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PubMed2004

Soft docking and multiple receptor conformations in virtual screening.

Protein conformational change is an important consideration in ligand-docking screens, but it is difficult to predict. A simple way to account for protein flexibility is to soften the criterion for steric fit between ligand and receptor. A more comprehensive but more expensive method would be to sample multiple receptor conformations explicitly. Here, these two approaches are compared. A "soft" scoring function was created by attenuating the repulsive term in the Lennard-Jones potential, allowing for a closer approach between ligand and protein. The standard, "hard" Lennard-Jones potential was used for docking to multiple receptor conformations. The Available Chemicals Directory (ACD) was screened against two cavity sites in the T4 lysozyme. These sites undergo small but significant conformational changes on ligand binding, making them good systems for soft docking. The ACD was also screened against the drug target aldose reductase, which can undergo large conformational changes on ligand binding. We evaluated the ability of the scoring functions to identify known ligands from among the over 200 000 decoy molecules in the database. The soft potential was always better at identifying known ligands than the hard scoring function when only a single receptor conformation was used. Conversely, the soft function was worse at identifying known leads than the hard function when multiple receptor conformations were used. This was true even for the cavity sites and was especially true for aldose reductase. To test the multiple-conformation method predictively, we screened the ACD for molecules that preferentially docked to the expanded conformation of aldose reductase, known to bind larger ligands. Six novel molecules that ranked among the top 0.66% of hits from the multiple-conformation calculation, but ranked relatively poorly in the soft docking calculation, were tested experimentally for enzyme inhibition. Four of these six inhibited the enzyme, the best with an IC(50) of 8 microM. Although ligands can get better scores in soft docking, the same is also true for decoys. The improved ranking of such decoys can come at the expense of true ligands.

Aldehyde Reductase↗

PubMed2006

A critical assessment of docking programs and scoring functions.

Docking is a computational technique that samples conformations of small molecules in protein binding sites; scoring functions are used to assess which of these conformations best complements the protein binding site. An evaluation of 10 docking programs and 37 scoring functions was conducted against eight proteins of seven protein types for three tasks: binding mode prediction, virtual screening for lead identification, and rank-ordering by affinity for lead optimization. All of the docking programs were able to generate ligand conformations similar to crystallographically determined protein/ligand complex structures for at least one of the targets. However, scoring functions were less successful at distinguishing the crystallographic conformation from the set of docked poses. Docking programs identified active compounds from a pharmaceutically relevant pool of decoy compounds; however, no single program performed well for all of the targets. For prediction of compound affinity, none of the docking programs or scoring functions made a useful prediction of ligand binding affinity.

Algorithms↗

PubMed2007

Comparison of shape-matching and docking as virtual screening tools.

Ligand docking is a widely used approach in virtual screening. In recent years a large number of publications have appeared in which docking tools are compared and evaluated for their effectiveness in virtual screening against a wide variety of protein targets. These studies have shown that the effectiveness of docking in virtual screening is highly variable due to a large number of possible confounding factors. Another class of method that has shown promise in virtual screening is the shape-based, ligand-centric approach. Several direct comparisons of docking with the shape-based tool ROCS have been conducted using data sets from some of these recent docking publications. The results show that a shape-based, ligand-centric approach is more consistent than, and often superior to, the protein-centric approach taken by docking.

Binding Sites↗

PubMed2006

Reversible, cooperative reactions of yeast vacuole docking.

Homotypic yeast vacuole fusion occurs in three stages: (i) priming reactions, which are independent of vacuole clustering, (ii) docking, in which vacuoles cluster and accumulate fusion proteins and fusion regulatory lipids at a ring-shaped microdomain surrounding the apposed membranes of each docked vacuole, where fusion will occur, and (iii) bilayer fusion/compartment mixing. These stages require vacuolar SNAREs, SNARE-chaperones, GTPases, effector complexes, and chemically minor but functionally important lipids. For each, we have developed specific ligands that block fusion and conditions that reverse each block. Using them, we test whether docking entails a linearly ordered series of catalytic events, marked by sequential acquisition of resistance to inhibitors, or whether docking subreactions are cooperative and/or reversible. We find that each fusion protein and regulatory lipid is needed throughout docking, indicative of a reversible or highly cooperative assembly of the fusion-competent vertex ring. In accord with this cooperativity, vertices enriched in one fusion catalyst are enriched in others. Docked vacuoles finally assemble SNARE complexes, yet still require physiological temperature and lipid rearrangements to complete fusion.

