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This article reviews the docking field starting from basic docking algorithms and describing the latest advances. We present the algorithmic framework and classify the state of-the-art methods. We point out the bottlenecks of the methods, like flexibility, absence of absolute scoring functions and explain what types of information can potentially be added to improve the results.
The TCR recognizes its peptide:MHC (pMHC) ligand by assuming a diagonal orientation relative to the MHC helices, but it is unclear whether and to what degree individual TCRs exhibit docking variations when contacting similar pMHC complexes. We analyzed monospecific and cross-reactive recognition by diverse TCRs of an immunodominant HVH-1 glycoprotein B epitope (HSV-8p) bound to two closely related MHC class I molecules, H-2K(b) and H-2K(bm8). Previous studies indicated that the pMHC portion likely to vary in conformation between the two complexes resided at the N-terminal part of the complex, adjacent to peptide residues 2-4 and the neighboring MHC side chains. We found that CTL clones sharing TCR beta-chains exhibited disparate recognition patterns, whereas those with drastically different TCRbeta-chains but sharing identical TCRalpha CDR3 loops displayed identical functional specificity. This suggested that the CDRalpha3 loop determines the TCR specificity in our model, the conclusion supported by modeling of the TCR over the actual HSV-8:K(b) crystal structure. Importantly, these results indicate a remarkable conservation in CDRalpha3 positioning, and, therefore, in docking of diverse TCRalphabeta heterodimers onto variant peptide:class I complexes, implying a high degree of determinism in thymic selection and T cell activation.
The functions of centrosomal protein ninein may be involved in microtubule minus end capping, centriole positioning, protein anchoring and microtubule nucleation, but the true physiological function of various human hNinein isoforms remains to be determined. Here we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. These hNinein isoforms exhibit centrosomal (concentrated) and noncentrosomal (aggregated) localization when GFP-tagged fusion proteins are expressed transiently in mammalian cells. In a kinase assay, we show that the CCII region of hNinein provides a differential phosphorylation site by GSK3beta. In addition, our data indicate that either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits gamma-tubulin to centrosomal or noncentrosomal hNinein-containing sites, implying that the gamma-tubulin localization may be hNinein-dependent. Further, our RNA interference experiment against all hNinein isoforms caused a significant decrease in the gamma-tubulin signal in the centrosome. In domain swapping, we clearly show that the CCIIX-CCIIY region provides docking sites for gamma-tubulin. Moreover, our data also show that nucleation of microtubules from the centrosome is significantly affected by the presence of either the full -length hNinein or CCIIX-CCIIY region overexpression. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit gamma-tubulin at centrosomal and noncentrosomal sites.
Water is known to play a significant role in the formation of protein-ligand complexes. In this paper, we focus on the influence of water molecules on the structure of protein-ligand complexes. We present an algorithmic approach, called the particle concept, for integrating the placement of single water molecules in the docking algorithm of FLEXX. FLEXX is an incremental construction approach to ligand docking consisting of three phases: the selection of base fragments, the placement of the base fragments, and the incremental reconstruction of the ligand inside the active site of a protein. The goal of the extension is to find water molecules at favorable places in the protein-ligand interface which may guide the placement of the ligand. In a preprocessing phase, favorable positions of water molecules inside the active site are calculated and stored in a list of possible water positions. During the incremental construction phase, water molecules are placed at the precomputed positions if they can form additional hydrogen bonds to the ligand. Steric constraints resulting from the water molecules as well as the geometry of the hydrogen bonds are used to optimize the ligand orientation in the active site during the reconstruction process. We have tested the particle concept on a series of 200 protein-ligand complexes. Although the average improvement of the prediction results is minor, we were able to predict water molecules between the protein and the ligand correctly in several cases. For instance in the case of HIV-1 protease, where a single water molecule between the protein and the ligand is known to be of importance in complex formation, significant improvements can be achieved.
