Homology modeling and docking studies of human Bcl-2L10 protein

Cancer, an unrestrained proliferation of cells, is one of the lead cause of death. Nearly 12.5 million people are diagnosed with cancer worldwide, 7.5 million people die of which 2.5 million cases are from India. Major cause for cancer is restriction of programmed cell death (apoptosis). Multiple signaling pathways regulate apoptosis. Bcl-2 (B - Cell Lymphomas-2) family proteins play a vital role as central regulators of apoptosis. Bcl-2L10, a novel anti-apoptotic protein, blocks apoptosis by mitochondrial dependent mechanism. The present study evaluates the 3D structure of Bcl-2L10 protein using homology modeling and aims to understand plausible functional and binding interactions between Bcl-2L10 with BH3 domain of BAX using protein - protein docking. The docking studies show binding of BH3 domain at Lys 110, Trp-111, Pro-115, Glu-119 and Asp-127 in the groove of BH 1, 2 and 3 domains of Bcl-2L10. Heterodimerization of anti-apoptotic Bcl-2 and BH3 domain of pro-apoptotic Bcl-2 proteins instigates apoptosis. Profound understanding of Bcl-2 pathway may prove useful in identification of future therapeutic targets for cancer.     

Two closely related human members of chitinase-like family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U373 cells but have the different influence on cell proliferation

The activation of extracellular signal-regulated kinases (ERK1/2) has been associated with specific outcomes. Sustained activation of ERK1/2 by nerve growth factor (NGF) is associated with translocation of ERKs to the nucleus of PC12 cells and precedes their differentiation into sympathetic-like neurons whereas transient activation by epidermal growth factor (EGF) leads to cell proliferation. It was demonstrated that different growth factors initiating the same cellular signaling pathways may lead to the different cell destiny, either to proliferation or to the inhibition of mitogenesis and apoptosis. Thus, further investigation on kinetic differences in activation of certain signal cascades in different cell types by biologically different agents are necessary for understanding the mechanisms as to how cells make a choice between proliferation and differentiation.It was reported that chitinase 3-like 1 (CHI3L1) protein promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts similarly to insulin-like growth factor 1 (IGF1). Both are involved in mediating the mitogenic response through the signal-regulated kinases ERK1/2. In addition, CHI3L1 which is highly expressed in different tumors including glioblastomas possesses oncogenic properties. As we found earlier, chitinase 3-like 2 (CHI3L2) most closely related to human CHI3L1 also showed increased expression in glial tumors at both the RNA and protein levels and stimulated the activation of the MAPK pathway through phosphorylation of ERK1/2 in 293 and U87 MG cells. The work described here demonstrates the influence of CHI3L2 and CHI3L1 on the duration of MAPK cellular signaling and phosphorylated ERK1/2 translocation to the nucleus. In contrast to the activation of ERK1/2 phosphorylation by CHI3L1 that leads to a proliferative signal (similar to the EGF effect in PC12 cells), activation of ERK1/2 phosphorylation by CHI3L2 (similar to NGF) inhibits cell mitogenesis and proliferation.     

Homology modeling and consensus protein disorder prediction of human filamin

Filamins are dimeric actin-binding proteins participating in the organization of the actin-based cytoskeleton. Their modular domain organization is made up of an N-terminal actin-binding domain composed of two CH domains followed by flexible rod regions that consist of 24 Ig-like domains. Homology modeling was used to model human filamin using Modeller 9v5. The resulting model assessed by Verify 3D and PROCHECK showed that the final model is reliable. The conformational disorder prediction of human filamin residues were also mapped on the validated structure of human filamin. Prediction of protein disorder in filamin structures will help structural biologists to find suitable targets to be analyzed and for understanding protein function.     

Homology modeling of human 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) based on the crystal structure of rabbit CYP2C5

Seventeen missense mutations of 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) that cause Vitamin D-dependent rickets type I (VDDR-I) have been identified. To understand the mechanism by which each mutation disrupts 1alpha-hydroxylase activity and to visualize the substrate-binding site, we performed the homology modeling of CYP27B1. The three-dimensional (3D) structure of CYP27B1 was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of a eukaryotic CYP. The 3D structure of CYP27B1 contains 17 helices and 6 beta-strands, and the overall structural folding is similar to the available structures of soluble CYPs as well as to the template CYP2C5. Mapping of the residues responsible for VDDR-I has provided much information concerning the function of each mutant. We have previously reported site-directed mutagenesis studies on several mutants of CYP27B1 causing VDDR-1, and suggested the role of each residue. All these suggestions are in good agreement with our 3D-model of CYP27B1. Furthermore, this model enabled us to predict the function of the other mutation residues responsible for VDDR-I.     

