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PubMed2002

The outer dynein arm-docking complex: composition and characterization of a subunit (oda1) necessary for outer arm assembly.

To learn more about how dyneins are targeted to specific sites in the flagellum, we have investigated a factor necessary for binding of outer arm dynein to the axonemal microtubules of Chlamydomonas. This factor, termed the outer dynein arm-docking complex (ODA-DC), previously was shown to be missing from axonemes of the outer dynein armless mutants oda1 and oda3. We have now partially purified the ODA-DC, determined that it contains equimolar amounts of M(r) approximately 105,000 and approximately 70,000 proteins plus a third protein of M(r) approximately 25,000, and found that it is associated with the isolated outer arm in a 1:1 molar ratio. We have cloned a full-length cDNA encoding the M(r) approximately 70,000 protein; the sequence predicts a 62.5-kDa protein with potential homologs in higher ciliated organisms, including humans. Sequencing of corresponding cDNA from strain oda1 revealed it has a mutation resulting in a stop codon just downstream of the initiator ATG; thus, it is unable to make the full-length M(r) approximately 70,000 protein. These results demonstrate that the ODA1 gene encodes the M(r) approximately 70,000 protein, and that the protein is essential for assembly of the ODA-DC and the outer dynein arm onto the doublet microtubule.

Algal Proteins↗
PubMed1999

Pex17p is required for import of both peroxisome membrane and lumenal proteins and interacts with Pex19p and the peroxisome targeting signal-receptor docking complex in Pichia pastoris.

Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Delta mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Delta mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal-receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.

Amino Acid Sequence↗
PubMed2001

The N-terminal domain of the t-SNARE Vam3p coordinates priming and docking in yeast vacuole fusion.

Homotypic fusion of yeast vacuoles requires a regulated sequence of events. During priming, Sec18p disassembles cis-SNARE complexes. The HOPS complex, which is initially associated with the cis-SNARE complex, then mediates tethering. Finally, SNAREs assemble into trans-complexes before the membranes fuse. The t-SNARE of the vacuole, Vam3p, plays a central role in the coordination of these processes. We deleted the N-terminal region of Vam3p to analyze the role of this domain in membrane fusion. The truncated protein (Vam3 Delta N) is sorted normally to the vacuole and is functional, because the vacuolar morphology is unaltered in this strain. However, in vitro vacuole fusion is strongly reduced due to the following reasons: Assembly, as well as disassembly of the cis-SNARE complex is more efficient on Vam3 Delta N vacuoles; however, the HOPS complex is not associated well with the Vam3 Delta N cis-complex. Thus, primed SNAREs from Vam3 Delta N vacuoles cannot participate efficiently in the reaction because trans-SNARE pairing is substantially reduced. We conclude that the N-terminus of Vam3p is required for coordination of priming and docking during homotypic vacuole fusion.

Adenosine Triphosphatases↗
PubMed2001

Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods.

Rab8 is a GTPase involved in membrane trafficking. In photoreceptor cells, rab8 is proposed to participate in the late stages of delivery of rhodopsin-containing post-Golgi membranes to the plasma membrane near the base of the connecting cilium. To test the function of rab8 in vivo, we generated transgenic Xenopus laevis expressing wild-type, constitutively active (Q67L), and dominant negative (T22N) forms of canine rab8 in their rod photoreceptors as green fluorescent protein (GFP) fusion proteins. Wild-type and constitutively active GFP-rab8 proteins were primarily associated with Golgi and post-Golgi membranes, whereas the dominant negative protein was primarily cytoplasmic. Expression of wild-type GFP-rab8 had minimal effects on cell survival and intracellular structures. In contrast, GFP-rab8T22N caused rapid retinal degeneration. In surviving peripheral rods, tubulo-vesicular structures accumulated at the base of the connecting cilium. Expression of GFP-rab8Q67L induced a slower retinal degeneration in some tadpoles. Transgene effects were transmitted to F1 offspring. Expression of the GFP-rab8 fusion proteins appears to decrease the levels of endogenous rab8 protein. Our results demonstrate a role for rab8 in docking of post-Golgi membranes in rods, and constitute the first report of a transgenic X. laevis model of retinal degenerative disease.