Catalysis↗

PubMed2004

TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells.

We imaged and analysed the motion of single insulin secretory granules near the plasma membrane in live pancreatic beta-cells, from normal and diabetic Goto-Kakizaki (GK) rats, using total internal reflection fluorescence microscopy (TIRFM). In normal rat primary beta-cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from 'newcomers'. In diabetic GK rat beta-cells, the number of fusion events from previously docked granules were markedly reduced, and, in contrast, the fusion from newcomers was still preserved. The dynamic change in the number of docked insulin granules showed that, in GK rat beta-cells, the total number of docked insulin granules was markedly decreased to 35% of the initial number after glucose stimulation. Immunohistochemistry with anti-insulin antibody observed by TIRFM showed that GK rat beta-cells had a marked decline of endogenous insulin granules docked to the plasma membrane. Thus our results indicate that the decreased number of docked insulin granules accounts for the impaired insulin release during the first phase of insulin release in diabetic GK rat beta-cells.

Animals↗

PubMed2000

A new role for a SNARE protein as a regulator of the Ypt7/Rab-dependent stage of docking.

The homotypic fusion of yeast vacuoles occurs in an ordered cascade of priming, docking, and fusion. The linkage between these steps has so far remained unclear. We now report that Vam7p (the vacuolar SNAP-23/25 homolog) signals from the cis-SNARE complex to Ypt7p (the vacuolar Rab/Ypt) to initiate the docking process. After Vam7p has been released from the cis-SNARE complex by Sec18p-mediated priming, it is still required for Ypt7p-dependent docking and it needs Ypt7p to remain on the vacuole. Thus, after priming, Vam7p is released from the vacuole altogether if Ypt7p has been extracted by Gdi1p or inactivated by antibody but is not released if docking is blocked simply by vacuole dilution; it is therefore Ypt7p function, and not docking per se, that retains Vam7p. In accord with this finding, cells deleted for the gene encoding Ypt7 have normal amounts of Vam7p but have little Vam7p on their isolated vacuoles. Interaction of Vam7p and Ypt7p is further indicated by two-hybrid analysis [Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., et al. (2000) Nature (London) 403, 623-627] and by the effect of Vam7p on the association of the Rab/Ypt-effector HOPS complex (homotypic fusion and vacuole protein sorting; Vam2p and Vam6p plus four vacuole protein sorting class C proteins) with Ypt7p. Vam7p provides a functional link between the priming step, which releases it from the cis-SNARE complex, and docking.

Membrane Proteins↗

PubMed2003

Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase.

Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTK-substrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substrate-docking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC50 = 21 microM) Csk phosphorylation of Src but only moderately inhibited (IC50 = 422 microM) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.

Alanine↗

PubMed1995

Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes.

Previously, we had purified a cytosolic protein complex, termed karyopherin, that functions in docking import substrate at the nuclear envelope in digitonin-permeabilized cells and also had molecularly cloned and sequenced its 97-kDa beta subunit. We now report that the karyopherin alpha subunit is the previously identified protein NPI-1/SRP-1 of hitherto uncertain function. Using purified recombinant karyopherin alpha or beta subunit, we showed that neither karyopherin alpha nor karyopherin beta alone was sufficient for docking of import substrate at the nuclear envelope. Docking occurred only when both subunits were present. Moreover, docking of import substrate by the two recombinant karyopherin subunits was productive, as it led to nuclear internalization of the docked substrate in the presence of additional, previously characterized cytosolic factors. In a binding assay using immobilized karyopherin alpha and beta subunits and import substrate as a ligand, we found that only karyopherin alpha bound ligand. We suggest that karyopherin beta functions as an adaptor that binds both to karyopherin alpha and to any of a large number of docking sites that are represented by a repetitive peptide motif containing nucleoporins on both the cytoplasmic and nucleoplasmic side of the nuclear pore complex (NPC), bidirectionally ferrying a complex of karyopherin alpha-substrate across the NPC.

Animals↗

PubMed2003

Docking site dynamics of ba3-cytochrome c oxidase from Thermus thermophilus.