Homology modeling of breast cancer resistance protein (ABCG2)

BCRP (also known as ABCG2, MXR, and ABC-P) is a member of the ABC family that transports a wide variety of substrates. BCRP is known to play a key role as a xenobiotic transporter. Since discovering its role in multidrug resistance, considerable efforts have been made in order to gain deeper understanding of BCRP structure and function. The recent study was aimed at predicting BCRP structure by creating a homology model. Based on sequence similarity with known structures of full-length, NB and TM domain of ABC transporters, TM, NB, and linker regions of BCRP were defined. The NB domain of BCRP was modeled using MalK as a template. Based on secondary structure prediction of BCRP and comparison of the transmembrane connecting regions of known structures of ABC transporters, the TM domain arrangement of BCRP was established and was found to resemble to that of the recently published crystal structure of Sav1866. Thus, an initial alignment of TM domain of BCRP was established using Sav1866 as a template. This alignment was subsequently refined using constrains derived from secondary structure and TM predictions and the final model was built. Finally, the complete homodimer ABCG2 model was generated using Sav1866 as template. Furthermore, known ligands of BCRP were docked to our model in order to define possible binding sites. The results of molecular dockings of known BCRP substrates to the BCRP model were in agreement with recently published experimental data indicating multiple binding sites in BCRP.     

Homology modeling of the Abl-SH3 domain

A tertiary structure model of the Abl-SH3 domain is predicted by using homology modeling techniques coupled to molecular dynamics simulations. Two template proteins were used, Fyn-SH3 and Spc-SH3. The refined model was extensively checked for errors using criteria based on stereochemistry, packing, solvation free-energy, accessible surface areas, and contact analyses. The different checking methods do not totally agree, as each one evaluates a different characteristic of protein structures. Several zones of the protein are more susceptible to incorporating errors. These include residues 13, 15, 35, 39, 45, 46, 50, and 60. An interesting finding is that the measurement of the Cα chirality correlated well with the rest of the criteria, suggesting that this parameter might be a good indicator of correct local conformation. Deviations of more than 4 degrees may be indicative of poor local structure. © 1994 Wiley-Liss, Inc.     

Determining the 3D structure of human ASC2 protein involved in apoptosis and inflammation

ASC2 structure has been well defined by 1141 NOE experimental restraints. The model consists of five alpha helices. alpha-Helices are connected by short random structure loops. The sole exception is the loop connecting helices 2 and 3, which has a 20-residue length. Folding generally agrees with the folding of recently published death domain structures in which alpha-helix structures have been reported. In spite of structural similarity, amino acid sequence homology with the most similar protein (ASC1) is just 64%. DD, DED, and CASP protein structures present six helices along their sequences; ASC2 presents 5 well-defined helices due to long distance restraints. However, a helical fragment was observed between amino acids 38 and 42 (representing helix 3) in the death domains when constructing the model.     

Homology modeling and structural analysis of human P-glycoprotein

Homology modeling and structural analysis of human P-glycoprotein (hP-gp) were performed with a software package the Molecular Operating Environment (MOE). A mouse P-gp (mP-gp; PDB code: 3G5U) was selected as a template for the 3D structure modeling of hP-gp. The modeled hP-gp showed significant 3D similarities at the drug biding site (DBS) to the mP-gp structure. The contact energy profiles of the hP-gp model were in good agreement with those of the mP-gp structure. Ramachandran plots revealed that only 3.5% of the amino acid residues were in the disfavored region for hP-gp. Further, docking simulations between 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) and the P-gp models revealed the similarity of the ligand-receptor bound location between the hP-gp and mP-gp models. These results indicate that the hP-gp model was successfully modeled and analyzed. To the best of our knowledge, this is the first report of a hP-gp model with a naturally occurring isothiocyanate, and our data verify that the model can be utilized for application to target hP-gp for the development of antitumor drugs.

Abbreviations

ABC - ATP-binding cassette, ASE-Dock - alpha sphere and excluded volume-based ligand-protein docking, DBS - drug biding site, MDR - multidrug resistance, MOE - Molecular Operating Environment, ITC - isothiocyanate, P-gp - P-glycoprotein.     

Homology modeling and substrate binding study of human CYP2C9 enzyme

The CYP2C subfamily of human liver P450 isozymes is of major importance in drug metabolism. The most abundant 2C isozyme, CYP2C9, regioselectively hydroxylates a wide variety of substrates. A major obstacle to understanding this specificity in human CYP2C9 is the absence of a 3D structure. A 3D model of CYP2C9 was built, assessed, and used to characterize explicit enzyme-substrate complexes using methods previously developed in our laboratory. The 3D model was assessed by determining its stability to unconstrained molecular dynamics and by comparison of specific properties with those of known protein structures. The CYP2C9 model was then used to characterize explicit enzyme complexes with three structurally and chemically diverse substrates: (S)-naproxen, phenytoin, and progesterone. Each substrate was found to bind to the enzyme with a favorable interaction energy and to remain in the binding site during unconstrained molecular dynamics. Moreover, the mode of binding of each substrate led to calculated preferred hydroxylation sites consistent with experiment. Binding-site residues identified for the models included Arg 105 and Arg97 as key cationic residues, as well as Asn 202, Asp 293, Pro 101, Leu 102, Gly 296, and Phe 476. Site-specific mutations are proposed for further integrated computational and experimental study.     