Animals↗
PubMed2006

The docking protein Cas links tyrosine phosphorylation signaling to elongation of cerebellar granule cell axons.

Crk-associated substrate (Cas) is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of the actin cytoskeletal organization and cell migration in fibroblasts. The function of Cas in neurons, however, is poorly understood. Here we report that Cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. During cerebellar development, Cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. Cas coimmunoprecipitates with Src family protein tyrosine kinases, Crk, and cell adhesion molecules and colocalizes with these proteins in granule cells. The axon extension of granule cells is inhibited by either RNA interference knockdown of Cas or overexpression of the Cas mutant lacking the YDxP motifs, which are tyrosine phosphorylated and thereby interact with Crk. These findings demonstrate that Cas acts as a key scaffold that links the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation.

Amino Acid Sequence↗
PubMed2006

ADP_EM: fast exhaustive multi-resolution docking for high-throughput coverage.

MOTIVATION: Efficient fitting tools are needed to take advantage of a fast growth of atomic models of protein domains from crystallography or comparative modeling, and low-resolution density maps of larger molecular assemblies. Here, we report a novel fitting algorithm for the exhaustive and fast overlay of partial high-resolution models into a low-resolution density map. The method incorporates a fast rotational search based on spherical harmonics (SH) combined with a simple translational scanning. RESULTS: This novel combination makes it possible to accurately dock atomic structures into low-resolution electron-density maps in times ranging from seconds to a few minutes. The high-efficiency achieved with simulated and experimental test cases preserves the exhaustiveness needed in these heterogeneous-resolution merging tools. The results demonstrate its efficiency, robustness and high-throughput coverage. AVAILABILITY: http://sbg.cib.csic.es/Software/ADP_EM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Algorithms↗
PubMed1998

Vam7p, a vacuolar SNAP-25 homolog, is required for SNARE complex integrity and vacuole docking and fusion.

The vacuole v-t-SNARE complex is disassembled by Sec17p/alpha-SNAP and Sec18p/NSF prior to vacuole docking and fusion. We now report a functional characterization of the vacuolar SNARE Vam7p, a SNAP-25 homolog. Although Vam7p has no hydrophobic domains, it is tightly associated with the vacuolar membrane. Vam7p is a constituent of the vacuole SNARE complex and is released from this complex by the Sec17p/Sec18p/ATP-mediated priming of the vacuoles. Even in the absence of the vacuolar v-SNARE Nyv1p, a subcomplex which includes Vam7p and the t-SNARE Vam3p is preserved. Vam7p is necessary for the stability of the vacuolar SNARE complex, since vacuoles from mutants deleted in VAM7 do not have a Vam3p-Nyv1p complex. Furthermore, Vam7p alone, in the absence of Nyv1p and Vam3p, cannot mediate fusion with wild-type vacuoles, whereas vacuoles with only Nyv1p or Vam3p alone can fuse with wild-type vacuoles in the absence of the other two SNAREs. Thus, Vam7p is important for the stable assembly and efficient function of the vacuolar SNARE complex and maintenance of the vacuolar morphology. This functional characterization of Vam7p suggests a general role for SNAP-25 homologs, not only on the plasma membrane but along the secretory pathway.

Adenosine Triphosphatases↗
PubMed2000

A phosphoserine-regulated docking site in the protein kinase RSK2 that recruits and activates PDK1.

