Interacting Amino Acid Residues Research Articles

Retinoid BMS411 (4-{[(5,5-dimethyl-8-phenyl-5,6-dihydronaphthalen-2-yl) carbonyl] amino} benzoic acid), a potential inhibitor of NS5A protein of hepatitis C virus, a candidate for combined therapy of hepatitis C infection.

Hepatitis C infection is a serious health issue worldwide caused by hepatitis C virus (HCV). There is an urgent need of search for new direct acting antiviral drugs due to the rapid development of drug resistance. The HCV NS5A protein is involved in creating resistance against antiviral therapy and there are also many reports that vitamin A deficiency is associated with non-responsiveness to antiviral treatment in HCV infected patients. So the present in silico study was aimed to find the relation between vitamin A deficiency and the NS5A protein's function in antiviral resistance. Structure of NS5A protein was predicted by using I-Tasser (Interactive Tasser). Previous data on conservative domains and dimer formation were confirmed by using a series of current computational methods. The structure was employed for molecular docking analysis to investigate the interaction of ligand BMS411 (4-[(5,5-dimethyl-8-phenyl-6H-naphthalene-2-carbonyl)amino]benzoic acid), a vitamin A related compound with NS5A protein. Docking analysis showed that retinoid BMS411 can bind to HCV NS5A protein and may act as inhibitor of this protein. The functionally interacting amino acid residues surrounding the ligand molecule were identified and were shown to be involved in the formation of binding pocket. The present study suggests that retinoids may play an important role in the improvement of the outcomes of antiviral therapy against HCV through interaction with NS5A and inhibition of this protein. It is of great importance to check and verify if other retinoids could act as NS5A inhibitors.

Peroxisome Proliferator-activated Receptors as Potential Targets for Carcinogenic Activity of Polychlorinated Biphenyls: A Computational Perspective.

Polychlorinated biphenyls (PCBs) are ubiquitous environment-contaminating synthetic chemicals that have been associated with increased risk of hepatic cancer, melanoma, non-Hodgkin lymphoma and cancer of many other body organs. Structural binding analyses of PCB 77 and PCB 118 with peroxisome proliferator-activated receptors (PPARα, PPARβ/δ and PPARγ) was performed to predict the association of PCBs with potential disruption of PPAR signaling pathways. The crystal structures of human PPARα, PPARβ/δ and PPARγ were obtained from the Protein Data Bank. Structures of PCB 77 and PCB 118 were obtained from PubChem database. Docking was performed using glide (Schrodinger) induced fit docking (IFD) module. The PCB 77 and PCB 118 interacted with PPARα, PPARβ/δ and PPARγ showing an overlapping of 40-58% interacting amino acid residues with synthetic co-complex agonists of the three PPARs. The binding affinity was higher for PCB 118 than for PCB 77 during docking interactions with each of the three PPARs. The consistent commonality of interacting residues for PCB 77 and PCB 118 with co-complex synthetic agonists of the PPARs together with good binding affinity suggested that the PPAR signaling pathway is a potential target for toxicologic activity of PCBs.

F901318 represents a novel class of antifungal drug that inhibits dihydroorotate dehydrogenase

There is an important medical need for new antifungal agents with novel mechanisms of action to treat the increasing number of patients with life-threatening systemic fungal disease and to overcome the growing problem of resistance to current therapies. F901318, the leading representative of a novel class of drug, the orotomides, is an antifungal drug in clinical development that demonstrates excellent potency against a broad range of dimorphic and filamentous fungi. In vitro susceptibility testing of F901318 against more than 100 strains from the four main pathogenic Aspergillus spp. revealed minimal inhibitory concentrations of ≤0.06 µg/mL-greater potency than the leading antifungal classes. An investigation into the mechanism of action of F901318 found that it acts via inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) in a fungal-specific manner. Homology modeling of Aspergillus fumigatus DHODH has identified a predicted binding mode of the inhibitor and important interacting amino acid residues. In a murine pulmonary model of aspergillosis, F901318 displays in vivo efficacy against a strain of A. fumigatus sensitive to the azole class of antifungals and a strain displaying an azole-resistant phenotype. F901318 is currently in late Phase 1 clinical trials, offering hope that the antifungal armamentarium can be expanded to include a class of agent with a mechanism of action distinct from currently marketed antifungals.

