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At least 613 records · Page 34Linked to original sources
PubMed1999

Identification of an extracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, a sequence critical for activation by ERK in vivo.

Glutathione S-transferase (GST)-fusion proteins containing the carboxyl-terminal tails of three p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, and RSK3) interacted with extracellular signal-regulated kinase (ERK) but not c-Jun-NH2-kinase (JNK) or p38 mitogen-activated protein kinase (MAPK). Within the carboxyl-terminal residues of the RSK isozymes is a region of high conservation corresponding to residues 722LAQRRVRKLPSTTL735 in RSK1. Truncation of the carboxyl-terminal 9 residues, 727VRKLPSTTL735, completely eliminated the interaction of the GST-RSK1 fusion protein with purified recombinant ERK2, whereas the truncation of residues 731PSTTL735 had no effect on the interaction with purified ERK2. ERK1 and ERK2 co-immunoprecipitated with hemagglutinin-tagged wild type RSK2 (HA-RSK2) in BHK cell cytosol. However, ERK did not co-immunoprecipitate with HA-RSK2((1-729)), a mutant missing the carboxyl-terminal 11 amino acids, similar to the minimal truncation that eliminated in vitro interaction of ERK with the GST-RSK1 fusion protein. Kinase activity of HA-RSK2 increased 6-fold in response to insulin. HA-RSK2((1-729)) had a similar basal kinase activity to that of HA-RSK2 but was not affected by insulin treatment. Immunoprecipitated HA-RSK2 and HA-RSK2((1-729)) could be activated to the same extent in vitro by active ERK2, demonstrating that HA-RSK2((1-729)) was properly folded. These data suggest that the conserved region of the RSK isozymes (722LAQRRVRKL730 of RSK1) provides for a specific ERK docking site approximately 150 amino acids carboxyl-terminal to the nearest identified ERK phosphorylation site (Thr573). Complex formation between RSK and ERK is essential for the activation of RSK by ERK in vivo. Comparison of the docking site of RSK with the carboxyl-terminal tails of other MAPK-activated kinases reveals putative docking sites within each of these MAPK-targeted kinases. The number and placement of lysine and arginine residues within the conserved region correlate with specificity for activation by ERK and p38 MAPKs in vivo.

Amino Acid Sequence↗
PubMed2004

The cytokine interleukin-1beta reduces the docking and fusion of insulin granules in pancreatic beta-cells, preferentially decreasing the first phase of exocytosis.

The prediabetic period in type I diabetes mellitus is characterized by the loss of first phase insulin release. This might be due to islet infiltration mediated by mononuclear cells and local release of cytokines, but the mechanisms involved are unknown. To determine the role of cytokines in insulin exocytosis, we have presently utilized total internal reflection fluorescence microscopy (TIRFM) to image and analyze the dynamic motion of single insulin secretory granules near the plasma membrane in live beta-cells exposed for 24 h to interleukin (IL)-1beta or interferon (IFN)-gamma. Immunohistochemistry observed via TIRFM showed that the number of docked insulin granules was decreased by 60% in beta-cells treated with IL-1beta, while it was not affected by exposure to IFN-gamma. This effect of IL-1beta was paralleled by a 50% reduction in the mRNA and the number of clusters of SNAP-25 in the plasma membrane. TIRF images of single insulin granule motion during a 15-min stimulation by 22 mm glucose in IL-1beta-treated beta-cells showed a marked reduction in the fusion events from previously docked granules during the first phase insulin release. Fusion from newcomers, however, was well preserved during the second phase of insulin release of IL-1beta-treated beta-cells. The present observations indicate that IL-1beta, but not IFN-gamma, has a preferential inhibitory effect on the first phase of glucose-induced insulin release, mostly via an action on previously docked granules. This suggests that beta-cell exposure to immune mediators during the course of insulitis might be responsible for the loss of first phase insulin release.

Animals↗
PubMed2000

Identification and analysis of the acyl carrier protein (ACP) docking site on beta-ketoacyl-ACP synthase III.

The molecular details that govern the specific interactions between acyl carrier protein (ACP) and the enzymes of fatty acid biosynthesis are unknown. We investigated the mechanism of ACP-protein interactions using a computational analysis to dock the NMR structure of ACP with the crystal structure of beta-ketoacyl-ACP synthase III (FabH) and experimentally tested the model by the biochemical analysis of FabH mutants. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The ACP interaction surface was defined by mutations that compromised FabH activity in the ACP-dependent assay but had no effect in the ACP-independent assay. ACP docked to a positively charged/hydrophobic patch adjacent to the active site tunnel on FabH, which included a conserved arginine (Arg-249) that was required for ACP docking. Kinetic analysis and direct binding studies between FabH and ACP confirmed the identification of Arg-249 as critical for FabH-ACP interaction. Our experiments reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of the fatty acid biosynthesis enzymes and the high degree of sequence conservation in helix II of ACP across species.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2003

Site of docking and fusion of insulin secretory granules in live MIN6 beta cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy.