Ligand trajectories trapped within a docking site or within an internal cavity near the active site of proteins are important issues toward the elucidation of the mechanism of reaction of such complex systems, in which activity requires the shuttling of oriented ligands to and from their active site. The ligand motion within ba3-cytochrome c oxidase from Thermus thermophilus has been investigated by measuring time-resolved step-scan Fourier transform infrared difference spectra of photodissociated CO from heme a3 at ambient temperature. Upon photodissociation, 15-20% of the CO is not covalently attached to CuB but is trapped within a docking site near the ring A of heme a3 propionate. Two trajectories of CO that are distinguished spectroscopically and kinetically (vCO = 2131 cm-1, td = 10-35 micros and vCO = 2146 cm-1, td = 85 micros) are observed. At later times (td = 110 micros) the docking site reorganizes about the CO and quickly establishes an energetic barrier that facilitates equilibration of the ligand with the protein solvent. The time-dependent shift of the CO trajectories we observe is attributed to a conformational motion of the docking site surrounding the ligand. The implications of these results with respect to the ability of the docking site to constrain ligand orientation and the reaction dynamics of the docking site are discussed herein.

Binding Sites↗

PubMed1998

A method for biomolecular structural recognition and docking allowing conformational flexibility.

In this work, we present an algorithm developed to handle biomolecular structural recognition problems, as part of an interdisciplinary research endeavor of the Computer Vision and Molecular Biology fields. A key problem in rational drug design and in biomolecular structural recognition is the generation of binding modes between two molecules, also known as molecular docking. Geometrical fitness is a necessary condition for molecular interaction. Hence, docking a ligand (e.g., a drug molecule or a protein molecule), to a protein receptor (e.g., enzyme), involves recognition of molecular surfaces. Conformational transitions by "hinge-bending" involves rotational movements of relatively rigid parts with respect to each other. The generation of docked binding modes between two associating molecules depends on their three dimensional structures (3-D) and their conformational flexibility. In comparison to the particular case of rigid-body docking, the computational difficulty grows considerably when taking into account the additional degrees of freedom intrinsic to the flexible molecular docking problem. Previous docking techniques have enabled hinge movements only within small ligands. Partial flexibility in the receptor molecule is enabled by a few techniques. Hinge-bending motions of protein receptors domains are not addressed by these methods, although these types of transitions are significant, e.g., in enzymes activity. Our approach allows hinge induced motions to exist in either the receptor or the ligand molecules of diverse sizes. We allow domains/subdomains/group of atoms movements in either of the associating molecules. We achieve this by adapting a technique developed in Computer Vision and Robotics for the efficient recognition of partially occluded articulated objects. These types of objects consist of rigid parts which are connected by rotary joints (hinges). Our method is based on an extension and generalization of the Hough transform and the Geometric Hashing paradigms for rigid object recognition. We show experimental results obtained by the successful application of the algorithm to cases of bound and unbound molecular complexes, yielding fast matching times. While the "correct" molecular conformations of the known complexes are obtained with small RMS distances, additional, predictive good-fitting binding modes are generated as well. We conclude by discussing the algorithm's implications and extensions, as well as its application to investigations of protein structures in Molecular Biology and recognition problems in Computer Vision.

Algorithms↗

PubMed2004

ClusPro: an automated docking and discrimination method for the prediction of protein complexes.

MOTIVATION: Predicting protein interactions is one of the most challenging problems in functional genomics. Given two proteins known to interact, current docking methods evaluate billions of docked conformations by simple scoring functions, and in addition to near-native structures yield many false positives, i.e. structures with good surface complementarity but far from the native. RESULTS: We have developed a fast algorithm for filtering docked conformations with good surface complementarity, and ranking them based on their clustering properties. The free energy filters select complexes with lowest desolvation and electrostatic energies. Clustering is then used to smooth the local minima and to select the ones with the broadest energy wells-a property associated with the free energy at the binding site. The robustness of the method was tested on sets of 2000 docked conformations generated for 48 pairs of interacting proteins. In 31 of these cases, the top 10 predictions include at least one near-native complex, with an average RMSD of 5 A from the native structure. The docking and discrimination method also provides good results for a number of complexes that were used as targets in the Critical Assessment of PRedictions of Interactions experiment. AVAILABILITY: The fully automated docking and discrimination server ClusPro can be found at http://structure.bu.edu

Algorithms↗

PubMed2006

Inherent limitations in protein-protein docking procedures.