Brain-derived neurotrophic factor (BDNF) is emerging as a key mediator of activity-dependent modifications of synaptic strength in the CNS. We investigated the hypothesis that BDNF enhances quantal neurotransmitter release by modulating the distribution of synaptic vesicles within presynaptic terminals using organotypic slice cultures of postnatal rat hippocampus. BDNF specifically increased the number of docked vesicles at the active zone of excitatory synapses on CA1 dendritic spines, with only a small increase in active zone size. In agreement with the hypothesis that an increased docked vesicle density enhances quantal neurotransmitter release, BDNF increased the frequency, but not the amplitude, of AMPA receptor-mediated miniature EPSCs (mEPSCs) recorded from CA1 pyramidal neurons in hippocampal slices. Synapse number, independently estimated from dendritic spine density and electron microscopy measurements, was also increased after BDNF treatment, indicating that the actions of BNDF on mEPSC frequency can be partially attributed to an increased synaptic density. Our results further suggest that all these actions were mediated via tyrosine kinase B (TrkB) receptor activation, established by inhibition of plasma membrane tyrosine kinases with K-252a. These results provide additional evidence of a fundamental role of the BDNF-TrkB signaling cascade in synaptic transmission, as well as in cellular models of hippocampus-dependent learning and memory.
We present a new method for representing the binding site of a protein receptor that allows the use of the DOCK approach to screen large ensembles of receptor conformations for ligand binding. The site points are constructed from templates of what we called "attached points" (ATPTS). Each template (one for each type of amino acid) is composed of a set of representative points that are attached to side-chain and backbone atoms through internal coordinates, carry chemical information about their parent atoms and are intended to cover positions that might be occupied by ligand atoms when complexed to the protein. This method is completely automatic and proved to be extremely fast. With the aim of obtaining an experimental basis for this approach, the Protein Data Bank was searched for proteins in complex with small molecules, to study the geometry of the interactions between the different types of protein residues and the different types of ligand atoms. As a result, well-defined patterns of interaction were obtained for most amino acids. These patterns were then used for constructing a set of templates of attached points, which constitute the core of the ATPTS approach. The quality of the ATPTS representation was demonstrated by using this method, in combination with the DOCK matching and orientation algorithms, to generate correct ligand orientations for >1000 protein--ligand complexes.
The interleukin-2 receptor alpha (IL-2Ralpha) forms, together with IL-2Rbeta and gammac chains, a high affinity IL-2 receptor that is important for IL-2 responsiveness and normal T cell function. Expression of the IL-2Ralpha gene by T cells is regulated mainly at the transcription level which is transiently activated by antigen and upregulated and then prolonged by stimulation with IL-2. The effect of IL-2 on the IL-2Ralpha gene depends on the activation of the transcription factor Stat5, which acts on an IL-2- responsive enhancer that consists of two Stat5 and an Elf1 binding site. To identify the functional domains of the IL-2 receptor required for the stimulation of IL-2Ralpha gene expression, we introduced, into the CTL44 T cell line, receptor chimeras between the extracellular domain of the IL-9 receptor and the cytoplasmic region of IL-2Rbeta. Analyzing the effect of mutations in the intracellular IL-2Rbeta segment, we found that a minimal receptor containing the Jak boxes and one intact Stat5 docking site (i.e. tyrosine 392 or 510) can, as expected, mediate Stat5 activation, but is unable to stimulate IL-2Ralpha expression. However, when this minimal receptor includes the region between the two tyrosines, its capacity to mediate IL-2Ralpha cell surface expression is restored. These data suggest that the segment between the two Stat5 docking sites of the IL-2Rbeta chain mediates signaling events that, together with Stat5 activation, are essential for the stimulation of IL-2Ralpha gene transcription.