To determine the site of insulin exocytosis in the pancreatic beta cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 beta cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomal-associated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mm KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl-beta-cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 beta cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.

Animals↗
PubMed2003

Docking interactions in the c-Jun N-terminal kinase pathway.

The c-Jun N-terminal kinase (JNK) signaling pathway is a major mediator of stress responses in cells. Similar to other mitogen-activated protein kinases (MAPKs), JNK activity is controlled by a cascade of protein kinases and by protein phosphatases, including dual-specificity MAPK phosphatases. Components of the JNK pathway associate with scaffold proteins that modulate their activities and cellular localization. The JNK-interacting protein-1 (JIP-1) scaffold protein specifically binds JNK, MAPK kinase 7 (MKK7), and members of the mixed lineage kinase (MLK) family, and regulates JNK activation in neurons. In this study we demonstrate that distinct regions within the N termini of MKK7 and the MLK family member dual leucine zipper kinase (DLK) mediate their binding to JIP-1. We have also identified amino acids in JNK required for: (a) binding to JIP-1 and for JIP-1-mediated JNK activation, (b) docking to MAPK kinase 4 (MKK4) and efficient phosphorylation by MKK4, and (c) docking to its substrate c-Jun and efficient c-Jun phosphorylation. None of the amino acids identified were essential for JNK docking to MKK7 or the dual-specificity phosphatase MAPK phosphatase 7 (MKP7). These findings uncover molecular determinants of JIP-1 scaffold complex assembly and demonstrate that there are overlapping, but also distinct, binding determinants within JNK that mediate interactions with scaffold proteins, activators, phosphatases, and substrates.

Amino Acid Sequence↗
PubMed2005

The C2B domain of rabphilin directly interacts with SNAP-25 and regulates the docking step of dense core vesicle exocytosis in PC12 cells.

Rabphilin is a membrane trafficking protein on secretory vesicles that consists of an N-terminal Rab-binding domain and C-terminal tandem C2 domains. The N-terminal part of rabphilin has recently been shown to function as an effector domain for both Rab27A and Rab3A in PC12 cells (Fukuda, M., Kanno, E., and Yamamoto, A. (2004) J. Biol. Chem. 279, 13065-13075), but the function of the C2 domains of rabphilin during secretory vesicle exocytosis is largely unknown. In this study we investigated the interaction between rabphilin and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors, VAMP-2/synaptobrevin-2, syntaxin IA, and SNAP-25) and SNARE-associated proteins (Munc18-1 and Munc13-1) and found that the C2B domain of rabphilin, but not of other Rab27A-binding proteins with tandem C2 domains (i.e. Slp1-5), directly interacts with a plasma membrane protein, SNAP-25. The interaction between rabphilin and SNAP-25 occurs even in the absence of Ca(2+) (EC(50) = 0.817 microm SNAP-25), but 0.5 mm Ca(2+) increases the affinity for SNAP-25 2-fold (EC(50) = 0.405 microm SNAP-25) without changing the B(max) value (1.06 mol of SNAP-25/mol of rabphilin). Furthermore, vesicle dynamics were imaged by total internal reflection fluorescence microscopy in a single PC12 cell expressing a lumen-targeted pH-insensitive yellow fluorescent protein (Venus), neuropeptide Y-Venus. Expression of the wild-type rabphilin in PC12 cells significantly increased the number of docked vesicles to the plasma membrane without altering the kinetics of individual secretory events, whereas expression of the mutant rabphilin lacking the C2B domain, rabphilin-DeltaC2B, decreased the number of docked vesicle or fusing at the plasma membrane. These findings suggest that rabphilin is involved in the docking step of regulated exocytosis in PC12 cells, possibly through interaction between the C2B domain and SNAP-25.

Animals↗
PubMed2006

Specificity of Prodan for the self-associating domain of spectrin: a molecular docking study.