MOTIVATION: The limited success rate of protein-protein docking procedures is generally attributed to structure differences between the bound and unbound states of the molecules. Herein we analyze a large dataset of protein-protein docking results and identify additional parameters that affect the performance of docking procedures. RESULTS: We find that the distinction between nearly correct models (NCMs) and decoys depends on the size of the interface to be predicted thus setting a limit to the prediction ability of docking procedures, particularly those in which the geometric complementarity descriptor is dominant. The geometric complementarity score in grid-based docking carries a large statistical error which further reduces the distinction between NCMs and decoys. We propose a method for correcting the statistical error and show that the distinction is improved when the docking models are ranked by statistically equivalent scores. AVAILABILITY: MolFit can be downloaded from our website http://www.weizmann.ac.il/Chemical_Research_Support/molfit. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Algorithms↗

PubMed1998

Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins.

ER-to-Golgi transport in yeast may be reproduced in vitro with washed membranes, purified proteins (COPII, Uso1p and LMA1) and energy. COPII coated vesicles that have budded from the ER are freely diffusible but then dock to Golgi membranes upon the addition of Uso1p. LMA1 and Sec18p are required for vesicle fusion after Uso1p function. Here, we report that the docking reaction is sensitive to excess levels of Sec19p (GDI), a treatment that removes the GTPase, Ypt1p. Once docked, however, vesicle fusion is no longer sensitive to GDI. In vitro binding experiments demonstrate that the amount of Uso1p associated with membranes is reduced when incubated with GDI and correlates with the level of membrane-bound Ypt1p, suggesting that this GTPase regulates Uso1p binding to membranes. To determine the influence of SNARE proteins on the vesicle docking step, thermosensitive mutations in Sed5p, Bet1p, Bos1p and Sly1p that prevent ER-to-Golgi transport in vitro at restrictive temperatures were employed. These mutations do not interfere with Uso1p-mediated docking, but block membrane fusion. We propose that an initial vesicle docking event of ER-derived vesicles, termed tethering, depends on Uso1p and Ypt1p but is independent of SNARE proteins.

Animals↗

PubMed2006

Information-driven protein-DNA docking using HADDOCK: it is a matter of flexibility.

Intrinsic flexibility of DNA has hampered the development of efficient protein-DNA docking methods. In this study we extend HADDOCK (High Ambiguity Driven DOCKing) [C. Dominguez, R. Boelens and A. M. J. J. Bonvin (2003) J. Am. Chem. Soc. 125, 1731-1737] to explicitly deal with DNA flexibility. HADDOCK uses non-structural experimental data to drive the docking during a rigid-body energy minimization, and semi-flexible and water refinement stages. The latter allow for flexibility of all DNA nucleotides and the residues of the protein at the predicted interface. We evaluated our approach on the monomeric repressor-DNA complexes formed by bacteriophage 434 Cro, the Escherichia coli Lac headpiece and bacteriophage P22 Arc. Starting from unbound proteins and canonical B-DNA we correctly predict the correct spatial disposition of the complexes and the specific conformation of the DNA in the published complexes. This information is subsequently used to generate a library of pre-bent and twisted DNA structures that served as input for a second docking round. The resulting top ranking solutions exhibit high similarity to the published complexes in terms of root mean square deviations, intermolecular contacts and DNA conformation. Our two-stage docking method is thus able to successfully predict protein-DNA complexes from unbound constituents using non-structural experimental data to drive the docking.

Computational Biology↗

PubMed2002

An ERK2 docking site in the Pointed domain distinguishes a subset of ETS transcription factors.

The ETS transcription factors perform distinct biological functions despite conserving a highly similar DNA-binding domain. One distinguishing property of a subset of ETS proteins is a conserved region of 80 amino acids termed the Pointed (PNT) domain. Using enzyme kinetics we determined that the Ets-1 PNT domain contains an ERK2 docking site. The docking site enhances the efficiency of phosphorylation of a mitogen-activated protein kinase (MAPK) site N-terminal to the PNT domain. The site enhances ERK2 binding rather than catalysis. Three hydrophobic residues are involved in docking, and the previously determined NMR structure indicates that these residues are clustered on the surface of the Ets-1 PNT domain. The docking site function is conserved in the PNT domain of the highly related Ets-2 but not in the ets family member GABPalpha. Ablation of the docking site in Ets-1 and Ets-2 prevented Ras pathway-mediated enhancement of the transactivation function of these proteins. This study provides structural insight into the function of a MAPK docking site and describes a unique activity for the PNT domain among a subset of ets family members.