Molecular docking and dynamic simulation studies of pentacyclictriterpenoids from euphorbiaceae Plants with snake venom phospolipase a2 And acostatin proteins

Health threat due to snake bite is one of the most ignored areas of active research vis-a-vis other domains of human healthcare studies. Fatalities due to snake bite are a major health threat affecting the lives of thousands of people every year in many developing countries. The prevalence of mortality caused by snake bite is taken as one of the serious challenges to solve the problem. The objective of the present study was computational analysis of proteins present in high and low poisonous snakes. Docking of inhibitors (taraxerol, taraxasterol, α-amyrin, β-amyrin, friedeline and oleanolic acid) with PLA2 enzyme from common cobra and acostatin from copper head snake for the identification of amino acid residues is crucial to analyze enzyme-inhibitor interaction. Molecular Docking with protein-inhibitors was performed using Schrodinger 16.2v. Glide score revealed that interaction of PLA2 with taraxerol and oleanolic acid showed a score of -1.43 and -1.74 respectively, whereas acostatin with taraxerol and taraxasterol showed the scores -1.78 and -2.00. The Molecular dynamic (MD) simulation studies were performed for docked complexes using Desmond tool. MD simulation of the complexes revealed stable nature of interactions. The present study identifies the interacting amino acid residues of snake venom proteins with ligands using bioinformatics tools and it will help in the development of a versatile drug in near future for treating snake bite

Characteristics of C-terminal, β-amyloid peptide binding fragment of neuroprotective protease inhibitor, cystatin C.

Cystatin C originally identified as a cysteine proteases inhibitor has a broad spectrum of biological roles ranging from inhibition of extracellular cysteine protease activities, bone resorption, and modulation of inflammatory responses to stimulation of fibroblasts proliferation. There is an increasing number of evidence to suggest that human cystatin C (hCC) might play a protective role in the pathophysiology of sporadic Alzheimer's disease. In vivo and in vitro results well documented the association of hCC with Aβ and the hCC-induced inhibition of Aβ fibril formation. In our earlier work, using a combination of selective proteolytic methods and MS spectroscopy, C-terminal fragment hCC(101-117) was identified as the Aβ-binding region. The fragment of Aβ peptide responsible for the complex formation with hCC was found in the middle, highly hydrophobic part, Aβ(17-24). Structures and affinities of both Aβ and hCC binding sites were characterized by the enzyme-linked immunosorbent assay-like assay, by surface plasmon resonance, and by nano-ESI-FTICR MS of the hCC-Aβ-binding peptide complexes. In the in vitro inhibition studies, the binding cystatin sequence, hCC(101-117), revealed the highest relative inhibitory effect toward Aβ-fibril formation. Herein, we present further studies on molecular details of the hCC-Aβ complex. With Ala substitution, affinity experiments, and enzyme-linked immunosorbent assay-like assays for the Aβ-binding fragment, hCC(101-117), and its variants, the importance of individual amino acid residues for the protein interaction was evaluated. The results were analyzed using hCC(101-117) nuclear magnetic resonance structural data with molecular dynamics calculations and molecular modeling of the complexes. The results point to conformational requirements and special importance of some amino acid residues for the protein interaction. The obtained results might be helpful for the design of low molecular compounds modulating the biological role of both proteins. Copyright © 2016 John Wiley & Sons, Ltd.

Interacting with GPCRs: Using Interaction Fingerprints for Virtual Screening.

The expanding number of crystal structures of G protein-coupled receptors (GPCRs) has increased the knowledge on receptor function and their ability to recognize ligands. Although structure-based virtual screening has been quite successful on GPCRs, scores obtained by docking are typically not indicative for ligand affinity. Methods capturing interactions between protein and ligand in a more explicit manner, such as interaction fingerprints (IFPs), have been applied as an addition or alternative to docking. Originally IFPs captured the interactions of amino acid residues with ligands with specific definitions for the various interaction types. More complex IFPs now capture atom-atom interactions, such as in SYBYL, or fragment-fragment co-occurrences such as in SPLIF. Overall, most of the IFPs have been studied in comparison with docking in retrospective studies. For GPCRs it remains unclear which IFP should be used, if at all, and in what manner. Thus, the performance between five different IFPs was compared on five different representative GPCRs, including several extensions of the original implementations,. Results show that the more detailed IFPs, SYBYL and SPLIF, perform better than the other IFPs (Deng, Credo, and Elements). SPLIF was further tuned based on the number of poses, fingerprint similarity coefficient, and using an ensemble of structures. Enrichments were obtained that were significantly higher than initial enrichments and those obtained by 2D-similarity. With the increase in available crystal structures for GPCRs, and given that IFPs such as SPLIF enhance enrichment in virtual screens, it is anticipated that IFPs will be used in conjunction with docking, especially for GPCRs with a large binding pocket.