The 90 kDa ribosomal S6 kinase-2 (RSK2) is a growth factor-stimulated protein kinase with two kinase domains. The C-terminal kinase of RSK2 is activated by ERK-type MAP kinases, leading to autophosphorylation of RSK2 at Ser386 in a hydrophobic motif. The N-terminal kinase is activated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) through phosphorylation of Ser227, and phosphorylates the substrates of RSK. Here, we identify Ser386 in the hydrophobic motif of RSK2 as a phosphorylation-dependent docking site and activator of PDK1. Treatment of cells with growth factor induced recruitment of PDK1 to the Ser386-phosphorylated hydrophobic motif and phosphorylation of RSK2 at Ser227. A RSK2-S386K mutant showed no interaction with PDK1 or phosphorylation at Ser227. Interaction with Ser386-phosphorylated RSK2 induced autophosphorylation of PDK1. Addition of a synthetic phosphoSer386 peptide (RSK2(373-396)) increased PDK1 activity 6-fold in vitro. Finally, mutants of RSK2 and MSK1, a RSK-related kinase, with increased affinity for PDK1, were constitutively active in vivo and phosphorylated histone H3. Our results suggest a novel regulatory mechanism based on phosphoserine-mediated recruitment of PDK1 to RSK2, leading to coordinated phosphorylation and activation of PDK1 and RSK2.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2001

SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis.

During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol-dependent membrane rafts since they are Triton X-100-soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion.

Animals↗
PubMed2002

High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site.

3-phosphoinositide dependent protein kinase-1 (PDK1) plays a key role in regulating signalling pathways by activating AGC kinases such as PKB/Akt and S6K. Here we describe the 2.0 A crystal structure of the PDK1 kinase domain in complex with ATP. The structure defines the hydrophobic pocket termed the "PIF-pocket", which plays a key role in mediating the interaction and phosphorylation of certain substrates such as S6K1. Phosphorylation of S6K1 at its C-terminal PIF-pocket-interacting motif promotes the binding of S6K1 with PDK1. In the PDK1 structure, this pocket is occupied by a crystallographic contact with another molecule of PDK1. Interestingly, close to the PIF-pocket in PDK1, there is an ordered sulfate ion, interacting tightly with four surrounding side chains. The roles of these residues were investigated through a combination of site-directed mutagenesis and kinetic studies, the results of which confirm that this region of PDK1 represents a phosphate-dependent docking site. We discuss the possibility that an analogous phosphate-binding regulatory motif may participate in the activation of other AGC kinases. Furthermore, the structure of PDK1 provides a scaffold for the design of specific PDK1 inhibitors.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1991

Molecular modelling of protein-carbohydrate interactions. Docking of monosaccharides in the binding site of concanavalin A.

A general procedure is described for addressing the computer simulation of protein-carbohydrate interactions. First, a molecular mechanical force field capable of performing conformational analysis of oligosaccharides has been derived by the addition of new parameters to the Tripos force field; it is also compatible with the simulation of protein. Second, a docking procedure which allows for a systematic exploration of the orientations and positions of a ligand into a protein cavity has been designed. This so-called 'crankshaft' method uses rotations and variations about/of virtual bonds connecting, via dummy atoms, the ligand to the protein binding site. Third, calculation of the relative stability of protein ligand complexes is performed. This strategy has been applied to search for all favourable interactions occurring between a lectin [concanavalin A (ConA)] and methyl alpha-D-mannopyranoside or methyl alpha-D-glucopyranoside. For each monosaccharide, different stable orientations and positions within the binding site can be distinguished. Among them, one corresponds to very favourable interactions, not only in terms of hydrogen bonding, but also in terms of van der Waals interactions. It corresponds precisely to the binding mode of methyl alpha-D-mannopyranoside into ConA as revealed by the 2.9 A resolution of the crystalline complex (Derewenda et al., 1989). Some implications of the present modelling study with respect to the molecular basis of the specificity of the interaction of lectins with various monosaccharides are presented.

Binding Sites↗
PubMed2006

LysM domains of Medicago truncatula NFP protein involved in Nod factor perception. Glycosylation state, molecular modeling and docking of chitooligosaccharides and Nod factors.

