 has been found to form a hydrophobic stacking interaction with the substrate in subsite +2, and its importance has been examined by means of site-directed mutagenesis. The mutant W168A retains about half of its catalytic activity, indicating that other residues also contribute to the binding affinity of subsite +2. Finally, residue D474 has been identified as the catalytic acid, interacting with the glycosidic oxygen between subsites -1 and +1. Mutating this residue results in complete loss of activity. These results, for the first time, provide an insight in the enzyme-substrate interactions that determine the activity and specificity of cellodextrin phosphorylases.     

Modeling the structure of mAb 14B7 bound to the anthrax protective antigen

The anthrax protective antigen (PA) is a key component of the tripartite anthrax toxin. Monoclonal antibody (mAb) 14B7 and its engineered, affinity-matured variants have been shown to be effective in blocking PA binding to cellular receptors and mitigating anthrax toxicity. Here, we perform computational structural modeling of the mAb 14B7-PA interaction. Our objectives are to determine the structure of the 14B7-PA complex, to deduce a structural explanation for the affinity maturation from the docking models, and to study the effect of inaccuracies in the antibody homology model on docking. We used the RosettaDock program to dock PA with the mAb 14B7 crystal structure or homology model. Our simulations generate two distinct binding orientations consistent with experimental residue mutations that diminish 14B7-PA binding. Furthermore, the models suggest new site-directed mutations to positively identify one of these two solutions as the correct 14B7-PA docking orientation. The models indicate that PA regions 648-660 and 712-720 may be important for 14B7 binding in addition to the known PA epitope, and the binding interfaces are similar to that seen in the PA complex with cellular receptor CMG2. Antibody residues involved in affinity maturation do not contact the antigen in the docking models, suggesting that affinity maturation in the 14B7 family does not result from direct enhancements of antibody-antigen contacts. Docking the homology model produces low-resolution representations of the crystal structure docking orientations, but homology model docking is frustrated by antibody H3 loop conformation errors. This work demonstrates the usefulness and limitations of computational structure prediction for the development of antibody therapeutics, and reemphasizes the need for flexible backbone docking algorithms to achieve high-resolution docking using homology models.     

Three-dimensional model of the human urotensin-II receptor: docking of human urotensin-II and nonpeptide antagonists in the binding site and comparison with an antagonist pharmacophore model

Human urotensin-II (hU-II) is a cyclic peptide that plays a central role in cardiovascular homeostasis and is considered to be the most potent mammalian vasoconstrictor identified to date. It is a natural ligand of the human urotensin-II (hUT-II) receptor, a member of the family of rhodopsin-like G-protein-coupled receptors. To understand the molecular interactions of hU-II and certain antagonists with the hUT-II receptor, a model of the hUT-II receptor in an active conformation with all its connecting loops was constructed by homology modeling. The initial model was placed in a pre-equilibrated lipid bilayer and re-equilibrated by several procedures of energy minimization and molecular dynamics simulations. Docking studies were performed for hU-II and for a series of nonpeptide hUT-II receptor antagonists in the active site of the modeled receptor structure. Results of the hU-II docking study are in agreement with our previous work and with experimental data showing the contribution of the extracellular loops II and III to ligand recognition. The docking of hU-II nonpeptide antagonists allows identification of key molecular interactions and confirms a previously reported hU-II antagonist pharmacophore model. The results of the present studies will be used in structure-based drug design for developing novel antagonists for the hUT-II receptor.     

玉米黑粉菌CYP51稀有密码子和mRNA二级结构分析及与杀菌剂戊唑醇分子对接

通过截短玉米黑粉菌CYP51(P450-14DM,UmCYP51)基因(去除编码跨膜区部分)和选取不同的表达载体,构建了9种重组表达质粒,在大肠杆菌中进行UmCYP51基因的表达,发现只有BL21(DE3)/pET32-Um-35重组表达工程菌获得了表达.对稀有密码子和mRNA翻译起始区二级结构进行分析,结果表明稀有密码子和mRNA翻译起始区二级结构对UmCYP51蛋白的表达都有影响.适用于稀有密码子表达的菌株Rosetta(DE3)不利于UmCYP51蛋白的表达;同时只有翻译起始区二级结构自由能值最低的重组载体pET32-Um-35可以表达.为了设计以UmCYP51为靶标的新型抗真菌抑制剂,基于最新解析的真核生物人类的CYP51晶体结构,利用同源模建的方法构建了UmCYP51的三维结构并进行了分子动力学模拟优化.通过与商品化杀菌剂戊唑醇进行分子对接获得了此类抑制剂与UmCYP51的理论结合方式,阐述了戊唑醇分子的杀菌机理,为开发新型的抗真菌抑制剂奠定了基础.     