A combined pharmacophore modeling, 3D QSAR, virtual screening, molecular docking, and ADME studies to identify potential HDAC8 inhibitors

The World Cancer Report 2014 shows that cancer is a leading cause of death globally, and cancer related death is likely to go up by about 70 % in the next two decades. Among several target receptors of cancer, histone deacetylases are the promising therapeutic target for many cancers. The current study deals with a primary goal of identification of novel non-hydroxamic acid histone deacetylase 8 inhibitors. In this context, six featured pharmacophoric hypotheses with hydrogen bond acceptors (A), hydrogen bond donor (D), and aromatic ring (R) were generated using 36 reported histone deacetylase 8 inhibitors. Virtual screening of database using two pharmacophore hypothesis AAAADR.161 and AAADDR.189 resulted 2000 hits each having fitness score >1.503. The pharmacophore AAADDR.189 yielded statistically significant three-dimensional quantitative structure-activity relationship model with training set (R 2: 0.9756, SD: 0.0680, F: 151.6, N: 26) and test set (Q 2: 0.6922, Pearson R: 0.8705, N: 10) molecules. The R 2 pred value of the model was 0.6951, which confirmed the good predictive ability of the model for external data set. Hits resulted from virtual screening and known inhibitors were subjected to molecular docking study to identify the binding affinity of inhibitors with active site amino acid residues. Finally, absorption, distribution, metabolism, and excretion study were undertaken to determine the drug likeness properties of identified hits. On the basis of fitness score, predicted activities, XP Glide score, interacting amino acid residues of known inhibitors and absorption, distribution, metabolism, and excretion properties, ten structurally diverse hits are reported in this paper as potential histone deacetylase 8 inhibitors which reduce the cost of histone deacetylase 8 inhibitor discovery and enhance the process prior synthesis.

Molecular recognition mechanism and motion of HCV NS3/4A protease with Faldaprevir analogue

Faldaprevir analogue molecule (FAM) has been reported to effectively inhibit the catalytic activity of HCV NS3/4A protease, making it a potential lead compound against HCV. A series of HCV NS3/4A protease crystal structures were analyzed by bioinformatics methods, and the FAM-HCV NS3/4A protease crystal structure was chosen for this study. A 20.4 ns molecular dynamics simulation of the complex consists of HCV NS3/4A protease and FAM was conducted. The key amino acid residues for interaction and the binding driving force for the molecular recognition between the protease and FAM were identified from the hydrogen bonds and binding free energy analyses. With the driving force of hydrogen bonds and van der Waals, FAM specifically bind to the active pocket of HCV NS3/4A protease, including V130-S137, F152-D166, D77-D79 and V55, which agreed with the experimental data. The effect of R155K, D168E/V and V170T site-directed mutagenesis on FAM molecular recognition was analyzed for their effect on drug resistance, which provided the possible molecular explanation of FAM resistance. Finally, the system conformational change was explored by using free energy landscape and conformational cluster. The result showed four kinds of dominant conformation, which provides theoretical basis for subsequent design of Faldaprevir analogue inhibitors based on the structure of HCV NS3/4A protease.

Endocrine Disruption: Computational Perspectives on Human Sex Hormone-Binding Globulin and Phthalate Plasticizers.