The hydrophobic fluorescent probe Prodan binds to the self-associating domain of spectrin with 1:1 stoichiometry. A model of the self-associating domain was generated based on its homology with other domains of spectrin. Prodan was then docked onto the model, and several sites with low interaction energy were identified. To verify whether the binding of Prodan is specific towards the self-associating domain of spectrin, it was docked on to several other domains of spectrin, having a known three-dimensional structure. Analysis of the docking results suggests that the binding of Prodan to the self-associating domain of spectrin will involve hydrophobic and hydrophilic groups of Prodan. The results clearly indicate the preference of Prodan for a particular binding site of the self-associating domain.

Amino Acid Sequence↗
PubMed2006

Computer-aided molecular design of highly potent HIV-1 RT inhibitors: 3D QSAR and molecular docking studies of efavirenz derivatives.

Ligand- and structure-based design approaches have been applied to an extended series of 74 efavirenz compounds effectively inhibiting wild type (WT) and mutant type (K103N) HIV-1 reverse transcriptase (RT). For ligand-based approach, three dimensional quantitative structure-activity relationship (3D-QSAR) methods, comparative molecular field analysis (CoMFA) and comparative similarity indices analysis (CoMSIA), were performed. The starting geometry of efavirenz was obtained from X-ray crystallographic data. The efavirenz derivatives were constructed and fully optimized by ab-initio molecular orbital method at HF/3-21G level. Reliable QSAR models for high predictive abilities were developed. Regarding WT and K103N inhibitions, CoMFA models with r2/cv = 0.651 and 0.678 and CoMSIA models with r2/cv = 0.662 and 0.743 were derived, respectively. The interpretation obtained from the models highlights different structural requirements for inhibition of WT and K103N HIV-1 RT. To elucidate potential binding modes of efavirenz derivatives in the binding pocket of WT and K103N HIV-1 RT, structure-based approach based on computational docking studies of selected efavirenz compounds were performed by using GOLD and FlexX programs. The results derived from docking analysis give additional information and further probe the inhibitor-enzyme interactions. The correlation of the results obtained from 3D QSAR and docking models validate each other and lead to better understanding of the structural requirements for the activity. Therefore, these integrated results are informative to provide key features and a helpful guideline for novel compound design active against HIV-1 RT.

Alkynes↗
PubMed2003

Evaluation of indole esters as inhibitors of p60(c-Src) receptor tyrosine kinase and investigation of the inhibition using receptor docking studies.

Several indole esters were tested as inhibitors of tyrosine kinase p60(c-Src). Compound (4) was found fairly active against the enzyme with IC50 = 1.34 microM. DOCK methodology was used to asses our inhibitors for their inhibitory potency against tyrosine kinase. The docking results showed that compounds (4), (25) and (26) were bound to the active site of the enzyme Lys 295 of p60(c-Src) tyrosine kinase. Both activity and docking studies showed a parallel result, with compound (4) having a better interaction with the enzyme active site and also greater activity than the other compounds, indicating a potential role as new lead inhibitor.

Amino Acid Sequence↗
PubMedDate not supplied

Docking ligands to vasopressin and oxytocin receptors via genetic algorithm.

The aim of the study was to computer-dock selected ligands to neurophyseal receptors in order to identify amino acid residues responsible for ligand-receptor interactions. To this aim, reliable oxytocin receptor (OTR) and arginine-vasopressin receptor (V1aR/V2R) models were built. The OTR-selective agonist [Thr4,Gly7]OT, the OTR-selective cyclohexapeptide antagonist L-366,948 and OT itself were docked via genetic algorithm to OTR, V1aR, and V2R and relaxed using a constrained simulated annealing protocol. For the analysis of receptor/ligand interactions a subset of initial conformations was chosen using energetic and steric criteria. All three ligands seem to prefer similar modes of binding to the receptors, manifested by repetitive residues of the receptors which directly interact with the ligands. Taking into account that many aspects of mechanisms of G protein-coupled receptor (GPCR) action are still unsolved, the results obtained with the docking simulations may propose future experimental research, especially in site-directed mutagenesis analysis and searching for key amino acid residues responsible for drug activities.

Algorithms↗
PubMed1999

PEX12 interacts with PEX5 and PEX10 and acts downstream of receptor docking in peroxisomal matrix protein import.