The establishment of the symbiosis between legume plants and rhizobial bacteria depends on the production of rhizobial lipo-chitooligosaccharidic signals (the Nod factors) that are specifically recognized by roots of the host plant. In Medicago truncatula, specific recognition of Sinorhizobium meliloti and its Nod factors requires the NFP (Nod factor perception) gene, which encodes a putative serine/threonine receptor-like kinase (RLK). The extracellular region of this protein contains three tandem lysin motifs (LysMs), a short peptide domain that is implicated in peptidoglycan or chitin binding in various bacterial or eukaryotic proteins, respectively. We report here the homology modeling of the three LysM domains of M. truncatula NFP based on the structure of a LysM domain of the Escherichia coli membrane-bound lytic murein transglycosidase D (MltD). Expression of NFP in a homologous system (M. truncatula roots) revealed that the protein is highly N-glycosylated, probably with both high-mannose and complex glycans. Surface analysis and docking calculations performed on the models of the three domains were used to predict the most favored binding modes for chitooligosaccharides and Nod factors. A convergent model can be proposed where the sulfated, O-acetylated lipo-chitooligosaccharidic Nod factor of S. meliloti binds in similar orientation to the three LysM domains of M. truncatula NFP. N-glycosylation is not expected to interfere with Nod factor binding in this orientation.

Amino Acid Motifs↗
PubMed2006

Activity-structure correlations in divergent lectin evolution: fine specificity of chicken galectin CG-14 and computational analysis of flexible ligand docking for CG-14 and the closely related CG-16.

Gene duplication and sequence divergence are driving forces toward establishing protein families. To examine how sequence changes affect carbohydrate specificity, the two closely related proto-type chicken galectins CG-14 and CG-16 were selected as models. Binding properties were analyzed using a highly sensitive solid-phase assay. We tested 56 free saccharides and 34 well-defined glycoproteins. The two galectins share preference for the II (Galbeta1-4GlcNAc) versus I (Galbeta1-3GlcNAc) version of beta-galactosides. A pronounced difference is found owing to the reactivity of CG-14 with histo-blood group ABH active oligosaccharides and A/B active glycoproteins. These experimental results prompted to determine activity-structure correlations by modeling. Computational analysis included consideration of the flexibility of binding partners and the presence of water molecules. It provided a comparative description of complete carbohydrate recognition domains, which had so far not been characterized in animal galectins. The structural models assigned II, I selectivity to a region downstream of the central Trp moiety. Docking revealed that the tetrasaccharides can be accommodated in their free-state low-energy conformations. CG-14's preference for A versus B epitopes could be attributed to a contact between His124 and the N-acetyl group of GalNAc. Regarding intergalectin comparison, the Ala53/Cys51 exchange affects the interaction potential of His54/His52. Close inspection of simulated dynamic interplay revealed reorientation of His124 at the site of the His124/Glu123 substitution, with potential impact on ligand dissociation. In summary, this study identifies activity differences and provides information on their relation to structural divergence, epitomizing the value of this combined approach beyond galectins.

Amino Acid Sequence↗
PubMed2006

Probing the catalytic center of porcine aminoacylase 1 by site-directed mutagenesis, homology modeling and substrate docking.

Three-dimensional structural models of porcine aminoacylase 1 (pACY1) were constructed by homology modeling and aligning the structures of members of the M20 peptidase family. After energy minimization and quality evaluation, the best model from the homology modeling was chosen for docking with the best substrate (N-acetyl-L-methionine). The most reasonable binding mode was found among a large number of predicted complexes by using clustering analysis and screening with expert knowledge. Structural analysis revealed that the zinc ion is not likely to bind to the substrate, and that Arg348 and Glu146 play vital roles in binding and catalysis. In the site-directed mutagenesis experiments, mutation of His79, Asp112, Glu147, Arg348, and Glu146, resulted in significant reductions of specific activity, while the wild-type pACY1 overexpressed in Rosetta (DE3) had almost as high a specific activity as the native enzyme. On the basis of these observations, we proposed a revised catalytic mechanism for this metalloenzyme.

Amidohydrolases↗
PubMed1997

Transfer RNA docking pair model in the ribosomal pre- and post-translocational states.