Phthalates are a class of high volume production chemicals used as plasticizers for household and industrial use. Several members of this chemical family have endocrine disrupting activity. Owing to ubiquitous environmental distribution and exposure of human population at all stages of life, phthalate contamination is a continuous global public health problem. Clinical and experimental studies have indicated that several phthalates are associated with adverse effects on development and function of human and animal systems especially the reproductive system and exposures during pregnancy and early childhood are by far of utmost concern. Sex hormone-binding globulin (SHBG) is a plasma carrier protein that binds androgens and estrogens and represents a potential target for phthalate endocrine disruptor function in the body. In the present study, the binding mechanism of the nine phthalates i.e. DMP, DBP, DIBP, BBP, DNHP, DEHP, DNOP, DINP, DIDP with human SHBG was delineated by molecular docking simulation. Docking complexes of the nine phthalates displayed interactions with 15–31 amino acid residues of SHBG and a commonality of 55–95% interacting residues between natural ligand of SHBG, dihydrotestosterone, and the nine phthalate compounds was observed. The binding affinity values were more negative for long chain phthalates DEHP, DNOP, DINP, and DIDP compared to short chain phthalates such as DMP and DBP. The Dock score and Glide score values were also higher for long chain phthalates compared to short chain phthalates. Hence, overlapping of interacting amino acid residues between phthalate compounds and natural ligand, dihydrotestosterone, suggested potential disrupting activity of phthalates in the endocrine homeostasis function of SHBG, with long chain phthalates expected to be more potent than the short chain phthalates.

Studies on the binding sites of IgG2 monoclonal antibodies recognized by terpyridine-based affinity ligands.

This investigation has examined the origin of the molecular recognition associated with the interaction of monoclonal IgG2's with terpyridine-based ligands immobilized onto agarose-derived chromatographic adsorbents. Isothermal titration calorimetric (ITC) methods have been employed to acquire thermodynamic data associated with the IgG2-ligand binding. These ITC investigations have documented that different enthalpic and entropic processes are involved depending on the nature of the chemical substituents in the core structure of the terpyridinyl moiety. In addition, molecular docking studies have been carried out with IgG2 structures with the objective to identify possible ligand binding sites and key interacting amino acid residues. These molecular docking experiments with the different terpyridine-based ligands have shown that all of the examined ligands can potentially undergo favorable interactions with a site located within the Fab region of the IgG2. However, another favorable binding site was also identified from the docking poses to exist within the Fc region of the IgG2 for some, but not all, of the ligands studied. These investigations have provided a basis to elucidate the unique binding properties and chromatographic behaviors shown by several substituted terpyridine ligands in their interaction with IgGs of different isotype. Copyright © 2016 John Wiley & Sons, Ltd.

Using Network Models of Proteins to Predict Functionally Linked Interfaces of Proteins (FLIPs) at the Residue Level

Proteins perform numerous cellular activities either individually or by interacting with other proteins. The function carried out by participating oligomers is representative of their structural stability and interaction specificity. One general goal of our research is to identify the properties of interacting amino acid residues that guide formation of Functionally Linked Interfaces of Proteins (FLIPs) upon oligomerization. In this work, proteins are represented as networks (Residue Interaction Networks, RINs) where residues are the nodes and residue proximity (both bonded and non-bonded interactions) define the edges. Prior studies of RINs by others have shown that network properties aid identification of active sites and residues important for protein folding. In our prior work, we could distinguish known FLIPs from Functionally unCorrelated structures (FunCs) using RINs with ∼73% accuracy and found FLIPs were generally much more organized than FunCs. In this work, we expand this concept to identify the threshold values of network features for individual residues participating in a FLIP interface such that potential FLIP interface regions can be predicted at the residue level.

COMPUTATIONAL STUDIES ON DIFFERENT TYPES OF APOPTOTIC PROTEINS DOCKED WITH A DIETARY FLAVONOID ERIODICTYOL IN COLON CANCER

