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PubMed1998

Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells depends critically on import of the viral preintegration complex into the nucleus. Recent evidence suggests that viral protein R (Vpr) plays a key regulatory role in this process by binding to karyopherin alpha, a cellular receptor for nuclear localization signals, and increasing its affinity for the nuclear localization signals. An in vitro binding assay was used to investigate the role of Vpr in docking of the HIV-1 preintegration complex (PIC) to the nuclear pore complex. Mutant HIV-1 PICs that lack Vpr were impaired in the ability to dock to isolated nuclei and recombinant nucleoporins. Although Vpr by itself associated with nucleoporins, the docking of Vpr+ PICs was dependent on karyopherin beta and was blocked by antibodies to beta. Vpr stabilized docking by preventing nucleoporin-stimulated dissociation of the import complex. These results suggest a biochemical mechanism for Vpr function in transport of the HIV-1 genome across the nuclear pore complex.

Cell Line↗
PubMed1999

The oligosaccharyltransferase complex from Saccharomyces cerevisiae. Isolation of the OST6 gene, its synthetic interaction with OST3, and analysis of the native complex.

The key step of N-glycosylation of proteins, an essential and highly conserved protein modification, is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). So far, eight genes have been identified in Saccharomyces cerevisiae that are involved in this process. Enzymatically active OST preparations from yeast were shown to be composed of four (Ost1p, Wbp1p, Ost3p, Swp1p) or six subunits (Ost2p and Ost5p in addition to the four listed). Genetic studies have disclosed Stt3p and Ost4p as additional proteins needed for N-glycosylation. In this study we report the identification and functional characterization of a new OST gene, designated OST6, that has homology to OST3 and in particular a strikingly similar membrane topology. Neither gene is essential for growth of yeast. Disruption of OST6 or OST3 causes only a minor defect in N-glycosylation, but an Deltaost3Deltaost6 double mutant displays a synthetic phenotype, leading to a severe underglycosylation of soluble and membrane-bound glycoproteins in vivo and to a reduced OST activity in vitro. Moreover, each of the two genes has also a specific function, since agents affecting cell wall biogenesis reveal different growth phenotypes in the respective null mutants. By blue native electrophoresis and immunodetection, a approximately 240-kDa complex was identified consisting of Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p, Swp1p, Ost2p, and Ost5p, indicating that probably all so far identified OST proteins are constituents of the OST complex. It is also shown that disruption of OST3 and OST6 leads to a defect in the assembly of the complex. Hence, the function of these genes seems to be essential for recruiting a fully active complex necessary for efficient N-glycosylation.

Amino Acid Sequence↗
PubMed2000

A novel nuclear receptor corepressor complex, N-CoR, contains components of the mammalian SWI/SNF complex and the corepressor KAP-1.

Transcriptional silencing by many transcription factors is mediated by the nuclear receptor corepressor (N-CoR). The mechanism by which N-CoR represses basal transcription involves the direct or indirect recruitment of histone deacetylases (HDACs). We have isolated two multiprotein N-CoR complexes, designated N-CoR-1 and N-CoR-2, which possess histone deacetylase activity that is mediated by distinct HDACs. Based on Western blotting using antibodies against known subunits, the only HDAC found in the N-CoR-1 complex was HDAC3. In contrast, N-CoR-2 contained predominantly HDAC1 and HDAC2 as well as several other subunits that are found in the Sin3A.HDAC complex. Using mass spectrometry and Western blotting, we have identified several novel components of the N-CoR-1 complex including the SWI/SNF-related proteins BRG1, BAF 170, BAF 155, BAF 47/INI1, and the corepressor KAP-1 that is involved in silencing heterochromatin. Indirect immunofluorescence has revealed that both KAP-1 and N-CoR colocalize throughout the nucleus. These results suggest that N-CoR is found in distinct multiprotein complexes, which are involved in multiple pathways of transcriptional repression.

Blotting, Western↗
PubMed2004

Real-time analysis of ternary complex on particles: direct evidence for partial agonism at the agonist-receptor-G protein complex assembly step of signal transduction.

We developed a novel and generalized approach to investigate G protein-coupled receptor molecular assemblies. We solubilized a fusion protein consisting of the beta(2)-adrenergic receptor and green fluorescent protein (GFP) for bead-based flow cytometric analysis. beta(2)-Adrenergic receptor GFP bound to dihydroalprenolol-conjugated beads, providing a K(d) for the fusion protein and, in competition with beta(2)-adrenergic receptor ligands, K(d) values for agonists and antagonists. Beads displaying chelated nickel bound purified hexahistidine-tagged G protein heterotrimers and, subsequently, the binary complex of agonist with beta(2)-adrenergic receptor GFP. The dose-response curves of ternary complex formation revealed maximal assembly for ligands previously classified as full agonists and reduced assembly for ligands previously classified as partial agonists. Guanosine 5'-3-O-(thio)triphosphate-induced dissociation rates of the ternary complex were the same for full and partial agonists. Soluble G protein, competing with ternary complexes on beads provided an affinity estimate of agonist-receptor complexes to G protein. When performed simultaneously, the two assemblies discriminated between agonist, antagonist or inactive molecule in a manner appropriate for high throughput, small volume drug discovery. The assemblies can be further generalized to other G protein coupled receptor protein-protein interactions.