3T3 Cells↗

PubMed2000

Docking structures of domains into maps from cryo-electron microscopy using local correlation.

Accurate maps of large macromolecular complexes can be calculated from cryo-electron micrographs of non-crystalline specimens to resolutions of about 10 A. A method to dock the atomic structures of domains solved by X-ray crystallography or nuclear magnetic resonance into cryo-EM maps is presented. Domains can be docked independently into large complexes without prior definition of the boundaries. No special symmetry is assumed and the procedure is suitable for general application to almost any system where a cryo-EM map (at 15 A resolution or better) of a complex has been obtained and the atomic structures of the components are available. This is achieved through use of a real-space density-matching procedure based on local correlation. A complete asymmetric unit search correlating a density object derived from the atomic coordinates and the density of the EM map is performed. The correlation coefficient is calculated locally in real space using only values of the search object and corresponding samples extracted from the EM map which are under the 'footprint' of the positioned search object. The procedure has been demonstrated by docking the domains of GroEL from the crystal structure into a cryo-EM map Fourier filtered to 12 or 15 A resolution. The correct positions were found without applying any additional constraints. A model of the oligomer built from the docked domains compared favourably with the known crystal structure, confirming the validity of the approach. The procedure is designed to facilitate the incorporation of additional constraints on the docking solutions, which could help to dock using lower resolution maps.

Algorithms↗

PubMed2005

Physicochemical and residue conservation calculations to improve the ranking of protein-protein docking solutions.

Many protein-protein docking algorithms generate numerous possible complex structures with only a few of them resembling the native structure. The major challenge is choosing the near-native structures from the generated set. Recently it has been observed that the density of conserved residue positions is higher at the interface regions of interacting protein surfaces, except for antibody-antigen complexes, where a very low number of conserved positions is observed at the interface regions. In the present study we have used this observation to identify putative interacting regions on the surface of interacting partners. We studied 59 protein complexes, used previously as a benchmark data set for docking investigations. We computed conservation indices of residue positions on the surfaces of interacting proteins using available homologous sequences and used this information to filter out from 56% to 86% of generated docked models, retaining near-native structures for further evaluation. We used a reverse filter of conservation score to filter out the majority of nonnative antigen-antibody complex structures. For each docked model in the filtered subsets, we relaxed the conformation of the side chains by minimizing the energy with CHARMM, and then calculated the binding free energy using a generalized Born method and solvent-accessible surface area calculations. Using the free energy along with conservation information and other descriptors used in the literature for ranking docking solutions, such as shape complementarity and pair potentials, we developed a global ranking procedure that significantly improves the docking results by giving top ranks to near-native complex structures.

Algorithms↗

PubMed2007

A highly conserved MAPK-docking site in Mst7 is essential for Pmk1 activation in Magnaporthe grisea.

In Magnaporthe grisea, the MST11-MST7-PMK1 MAP kinase (MAPK) cascade is essential for appressorium formation and plant infection. Although expressing a dominant active MST7 allele results in Pmk1 activation in the absence of Mst11 and improper regulation of appressorium formation, the direct interaction between Mst7 and Pmk1 is not observed in yeast two-hybrid assays. Thus, it is not clear how Mst7 transmits the upstream signals to Pmk1. Like its homologues from other ascomycetes, Mst7 contains a putative MAPK-docking site (12-20) at its N-terminus. Deletion of this MAPK-docking site had no obvious effect on the expression of MST7 but blocked appressorium formation and plant infection. The kinase activity of Mst7 was not affected by the docking site deletion but Mst7(Delta12-20) failed to activate Pmk1. Mutations in the putative docking region of Pmk1 also abolished appressorium formation. In both co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays, the direct interaction between Mst7 and Pmk1 was detected only during appressorium formation. Deletion of the MAPK-docking site of Mst7 eliminated the Mst7-Pmk1 interaction in M. grisea. These data indicate that the MAPK-docking site of Mst7 is essential for its association and activation of downstream Pmk1, and the Mst7-Pmk1 interaction is enhanced or stabilized during appressorium formation.

Amino Acid Sequence↗