ABSTRACTObjective: In this study, to evaluate the computational studies of eriodictyol docked with apoptotic proteins.Methods: AutoDock Vina and Molecular Graphics Laboratory tools were used to determine the interaction between proteins and eriodictyol.Discovery studio visualizer and PyMOL were used to determine the interaction of amino acid residues between apoptotic proteins and eriodictyol.Results: The obtained results revealed more binding affinities of p53, caspase 8, B-cell lymphoma (Bcl)-2, Bcl-2 - associated X protein (BAX), andadenomatous polyposis coli (APC) of −10.6, −10.9, −9.0, −9.5, and 7.2 kcal/mol, respectively. Interaction of hydrophobic polar contacts of amino acidresidues of p53 (CYS-277 and ALA-276), caspase 8 [THR-467, THR-337 (2), and GLU-396 (2)], Bcl-2 [ARG-103 (3), ALA-104 (2), and PHE-105, TYR101],BAX[GLY-108,TRY-107, ASN-106, and GLN-155 (2)], and APC [GLU-40 (2) and LEU-37 (2)] were notified between macromolecules and smallmolecules. The calculation of root-mean-square deviation of proteins and eriodictyol showed the lower binding energies to be 11.6, 12.5, 15.9, 19.6,and 15.8 and the upper binding energies to be 12.4, 15.3, 16.9, 20.7, and 18, respectively. The homology of binding energies was determined below 2Å which is computationally less expensive and easily determined the hydrophobic polar contacts.Conclusions: The homology of binding energies was determined below 2Å which is computationally less expensive and easily determinedthe hydrophobic polar contacts. The results were proved that the eriodictyol highly interacted with apoptotic proteins. It might be a stronganti-inflammatory activity of colon cancer. In future, computational molecular docking studies should aid further in vitro and in vivo studies.Keywords: AutoDock Vina, p53, Eriodictyol, Caspase 8, B-cell lymphoma-2, Bcl-2 - associated X protein, Adenomatous polyposis coli.

Insight into recognition between aminoacyl carrier protein and its binding partner

Acyl carrier-protein (ACP) is one of the most promiscuous protein in terms of protein-protein interactions within the cell. Common to both, primary and secondary cell metabolic pathways, ACP acts as a central acceptor of various intermediate products of fatty acid, polyketide and nonribosomal peptide synthesis pathways. Thus, it's quite puzzling how ACP selects correct protein partner among many possible upstream and downstream binding partners. To address this question we choose recently described protein-protein complex formed between aminoacyl carrier protein from Bradyrhizobium japonicum (Bj CP) and Bj Gly:CP ligase 1. We performed molecular dynamics simulations, MM-GBSA binding free energy calculations, alanine scanning mutagenesis, and multiple sequence alignment to get deeper insight into relevant protein-protein interactions between three forms of the aminoacyl carrier protein and their partner protein Bj Gly:CP ligase 1. Our result are in line with experimental findings and indicate that correct protein-protein communication is regulated through specific non-polar contribution of the interacting amino acid residues placed at the interface between aminoacyl carrier protein and partner Bj Gly:CP ligase 1. In silico alanine scanning mutagenesis along with multiple sequence alignment aided in classifying interacting residues as universally required for aminoacyl carrier protein binding and those that determine the selectivity.

Identification of potent histone deacetylase 8 inhibitors using pharmacophore-based virtual screening, three-dimensional quantitative structure–activity relationship, and docking study

Identification of potent histone deacetylase 8 inhibitors using pharmacophore-based virtual screening, three-dimensional quantitative structure–activity relationship, and docking study Tanusree Debnath,1 Swapan Majumdar,2 Arunasree M Kalle,3 Vema Aparna,4 Sudhan Debnath1 1Department of Chemistry, MBB College, Agartala, Tripura, 2Department of Chemistry, Tripura University, Suryamanninagar, Agartala, Tripura, 3University of Hyderabad Gachibowli, Hyderabad, 4Sree Chaitanya Institute of Pharmaceutical Sciences, Karimnagar, India Abstract: In recent years, histone deacetylases (HDACs) have been considered one of the promising targets for cancer chemotherapy. In the present study, a six-featured pharmacophore model with two hydrogen bond acceptors (AA), two hydrogen bond donors (DD), and two aromatic rings (RR) was developed. A predictive three-dimensional quantitative structure–activity relationship model was generated using the pharmacophore models obtained. The model has an excellent correlation coefficient and good predictive ability, as shown by the significant statistical parameters for both the training set (R2=0.9565, standard deviation =0.1171, F=99, and number of ligands in training set =21) and test set (Q2=0.8468, Pearson's R=0.9363, number of ligands in test set =9) molecules. The pharmacophore model was employed for the virtual screening of molecules with HDAC8 activity. The screening resulted in 366 hits with predicted activity as HDAC8 inhibitors. The hits obtained from the virtual screening were subjected to a molecular docking study to identify the potent inhibitors that binds to the active site with high affinity. The molecular docking study of known inhibitors and their analysis showed that the crucial interacting amino acid residues of HDAC8 are TYR-306, HIS-142, PHE-152, TYR-100, HIE-180, PHE-207, and Zn-388. On the basis of fitness score, predicted activities, XP Glide score, ADME results, and interacting amino acid residues, ten structurally diverse hits were reported in this paper as HDAC8 inhibitors. Keywords: pharmacophore, atom-based 3D QSAR, virtual screening, docking, ADME, HDAC8 inhibitors