A consensus has been reached that the conformation of the anticodon-codon interactions of two adjacent tRNA molecules on the ribosome is a Sundaralingam-type (S-type). Even if it is kept to the S-type, there are still various possibilities. Various experimental data have been supporting an idea that the conformation of A-site tRNA is different from that of P-site tRNA. Those data as well as the recent result of Brimacombe and co-workers that U20:1 of lupin tRNAmMetbound to the A-site was cross-linked to a region, 875-905, of 23S rRNA in combination with the other recent findings of Nierhaus and co-workers about the spin-contrast method of neutron diffraction of the ribosome and the better accessible nucleotide patterns of phosphorothioated tRNAs on the ribosome have led to a new tRNA docking pair model, in which the highly conserved G18 and G19 of D-loop in A-site tRNA and C56 and C61 of TpsiC-loop in P-site tRNA base pair along with the conventional base pairs of adjacent codon-anticodon interactions. This A-P tRNA pair model can be translocated to the P-E tRNA pair model without changing the conformation except the ACCA termini, keeping the position of the growing nascent polypeptide chain.

Base Composition↗
PubMed2006

kinDOCK: a tool for comparative docking of protein kinase ligands.

KinDOCK is a new web server for the analysis of ATP-binding sites of protein kinases. This characterization is based on the docking of ligands already co-crystallized with other protein kinases. A structural library of protein kinase-ligand complexes has been extracted from the Protein Data Bank (PDB). This library can provide both potential ligands and their putative binding orientation for a given protein kinase. After protein-protein structural superposition, the ligands are transferred from the template complexes to the target protein kinase. The resulting complexes are evaluated using the program SCORE to compute a theoretical affinity. They can be dynamically visualized to allow a rapid mapping of important steric clashes and potential substitutions relevant for specificity and affinity. These characteristics allow a quick characterization of protein kinase active sites including conformation changes potentially required to accommodate particular ligands. Additionally, promising pharmacophores can be identified in the focussed library. These features will help to rationalize or optimize virtual screening (VS) on larger chemical compound libraries. The server and its documentation are freely available at http://abcis.cbs.cnrs.fr/kindock/.

Adenosine Triphosphate↗
PubMed1999

An analysis of conformational changes on protein-protein association: implications for predictive docking.

Conformational changes on complex formation have been measured for 39 pairs of structures of complexed proteins and unbound equivalents, averaged over interface and non-interface regions and for individual residues. We evaluate their significance by comparison with the differences seen in 12 pairs of independently solved structures of identical proteins, and find that just over half have some substantial overall movement. Movements involve main chains as well as side chains, and large changes in the interface are closely involved with complex formation, while those of exposed non-interface residues are caused by flexibility and disorder. Interface movements in enzymes are similar in extent to those of inhibitors. All eight of the complexes (six enzyme-inhibitor and two antibody-antigen) that have structures of both components in an unbound form available show some significant interface movement. However, predictive docking is successful even when some of the largest changes occur. We note however that the situation may be different in systems other than the enzyme-inhibitors which dominate this study. Thus the general model is induced fit but, because there is only limited conformational change in many systems, recognition can be treated as lock and key to a first approximation.

Algorithms↗
PubMed2001

Protein docking using continuum electrostatics and geometric fit.

The computer program DOT quickly finds low-energy docked structures for two proteins by performing a systematic search over six degrees of freedom. A novel feature of DOT is its energy function, which is the sum of both a Poisson-Boltzmann electrostatic energy and a van der Waals energy, each represented as a grid-based correlation function. DOT evaluates the energy of interaction for many orientations of the moving molecule and maintains separate lists scored by either the electrostatic energy, the van der Waals energy or the composite sum of both. The free energy is obtained by summing the Boltzmann factor over all rotations at each grid point. Three important findings are presented. First, for a wide variety of protein-protein interactions, the composite-energy function is shown to produce larger clusters of correct answers than found by scoring with either van der Waals energy (geometric fit) or electrostatic energy alone. Second, free-energy clusters are demonstrated to be indicators of binding sites. Third, the contributions of electrostatic and attractive van der Waals energies to the total energy term appropriately reflect the nature of the various types of protein-protein interactions studied.

Computer Simulation↗