1H-NMR studies on a potent and selective antagonist at human melanocortin receptor 4 (hMC-4R)

Melanocortin receptor 4 (MC-4R) is involved in the regulation of energy balance and body weight, and recognizes alpha-, beta-, and gamma-melanocyte stimulating hormones (alpha-, beta-, gamma-MSH). In the search for compounds that regulate food intake and body weight, two synthetic lactam-derivative ligands of alpha-MSH were discovered, MTII and SHU9119. MTII is an agonist and reduces food intake in rats, whereas SHU9119 is an antagonist, and increases food intake and body weight in rats. MTII and SHU9119 are nonselective compounds to MC-4R. To enhance the potency and selectivity at the human MC-4R (hMC-4R), we recently synthesized analogs of SHU9119 (M. A. Bednarek, T. MacNeil, R. N. Kalyani, R. Tang, Van der L. H. T. Ploeg, and D. H. Weinberg, Journal of Medicinal Chemistry, 2001, Vol. 44, pp. 401-409), wherein compound 1 was the most selective for hMC-4R. Replacement of D-Nal by L-Nal in compound 1 made compound 2 weakly active. Comparison of the structures by NMR and molecular modeling of compounds 1 and 2 vs SHU9119 and MTII indicated that, even though they existed as an average of several conformations in solution, there were distinctions in their structures. The gamma-methylene protons of Arg in compound 1 were nonequivalent and shielded probably by the aromatic ring of Nal. The NHi-NHi+1 NOE cross peaks and the temperature coefficients of the amide protons around the "essential core" Nal/Phe7-Arg8-Trp9, required for high affinity and high selectivity at hMC-4R, were indicative of a possible turn structure for these compounds but with differences in their NOE strengths and temperature coefficient values. Molecular modeling of these compounds based on their NMR data showed that the essential core appeared as a "V" shape with two different orientations, one for compound 1 and some of the conformers of SHU9119 and MTII, and the other for compound 2 and some other conformers of SHU9119 and MTII. The remaining conformers of SHU9119 and MTII, which did not map to compound 1 or 2, suggested that they were outside of the hMC-4R binding envelop. These observations may lead to conjectures as to why compound 1 is highly active and selective toward hMC-4R.     

Association of melanocortin 4 receptor gene variation with satiation and gastric emptying in overweight and obese adults

Melanocortin 4 receptor (MC4R) has a major role in energy homeostasis. The rs17782313 polymorphism, mapped 188 kb downstream from MC4R, has been associated with satiety, higher body mass index (BMI) and total calorie intake in adults. To assess the association of rs17782313 with gastric functions, satiation, or satiety, we studied 178 predominantly Caucasian overweight and obese people: 120 females, 58 males; mean BMI 33.4 ± 5.3 kg/m² (SD); age 37.7 ± 11.2 years. Quantitative traits assessed were gastric emptying (GE) of solids and liquids; fasting and postprandial gastric volume; satiation by maximum tolerated volume and 4 symptoms by 100-mm visual analog scales (VAS); and satiety by ad libitum buffet meal. Associations of genotype and quantitative traits were assessed by analysis of covariance (using gender and BMI as covariates), based on a dominant [TC (n = 72) − CC (n = 12) vs. TT (n = 94)] genetic model. rs17782313(C) was associated with postprandial satiation symptoms (median Δ total VAS 26.5 mm, p = 0.036), reduced proportion of solid GE at 2 h (median Δ 6.7 %, p = 0.008) and 4 h (median Δ 3.2 %, p = 0.006), and longer t_½ (median Δ 6 min, p = 0.034). Associations of rs17782313 with obesity may be explained by reduced satiation and GE. The role of MC4R mechanisms in satiation and gastric function deserves further study.     

Probing ligand binding modes of human cytochrome P450 2J2 by homology modeling, molecular dynamics simulation, and flexible molecular docking

Cytochrome P450 (P450) 2J2 catalyzes epoxidation of arachidonic acid to eicosatrienoic acids, which are related to a variety of diseases such as coronary artery disease, hypertension, and carcinogenesis. Recent experimental data also suggest that P450 2J2 could be a novel biomarker and a potential target for cancer therapy. However, the active site topology and substrate specificity of this enzyme remain unclear. In this study, a three-dimensional model of human P450 2J2 was first constructed on the basis of the crystal structure of human P450 2C9 in complex with a substrate using homology modeling method, and refined by molecular dynamics simulation. Flexible docking approaches were then employed to dock four ligands into the active site of P450 2J2 in order to probe the ligand-binding modes. By analyzing the results, active site architecture and certain key residues responsible for substrate specificity were identified on the enzyme, which might be very helpful for understanding the enzyme's biological role and providing insights for designing novel inhibitors of P450 2J2.