Interaction model of steviol glycosides from Stevia rebaudiana (Bertoni) with sweet taste receptors: A computational approach

Docking studies were performed on natural sweeteners from Stevia rebaudiana by constructing homology models of T1R2 and T1R3 subunits of human sweet taste receptors. Ramachandran plot, PROCHECK results and ERRAT overall quality factor were used to validate the quality of models. Furthermore, docking results of steviol glycosides (SG’s) were correlated significantly with data available in the literature which enabled to predict the exact sweetness rank order of SG’s. The binding pattern indicated that Asn 44, Ans 52, Ala 345, Pro 343, Ile 352, Gly 346, Gly 47, Ala 354, Ser 336, Thr 326 and Ser 329 are the main interacting amino acid residues in case of T1R2 and Arg 56, Glu 105, Asp 215, Asp 216, Glu 148, Asp 258, Lys 255, Ser 104, Glu 217, Leu 51, Arg 52 for T1R3, respectively. Amino acids interact with SG’s mainly by forming hydrogen bonds with the hydroxyl group of glucose moieties. Significant variation in docked poses of all the SG’s were found. In this study, we have proposed the mechanism of the sweetness of the SG’s in the form of multiple point stimulation model by considering the diverse binding patterns of various SG’s, as well as their structural features. It will give further insight in understanding the differences in the quality of taste and will be used to improve the taste of SG’s using semi-synthetic approaches.

Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1.

Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate co-adipate (PBSA), it produces RolA, a hydrophobin, and CutL1, a PBSA-degrading cutinase. Secreted RolA attaches to the surface of the PBSA particles and recruits CutL1, which then condenses on the particles and stimulates the hydrolysis of PBSA. Here, we identified amino acid residues that are required for the RolA-CutL1 interaction by using site-directed mutagenesis. We quantitatively analyzed kinetic profiles of the interactions between RolA variants and CutL1 variants by using a quartz crystal microbalance (QCM). The QCM analyses revealed that Asp142, Asp171 and Glu31, located on the hydrophilic molecular surface of CutL1, and His32 and Lys34, located in the N-terminus of RolA, play crucial roles in the RolA-CutL1 interaction via ionic interactions. RolA immobilized on a QCM electrode strongly interacted with CutL1 (K(D) = 6.5 nM); however, RolA with CutL1 variants, or RolA variants with CutL1, showed markedly larger KD values, particularly in the interaction between the double variant RolA-H32S/K34S and the triple variant CutL1-E31S/D142S/D171S (K(D) = 78.0 nM). We discuss a molecular prototype model of hydrophobin-based enzyme recruitment at the solid-water interface.

In Silico-based Study of Cytochrome P450 and Multidrug Resistance Protein 1 from Docking Perspective to Understand Kidney Failure

Background: The need to identify causes of drug induced kidney failure has been underscored by International Conference on Harmonization (ICH) regulated agencies. In our earlier studies on adverse drug reaction (ADR) reported in Canada Vigilance Adverse Reaction Online Database it was observed that drugs azathioprine, clozaril/clozapine, diclofenac sodium, diflucan/ fluconazole, furosemide, indomethacin, metformin, micardis/ telmisartan, viread/tenofovir, and zyprexa/olanzapine lead to kidney failure. Method: Attempts have been made to understand the physiological process via bioinformatics perspective. This was done by active site identification for cytochrome P450 along with multidrug resistance protein 1 (MRP1). Docking against the drugs in these proteins that are categorically involved in drug binding based on their pharmacological actions are as per drug bank annotations. Results: Cytochrome P450 2C19 protein showed better interactions with drug indomethacin with a maximum score of –119.2 kcal/mol followed by drug clozaril with a score of –102.5 kcal/mol. This was finally followed by of drug zyprexa with a score of –101.0 kcal/mol. The residues which are actively involved with the drug indomethacin include Arg97 and Arg433. Drug clozaril shows interaction with Ala297. For drug zyprexa the residues like Arg97, Ala297 and Cys435 interact with the protein. For MRP1, even though it showed better binding scores for drugs azathioprine, indomethacin, diflucan and furosemide. But still, they are not able to interact within the pocket, leaving it empty during docking studies. Conclusion: Through this study, it was possible to identify active site pocket in the related proteins and the interacting amino acid residues of cytochrome P450 that may contribute to drug induced kidney failure.