Homology modeling and molecular dynamics simulations of transmembrane domain structure of human neuronal nicotinic acetylcholine receptor

A three-dimensional model of the transmembrane domain of a neuronal-type nicotinic acetylcholine receptor (nAChR), (alpha4)2(beta2)3, was constructed from a homology structure of the muscle-type nAChR recently determined by cryo-electron microscopy. The neuronal channel model was embedded in a fully hydrated DMPC lipid bilayer, and molecular-dynamics simulations were performed for 5 ns. A comparative analysis of the neuronal- versus muscle-type nAChR models revealed many conserved pore-lining residues, but an important difference was found near the periplasmic mouth of the pore. A flickering salt-bridge of alpha4-E266 with its adjacent beta2-K260 was observed in the neuronal-type channel during the course of the molecular-dynamics simulations. The narrowest region, with a pore radius of approximately 2 A formed by the salt-bridges, does not seem to be the restriction site for a continuous water passage. Instead, two hydrophobic rings, formed by alpha4-V259, alpha4-L263, and the homologous residues in the beta2-subunits, act as the gates for water flow, even though the region has a slightly larger pore radius. The model offers new insight into the water transport across the (alpha4)2(beta2)3 nAChR channel, and may lead to a better understanding of the structures, dynamics, and functions of this family of ion channels.     

Insights from modeling the 3D structure of DNA-CBF3b complex

A 3-dimensional model for a DNA-protein interaction has been developed. The protein component is the 61-residue fragment (res. 11-71) of subunit b of the yeast centromeric DNA binding factor 3, CBF3b. The CBF3b fragments bind to the 17 base pairs (5'-CGGAGGACTGTCCTCCG-3') as a symmetric homodimer, with each folded into three distinct conformations: a compact, zinc-binding domain (res. 11-44); an extended linker (res. 45-57); and an alpha-helical dimerization element (res. 58-71). The DNA fragment in the complex is featured by a relatively straight conformation with only slight deviation from a standard B-structure, and a large part of the major groove not blocked by the protein. The large DNA open area provides the necessary space for the other subunits of CBF3 to bind coordinately with CBF3b, fully consistent with the observation that the cooperation of all four CBF3 components is absolutely required to constitute an activity that specifically interacts with centromere DNA. The model also provides a footing for further considering the possible binding arrangements of the other three subunits, namely CBF3a, CBF3c, and CBF3d.     

Secretion of a chitinase-like protein in embryogenic suspension cultures of Dactylis glomerata L.

A chitinase-like 32 kDa acidic protein with a potential chitinase activity has been identified in the medium of embryogenic suspension cultures of Dactylis glomerata L. using an antiserum raised against endochitinase EP3 from Daucus carota L. The presence of this protein discriminates between Dactylis glomerata L. embryogenic and nonembryogenic suspension cultures and thus could be possibly used as a marker for embryogenic potential.     

In silico 3D structure modeling and inhibitor binding studies of human male germ cell-associated kinase

Human male germ cell-associated kinase (hMAK) is an androgen-inducible gene in prostate epithelial cells, and it acts as a coactivator of androgen receptor signaling in prostate cancer. The 3D structure of the hMAK kinase was modeled based on the crystal structure of CDK2 kinase using comparative modeling methods, and the ATP-binding site was characterized. We have collected five inhibitors of hMAK from the literature and docked into the ATP-binding site of the kinase domain. Solvated interaction energies (SIE) of inhibitor binding are calculated from the molecular dynamics simulations trajectories of protein–inhibitor complexes. The contribution from each active site residue in hMAK toward inhibitor binding revealed the nature and extent of interactions between inhibitors and individual residues. The main chain atoms of Met79 invariably form hydrogen bonds with all five inhibitors. The amino acids Leu7, Val15, and Leu129 stabilize the inhibitors via CH–pi interactions. The Asp140 in the active site and Glu77 in hinge region show characteristic hydrogen bonding interactions with inhibitors. From SIE, the residue-wise interactions revealed the nature of non-bonding contacts and modifications required to increase the inhibitor activity. Our work provides 3D model structure of hMAK and molecular basis for the mechanisms of hMAK inhibition at atomic level that aid in designing new potent inhibitors.     