Gamma-secretase cleavage is the final enzymatic step generating beta-amyloid via intramembranous cleavage of the amyloid precursor protein (APP). Presenilin (PS), initially identified as a gene in which mutations account for the vast majority of early-onset autosomal dominant Alzheimer's disease, is a major component of gamma-secretase. Enzymatic activity also depends on nicastrin, Aph-1, and Pen-2. We propose a model in which gamma-secretase components assemble, interact with substrates initially at a docking site, and then cleave and release substrates. To test this model, we developed a novel morphological technique on the basis of advanced fluorescence microscopy methods, fluorescence lifetime imaging microscopy (FLIM). FLIM allows us to examine protein-protein "proximity" in intact cells. We show that, although the strongest colocalization of APP and PS1 is in the perinuclear area, the strongest interactions detected by FLIM are at or near the cell surface. We also found that APP-PS1 interactions occur even when gamma-secretase inhibitors or "dominant-negative" PS1 mutations are used to block gamma-secretase activity. Finally, using nicastrin RNA interference, we demonstrate that nicastrin is critical for APP association with PS1. We interpret these results to suggest that there is a noncatalytic docking site closely associated with PS1-gamma-secretase.
Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed.
AIM: To understand pharmacophore properties of styrylquinoline derivatives and to design inhibitors of HIV-1 integrase. METHODS: Comparative molecular field analysis (CoMFA) was performed to analyze three-dimensional quantitative structure-activity relationship (3D-QSAR) of styrylquinoline derivatives. Thirty-eight compounds were randomly divided into a training set of 28 compounds and a test set of 10 compounds. The stability of 3D-QSAR models was proved by the analysis of cross-validated and non-cross-validated methods. Moreover, the binding mode of these compounds and integrase was constructed by AutoDock program. RESULTS: The CoMFA model of the training compounds was reasonably predicted with cross-validated coefficient (q2) and conventional (r2) values (up to 0.696 and 0.754). Then the model was validated by the test set. The resulting CoMFA maps visualized structural requirements for the biological activity of these inhibitors. Docking results showed that a carboxyl group at C-7 and a hydroxyl group at C-8 in the quinoline subunit, bound closely to the divalent metal cofactor (Mg2+) around the integrase catalytic site. Moreover, there is a linear correlation between the binding energy of the inhibitors with integrase and their inhibitory effect. CONCLUSIONS: The present study indicated that the CoMFA model together with docking results could give us helpful hints for drug design as well as interpretation of the binding affinity between these inhibitors and integrase.
UNLABELLED: Direct radiolabeling of proteins can result in the loss of targeting activity, requires highly customized procedures, and yields heterogeneous products. Here we describe a novel imaging complex comprised of a standardized (99m)Tc-radiolabeled adapter protein noncovalently bound to a "Docking tag" fused to a "Targeting protein". The assembly of this complex is based on interactions between human 109-amino acid (HuS) and 15-amino acid (Hu-tag) fragments of ribonuclease I, which serve as an "Adapter protein" and a Docking tag, respectively. METHODS: HuS modified with hydrazinonicotinamide (HYNIC) was radiolabeled using (99m)Tc-tricine to a specific activity of 3.4-7.4 MBq/microg. Protein complexes were then formed by mixing (99m)Tc-HuS with equimolar amounts of either Hu-tagged VEGF(121) (Hu-VEGF [vascular endothelial growth factor]) or Hu-tagged anti-VEGFR-2 single-chain antibody (Hu-P4G7) and incubating on ice for 15 min. 4T1 luc/gfp luciferase-expressing murine mammary adenocarcinoma cells (1 x 10(4)) were implanted subcutaneously or injected intravenously into BALB/c mice. Bioluminescent imaging (BLI) was performed 10 d later. Immediately after BLI visualization of tumor, 18.5-37 MBq of tracer (5-10 microg of protein) were injected via tail vein. One hour later planar or SPECT images were obtained, followed by killing the mice. RESULTS: There was significantly (P = 0.0128) increased uptake of (99m)Tc-HuS/Hu-VEGF (n = 10) within subcutaneous tumor as compared with (99m)Tc-HuS/Hu-P4G7 (n = 5) at biodistribution assay (2.68 +/- 0.75 vs. 1.8 +/- 0.21; tumor-to-subcutaneous tissue [ratio of specific activities], respectively), despite similar molecular weights. The focal (99m)Tc-HuS/Hu-VEGF uptake seen on planar images (3.44 +/- 1.16 [tumor to soft-tissue background]) corresponded directly to the locations of tumor observed by BLI. Region of interest analyses of SPECT images revealed a significant increase of (99m)Tc-HuS/Hu-VEGF (n = 5) within the lungs with BLI-detectable pulmonary tumor nodules as compared with controls (n = 4) (right: 4.47 +/- 2.07 vs. 1.79 +/- 0.56; left: 3.66 +/- 1.65 vs. 1.62 +/- 0.45, tumor lung [counts/pixel]/normal lung [counts/pixel], respectively). CONCLUSION: (99m)Tc-HuS/Hu-VEGF complex is stable for at least 1 h in vivo and can be effectively used to image mouse tumor neovasculature in lesions as small as several millimeters in soft tissue. We expect that a similar approach can be adapted for in vivo delivery of other targeting proteins of interest without affecting their bioactivity.
Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y1-Y5 and y6. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.
Protein kinases represent a major class of drug targets for the pharmaceutical industry, and the identification of kinase inhibitors with novel and diverse chemotypes is therefore a high priority. Virtual screening methods are a primary source for the discovery of lead molecules for drug development, with high-throughput docking algorithms being among the most extensively used of these methods. A number of developments in virtual screening technologies have resulted in more effective computer-based compound screening of potential kinase inhibitors. These developments include the following: (i) conformational search methods for pose generation; (ii) improvements in the prediction of protein-ligand binding energy through the use of scoring functions; (iii) the use of interaction filters for identifying ligand poses with known binding determinants; and (iv) the impact of binding site flexibility upon high-throughput docking success. This review discusses the application of these methods in the context of the discovery of kinase adenosine triphosphate antagonists.
Cortisol responses of Dorset and Scottish Blackface lambs were investigated at six ages between birth and seven days. There were four treatments: control handling and blood sampling (n = 52), tail docking (T) (n = 57), castration plus tail docking (CT) (n = 54), and intravenous adrenocorticotrophin (ACTH) injection (n = 60). Most lambs exhibited transient rises in plasma cortisol concentrations after treatment and the results in individuals were expressed as the area under the cortisol curve (integrated response). Postnatal changes in the integrated responses of control, T and CT lambs differed significantly between the two breeds; in Dorsets they increased markedly between four hours and one day and then decreased, whereas in Scottish Blackface lambs there were no significant changes. The cortisol responses of ACTH lambs decreased markedly between four hours and seven days in both breeds. The results shed light on postnatal changes in the activity of the hypothalamic-pituitary-adrenal axis and suggest for both breeds that the average increments in ACTH secretion caused by noxious stimuli increased markedly during the first one to three days after birth. Whether this reflected parallel increases in the levels of distress experienced by the lambs remains to be clarified.
We present an algorithm for placing flexible molecules in active sites of proteins. The two major goals in the development of our docking program, called FLEXX, are the explicit exploitation of molecular flexibility of the ligand and the development of a model of the docking process that includes the physico-chemical properties of the molecules. The algorithm consists of three phases: The selection of a base fragment, the placement of the base fragment in the active site, and the incremental construction of the ligand inside the active site. Except for the selection of the base fragment, the algorithm runs without manual intervention. The algorithm is tested by reproducing 11 receptor-ligand complexes known from X-ray crystallography. In all cases, the algorithm predicts a placement of the ligand which is similar to the crystal structure (about 1.5 A RMS deviation or less) in a few minutes on a workstation, assuming that the receptor is given in the bound conformation.