Fluorous photoaffinity labeling to probe protein-small molecule interactions.

Identifying cellular targets of bioactive small molecules is essential for their applications as chemical probes or drug candidates. Of equal importance is to determine their "off-target" interactions, which usually account for unwanted properties including toxicity. Among strategies to profile small molecule-interacting proteins, photoaffinity labeling has been widely used because of its distinct advantages such as sensitivity. When combined with mass spectrometry, this approach can provide additional structural and mechanistic information, such as drug-target stoichiometry and exact interacting amino acid residues. We have described a novel fluorous photoaffinity labeling approach, in which a fluorous tag is incorporated into the photoaffinity labeling reagent to enable the enrichment of the labeled species from complex mixtures for analysis. This new feature likely makes the fluorous photoaffinity labeling approach suitable to identify transient interactions, and low-abundant, low-affinity interacting proteins in a cellular environment.

A crystallographic study of the Toho-1 β-lactamase acylation mechanism

β-lactam antibiotics have been used effectively over several decades against many types of highly virulent bacteria. The predominant cause of resistance to these antibiotics in Gram-negative bacterial pathogens is the production of serine β-lactamase enzymes. A key aspect of the class A serine β-lactamase mechanism that remains unresolved and controversial is the identity of the residue acting as the catalytic base during the acylation reaction. Multiple mechanisms have been proposed for the formation of the acyl-enzyme intermediate that are predicated on understanding the protonation states and hydrogen-bonding interactions among the important residues involved in substrate binding and catalysis of these enzymes. For resolving a controversy of this nature surrounding the catalytic mechanism, neutron crystallography is a powerful complement to X-ray crystallography that can explicitly determine the location of deuterium atoms in proteins, thereby directly revealing the hydrogen-bonding interactions of important amino acid residues. Neutron crystallography was used to unambiguously reveal the ground-state active site protonation states and the resulting hydrogen-bonding network in two ligand-free Toho-1 β-lactamase mutants which provided remarkably clear pictures of the active site region prior to substrate binding and subsequent acylation [1,2] and an acylation transition-state analog, benzothiophene-2-boronic acid (BZB), which was also isotopically enriched with 11B. The neutron structure revealed the locations of all deuterium atoms in the active site region and clearly indicated that Glu166 is protonated in the BZB transition-state analog complex. As a result, the complete hydrogen-bonding pathway throughout the active site region could then deduced for this protein-ligand complex that mimics the acylation tetrahedral intermediate [3].

Structural and mutagenic analysis of the RM controller protein C.Esp1396I.

Bacterial restriction-modification (RM) systems are comprised of two complementary enzymatic activities that prevent the establishment of foreign DNA in a bacterial cell: DNA methylation and DNA restriction. These two activities are tightly regulated to prevent over-methylation or auto-restriction. Many Type II RM systems employ a controller (C) protein as a transcriptional regulator for the endonuclease gene (and in some cases, the methyltransferase gene also). All high-resolution structures of C-protein/DNA-protein complexes solved to date relate to C.Esp1396I, from which the interactions of specific amino acid residues with DNA bases and/or the phosphate backbone could be observed. Here we present both structural and DNA binding data for a series of mutations to the key DNA binding residues of C.Esp1396I. Our results indicate that mutations to the backbone binding residues (Y37, S52) had a lesser affect on DNA binding affinity than mutations to those residues that bind directly to the bases (T36, R46), and the contributions of each side chain to the binding energies are compared. High-resolution X-ray crystal structures of the mutant and native proteins showed that the fold of the proteins was unaffected by the mutations, but also revealed variation in the flexible loop conformations associated with DNA sequence recognition. Since the tyrosine residue Y37 contributes to DNA bending in the native complex, we have solved the structure of the Y37F mutant protein/DNA complex by X-ray crystallography to allow us to directly compare the structure of the DNA in the mutant and native complexes.