Macromolecular structure modeling from 3D EM using VolRover 2.0

We review tools for structure identification and model-based refinement from three-dimensional electron microscopy implemented in our in-house software package, VOLROVER 2.0. For viral density maps with icosahedral symmetry, we segment the capsid, polymeric, and monomeric subunits using techniques based on automatic symmetry detection and multidomain fast marching. For large biomolecules without symmetry information, we again use our multidomain fast-marching method with manual or fit-based multiseeding to segment meaningful substructures. In either case, we subject the resulting segmented subunit to secondary structure detection when the EM resolution is sufficiently high, and rigid-body structure fitting when the corresponding X-ray structure is available. Secondary structure elements are identified by three techniques: our earlier volume-based and boundary-based skeletonization methods as well as a new method, currently in development, based on solving the grassfire flow equation. For rigid-body fitting, we adapt our earlier fast Fourier-based correlation scheme F2Dock. Our reported segmentation, secondary structure elements identification, and rigid-body fitting techniques, implemented in VOLROVER 2.0 are applied to the PSB 2011 cryo-EM modeling challenge data, and our results are briefly compared to similar results submitted from other research groups. The comparisons show that our techniques are equally capable of segmenting relatively accurate subunits from a viral or protein assembly, and that high segmentation quality leads in turn to higher-quality results of secondary structure elements identification and correlation-based rigid-body fitting. ? 2012 Wiley Periodicals, Inc. Biopolymers 97: 709-731, 2012.     

Motif3D: Relating protein sequence motifs to 3D structure

Motif3D is a web-based protein structure viewer designed to allow sequence motifs, and in particular those contained in the fingerprints of the PRINTS database, to be visualised on three-dimensional (3D) structures. Additional functionality is provided for the rhodopsin-like G protein-coupled receptors, enabling fingerprint motifs of any of the receptors in this family to be mapped onto the single structure available, that of bovine rhodopsin. Motif3D can be used via the web interface available at: http://www.bioinf.man.ac.uk/dbbrowser/motif3d/motif3d.html.     

Homology modeling of nematode Caenorhabditis elegans CED3 protein-inhibitor complex

CED3 protein, the product of a gene necessary for programmed cell death in the nematode Caenorhabditis elegans, is related to a highly specific cysteine protease family i.e., caspases. A tertiary-structural model has been constructed of a complex of the CED3 protein with tetrapeptide-aldehyde inhibitor, Ac-DEVD-CHO. The conformation of CED3 protein active site and the general binding features of inhibitor residues are similar to those observed in other caspases. The loop segment (Phe380-Pro387) binds with the P4 Asp in a different fashion compared to caspase-3. The comparative modeling of active sites from caspase-3 and CED3 protein indicated that although these enzymes require Asp at the position P4, variation could occur in the binding of this residue at the S4 subsite. This model allowed the definition of substrate specificity of CED3 protein from the structural standpoint and provided insight in designing of mutants for structure-function studies of this classical caspase homologue.     

节杆菌K1108乙内酰脲酶三维结构的模建和分析

利用同源模建技术,以节杆菌DSM3745乙内酰脲酶的晶体结构为模板,模建了节杆菌K1108乙内酰脲酶的三维结构。模建的节杆菌K1108乙内酰脲酶结构由一个中心的(α/β)g捅状结构域和富含β-折叠的结构域两个区域组成,富含β-折叠的结构域在中心(α/β)g捅状结构域的侧面,由分子的N端和C端组成。根据K1108乙内酰脲酶和其它酶在结构和活性部位的保守性,确定了K1108乙内酰脲酶的底物结合部位,并对酶的活性中心的特征进行了分析,对L-Hyd的底物选择性进行了解释。     

I-TASSER server for protein 3D structure prediction

Background  

Prediction of 3-dimensional protein structures from amino acid sequences represents one of the most important problems in computational structural biology. The community-wide Critical Assessment of Structure Prediction (CASP) experiments have been designed to obtain an objective assessment of the state-of-the-art of the field, where I-TASSER was ranked as the best method in the server section of the recent 7th CASP experiment. Our laboratory has since then received numerous requests about the public availability of the I-TASSER algorithm and the usage of the I-TASSER predictions.     

米根霉富马酸酶三维结构的同源建模与分析

利用Modeller7v7软件对米根霉（Rhizopus oryzoe）富马酸酶（fumarase）进行了三级结构的同源建模并对结果的空间和能量上的合理性进行了验证,进一步对酶的结构域和催化活性位点进行了研究。结果表明：富马酸酶由三个结构域组成,中心区域为一个由五个几乎平行的α螺旋组成的独特的束型结构,其催化活性位点是由三个亚基上的氨基酸相互靠近共同组成的。为以后有针对性的进行富马酸酶的定点突变提高富马酸产量提供分子水平上的理论指导。