Peroxisomal matrix protein import requires PEX12, an integral peroxisomal membrane protein with a zinc ring domain at its carboxy terminus. Mutations in human PEX12 result in Zellweger syndrome, a lethal neurological disorder, and implicate the zinc ring domain in PEX12 function. Using two-hybrid studies, blot overlay assays, and coimmunoprecipitation experiments, we observed that the zinc-binding domain of PEX12 binds both PEX5, the PTS1 receptor, and PEX10, another integral peroxisomal membrane protein required for peroxisomal matrix protein import. Furthermore, we identified a patient with a missense mutation in the PEX12 zinc-binding domain, S320F, and observed that this mutation reduces the binding of PEX12 to PEX5 and PEX10. Overexpression of either PEX5 or PEX10 can suppress this PEX12 mutation, providing genetic evidence that these interactions are biologically relevant. PEX5 is a predominantly cytoplasmic protein and previous PEX5-binding proteins have been implicated in docking PEX5 to the peroxisome surface. However, we find that loss of PEX12 or PEX10 does not reduce the association of PEX5 with peroxisomes, demonstrating that these peroxins are not required for receptor docking. These and other results lead us to propose that PEX12 and PEX10 play direct roles in peroxisomal matrix protein import downstream of the receptor docking event.

Alleles↗
PubMed2000

The docking stage of yeast vacuole fusion requires the transfer of proteins from a cis-SNARE complex to a Rab/Ypt protein.

The homotypic fusion of yeast vacuoles requires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown. We find that a large multisubunit protein called the Vam2/6p complex is bound to cis-paired SNAP receptors (SNAREs) on isolated vacuoles. This association of the Vam2/6p complex with the cis-SNARE complex is disrupted during priming. The Vam2/6p complex then binds to Ypt7p, a guanosine triphosphate binding protein of the Rab family, to initiate productive contact between vacuoles. Thus, cis-SNARE complexes can contain Rab/Ypt effectors, and these effectors can be mobilized by NSF/Sec18p-driven priming, allowing their direct association with a Rab/Ypt protein to activate docking.

Adaptor Proteins, Vesicular Transport↗
PubMed2006

Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures.

Neisseria meningitidis elicits the formation of membrane protrusions on vascular endothelial cells, enabling its internalization and transcytosis. We provide evidence that this process interferes with the transendothelial migration of leukocytes. Bacteria adhering to endothelial cells actively recruit ezrin, moesin, and ezrin binding adhesion molecules. These molecules no longer accumulate at sites of leukocyte-endothelial contact, preventing the formation of the endothelial docking structures required for proper leukocyte diapedesis. Overexpression of exogenous ezrin or moesin is sufficient to rescue the formation of docking structures on and leukocyte migration through infected endothelial monolayers. Inversely, expression of the dominant-negative NH(2)-terminal domain of ezrin markedly inhibits the formation of docking structures and leukocyte diapedesis through noninfected monolayers. Ezrin and moesin thus appear as pivotal endothelial proteins required for leukocyte diapedesis that are titrated away by N. meningitidis. These results highlight a novel strategy developed by a bacterial pathogen to hamper the host inflammatory response by interfering with leukocyte-endothelial cell interaction.

Cell Adhesion↗
PubMed2005

A docking analysis of the statistical physics of protein-protein recognition.

We describe protein-protein recognition within the frame of the random energy model of statistical physics. We simulate, by docking the component proteins, the process of association of two proteins that form a complex. We obtain the energy spectrum of a set of protein-protein complexes of known three-dimensional structure by performing docking in random orientations and scoring the models thus generated. We use a coarse protein representation where each amino acid residue is replaced by its Voronoï cell, and derive a scoring function by applying the evolutionary learning program ROGER to a set of parameters measured on that representation. Taking the scores of the docking models to be interaction energies, we obtain energy spectra for the complexes and fit them to a Gaussian distribution, from which we derive physical parameters such as a glass transition temperature and a specificity transition temperature.

Biophysics↗
PubMed2005

Combinatorial docking approach for structure prediction of large proteins and multi-molecular assemblies.

Protein folding and protein binding are similar processes. In both, structural units combinatorially associate with each other. In the case of folding, we mostly handle relatively small units, building blocks or domains, that are covalently linked. In the case of multi-molecular binding, the subunits are relatively large and are associated only by non-covalent bonds. Experimentally, the difficulty in the determination of the structures of such large assemblies increases with the complex size and the number of components it contains. Computationally, the prediction of the structures of multi-molecular complexes has largely not been addressed, probably owing to the magnitude of the combinatorial complexity of the problem. Current docking algorithms mostly target prediction of pairwise interactions. Here our goal is to predict the structures of multi-unit associations, whether these are chain-connected as in protein folding, or separate disjoint molecules in the assemblies. We assume that the structures of the single units are known, either through experimental determination or modeling. Our aim is to combinatorially assemble these units to predict their structure. To address this problem we have developed CombDock. CombDock is a combinatorial docking algorithm for the structural units assembly problem. Below, we briefly describe the algorithm and present examples of its various applications to folding and to multi-molecular assemblies. To test the robustness of the algorithm, we use inaccurate models of the structural units, derived either from crystal structures of unbound molecules or from modeling of the target sequences. The algorithm has been able to predict near-native arrangements of the input structural units in almost all of the cases, suggesting that a combinatorial approach can overcome the imperfect shape complementarity caused by the inaccuracy of the models. In addition, we further show that through a combinatorial docking strategy it is possible to enhance the predictions of pairwise interactions involved in a multi-molecular assembly.