Personal monitoring of noise exposure was conducted in a small dry dock, where mainly tug-boats and high sea fishing-boats are refitted. Manual tools were the prevalent noise sources. Noise exposure could be affected and amplified by factors extraneous to workers' tasks. The workers might be employed in different jobs simultaneously also in confined and small areas, and were mainly involved in non-routine tasks. Six different tasks were investigated: dry-dock supervisor, welder/ship-wrights, painters, electricians, woodworkers and unskilled workers. The number of workers employed in the tasks, with the exclusion of the supervisor, might change from day to day according to the particular work phase carried out on the boat. Daily personal exposure was measured using Larson Davis mod. 700 integrating sound level meters. The all shift time history of short equivalent continuous sound level with 10 s integrating time was collected for each selected worker. A 1/2 inch microphone was attached to the helmet at a distance of 11 +/- 1 cm from the right outer ear. Measurements were repeated over 13 days and randomly distributed over about four months. The number of data-points collected was nine for the supervisor, twenty-seven for the welders, eighteen for the painters, twelve for the electricians, six for the woodworkers and eleven for the unskilled workers. Ranges of the daily noise exposure level were from 15 to 30 dBA for all tasks, with the exclusion of the woodworkers. The time histories relative to workers grouped in the same task were matched to verify the correlation. The daily exposure levels derived from correlated samples were substituted by their arithmetic mean. The analysis of variance applied to experimental distributions of daily exposure for four of the welders indicates that the difference of means (p > 0.3) is not significant. A similar conclusion is reached for painters when only this task is considered. If we take into account the exposures of painters employed also in different tasks, the probability is included in a range of lower significance, between 0.1 and 0.05. The experimental distributions of daily noise exposure for all tasks fit the normal distribution. The Kolmogorov-Smirnov test was used at a p > 0.1 confidence level. These results may have an important role in defining sampling strategies for collecting noise exposure data both for compliance and epidemiological purposes.
The automated stochastic docking procedure BioDock has been applied to a series of inhibitors of PGH synthase, the key enzyme in the synthesis of eicosanoids from arachidonic acid. Some PGHS-2 selective inhibitors have been docked to the structure of the ovine PGHS-1 enzyme, as recently obtained by means of X-ray crystallographic analysis, in order to highlight possible structural bases for selectivity.
IL-4 is a cytokine with important antiapoptotic activity. We have analyzed the role that tyrosine-containing domains within the cytoplasmic tail of IL-4R alpha play in IL-4-mediated protection from apoptosis. 32D cells expressing a wt huIL-4R alpha or one truncated at aa 557 were protected by huIL-4 from apoptosis while cells expressing a receptor truncated at aa 657 were not, suggesting that the carboxyl-terminal domain signals protection from apoptosis. However, changing Y713 within this region to phenylalanine had no effect. To analyze the contribution of tyrosine-containing domains independently, we transplanted regions of the huIL-4R alpha to a truncated form of the huIL-2R beta that could not signal protection from apoptosis. Transplantation of the huIL-4R alpha domains containing Y497 or Y713 partially prevented cell death and together signaled protection from apoptosis in response to IL-2 as well as the wt IL-2R beta. Mutation of Y497 and Y713 to phenylalanine inhibited protection. In contrast, transplantation of the domain containing the potential STAT6-docking tyrosines alone had no effect, yet it inhibited the protection mediated by the other domains. Although IL-4R alpha signals Shc and SH2-containing inositol phosphatase (SHIP) phosphorylation, we could not establish an association between their activation and protection from apoptosis. Taken together, this study suggests that the domains of the huIL-4R alpha containing Y497 and Y713 positively regulate protection from apoptosis while the domain containing the STAT6 docking sites suppresses this protection, and that additional signaling molecules other than insulin receptor substrate-1 (IRS1), Shc, or SHIP may be involved in antiapoptotic signaling.