Algorithms↗
PubMed1999

Congruent docking of dimeric kinesin and ncd into three-dimensional electron cryomicroscopy maps of microtubule-motor ADP complexes.

We present a new map showing dimeric kinesin bound to microtubules in the presence of ADP that was obtained by electron cryomicroscopy and image reconstruction. The directly bound monomer (first head) shows a different conformation from one in the more tightly bound empty state. This change in the first head is amplified as a movement of the second (tethered) head, which tilts upward. The atomic coordinates of kinesin.ADP dock into our map so that the tethered head associates with the bound head as in the kinesin dimer structure seen by x-ray crystallography. The new docking orientation avoids problems associated with previous predictions; it puts residues implicated by proteolysis-protection and mutagenesis studies near the microtubule but does not lead to steric interference between the coiled-coil tail and the microtubule surface. The observed conformational changes in the tightly bound states would probably bring some important residues closer to tubulin. As expected from the homology with kinesin, the atomic coordinates of nonclaret disjunctional protein (ncd).ADP dock in the same orientation into the attached head in a map of microtubules decorated with dimeric ncd.ADP. Our results support the idea that the observed direct interaction between the two heads is important at some stages of the mechanism by which kinesin moves processively along microtubules.

Adenosine Diphosphate↗
PubMed1995

An automated computer vision and robotics-based technique for 3-D flexible biomolecular docking and matching.

The generation of binding modes between two molecules, also known as molecular docking, is a key problem in rational drug design and biomolecular recognition. Docking a ligand, e.g., a drug molecule or a protein molecule, to a protein receptor, involves recognition of molecular surfaces as molecules interact at their surface. Recent studies report that the activity of many molecules induces conformational transitions by 'hinge-bending', which involves movements of relatively rigid parts with respect to each other. In ligand-receptor binding, relative rotational movements of molecular substructures about their common hinges have been observed. For automatically predicting flexible molecular interactions, we adapt a new technique developed in Computer Vision and Robotics for the efficient recognition of partially occluded articulated objects. These type of objects consist of rigid parts which are connected by rotary joints (hinges). Our approach is based on an extension and generalization of the Geometric Hashing and Generalized Hough Transform paradigm for rigid object recognition. Unlike other techniques which match each part individually, our approach exploits forcefully and efficiently enough the fact that the different rigid parts do belong to the same flexible molecule. We show experimental results obtained by an implementation of the algorithm for rigid and flexible docking. While the 'correct', crystal-bound complex is obtained with a small RMSD, additional, predictive 'high scoring' binding modes are generated as well. The diverse applications and implications of this general, powerful tool are discussed.

Algorithms↗
PubMed1998

Estimation and filtering of potential protein-protein docking positions.

MOTIVATION: Software systems predicting automatically whether and how two proteins may interact are highly desirable, both for understanding biological processes and for the rational design of new proteins. As a part of a future complete solution to this problem, a bundle of programs is presented designed (i) to estimate initial docking positions for a given pair of docking candidates, (ii) to adjust them, and (iii) to filter them, thus preparing more detailed computations of free energies. RESULTS: The system is evaluated on a test set of 51 co-crystallized complexes aiming at redocking the subunits. It works completely automatically and the evaluation is performed using one single set of parameters for all complexes in the test set. The number of solutions is fixed to 50 positions with a median CPU time of 26 min. For 30 complexes, these contain a near-correct solution with root mean square deviation ( RMSD ) </=5.0 A, which is ranked first in five cases. For all complexes, the best solution is scored on rank 16 as the worst case, and has a median RMSD of 4.3 A. Alternatively to this initial estimation of docking positions, a global sampling of rotations was tested. Whereas this yields top-ranked solutions with RMSD </=3.0 A for all 51 complexes, the median CPU time increases to 11 h. This shows that this blind sampling is not feasible for most applications. AVAILABILITY: The system and its components are available on request from the authors. CONTACT: friedric@techfak.uni-bielefeld or posch@techfak.uni-bielefeld.de

Algorithms↗