Adrenergic beta-2 Receptor Agonists↗
PubMed2005

Assembly of an RNA-protein complex. Binding of NusB and NusE (S10) proteins to boxA RNA nucleates the formation of the antitermination complex involved in controlling rRNA transcription in Escherichia coli.

Analytical ultracentrifugation and fluorescence anisotropy methods have been used to measure the equilibrium parameters that control the formation of the core subcomplex of NusB and NusE proteins and boxA RNA. This subcomplex, in turn, nucleates the assembly of the antitermination complex that is involved in controlling the synthesis of ribosomal RNA in Escherichia coli and that also participates in forming the N protein-dependent antitermination complex in lambdoid phage synthesis. In this study we determined the dissociation constants (K(d) values) for the individual binary interactions that participate in the assembly of the ternary NusB-NusE-boxA RNA subassembly, and we showed that multiple equilibria, involving both specific and nonspecific binding, are involved in the assembly pathway of this protein-RNA complex. The measured K(d) values were used to model the in vitro assembly reaction and combined with in vivo concentration data to simulate the overall control of the assembly of this complex in E. coli at two different cellular growth rates. The results showed that at both growth rates assembly proceeds via the initial formation of a weak but specific NusB-boxA complex, which is then stabilized by NusE binding. We showed that NusE also binds nonspecifically to available single-stranded RNA sequences and that such nonspecific protein binding to RNA can help to regulate crucial interactions in the assembly of the various macromolecular machines of gene expression.

Anisotropy↗
PubMed2001

Radiosensitivity of DNA in a specific protein-DNA complex: the lac repressor-lac operator complex.

PURPOSE: To calculate the probability of radiation-induced frank strand breakage (FSB) at each nucleotide in the Escherichia coli lac repressor-lac operator system using a simulation procedure. To compare calculated and experimental results. To asses the contribution of DNA conformational changes and of the masking by the protein to DNA protection by the repressor. MATERIALS AND METHODS: Two structures of the complex were extracted from the PDB databank: crystallography- and NMR-based structures. Calculations were made of the accessibility of the atoms mainly involved in strand breakage (H4' and H5') to O&Hdot; and of the FSB probabilities, along: (1) DNA in the complex; (2) DNA in the complex depleted of the repressor; and (3) a linear DNA having the same sequence. An 80bp fragment bearing the operator was irradiated alone or in presence of the repressor. The relative probabilities of FSB at each nucleotide were determined using sequencing gel electrophoresis. RESULTS: Calculations predict modulation of the accessibility of H4' and H5' atoms and of the probabilities of FSB along the DNA fragments of complexes. This is due to the protein-induced conformational change and to masking by bound protein. The best agreement with the experimental FSB was observed for calculations that use the crystallography-based structure. CONCLUSIONS: For specific DNA-protein complexes, our calculations can predict the protein radiolytic footprints on DNA. They show the significant contribution of the protein-induced DNA conformational change to DNA protection.

Bacterial Proteins↗
PubMed1990

Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.

Nuclear pore complexes (NPCs) prepared from Xenopus laevis oocyte nuclear envelopes were studied in "intact" form (i.e., unexposed to detergent) and after detergent treatment by a combination of conventional transmission electron microscopy (CTEM) and quantitative scanning transmission electron microscopy (STEM). In correlation-averaged CTEM pictures of negatively stained intact NPCs and of distinct NPC components (i.e., "rings," "spoke" complexes, and "plug-spoke" complexes), several fine structural features arranged with octagonal symmetry about a central axis could reproducibly be identified. STEM micrographs of unstained/freeze-dried intact NPCs as well as of their components yielded comparable but less distinct features. Mass determination by STEM revealed the following molecular masses: intact NPC with plug, 124 +/- 11 MD; intact NPC without plug, 112 +/- 11 MD; heavy ring, 32 +/- 5 MD; light ring, 21 +/- 4 MD; plug-spoke complex, 66 +/- 8 MD; and spoke complex, 52 +/- 3 MD. Based on these combined CTEM and STEM data, a three-dimensional model of the NPC exhibiting eightfold centrosymmetry about an axis perpendicular to the plane of the nuclear envelope but asymmetric along this axis is proposed. This structural polarity of the NPC across the nuclear envelope is in accord with its well-documented functional polarity facilitating mediated nucleocytoplasmic exchange of molecules and particles.

Animals↗
PubMed1998

Vinculin is part of the cadherin-catenin junctional complex: complex formation between alpha-catenin and vinculin.

In epithelial cells, alpha-, beta-, and gamma-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. alpha-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell-matrix and cell-cell contacts, alpha-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell-cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and alpha-catenin. We show that alpha-catenin colocalizes at cell-cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH2-terminal head binds to alpha-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The Kd of the complex was determined to 2-4 x 10(-7) M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of alpha-catenin is involved in this interaction. Complex formation of vinculin and alpha-catenin was challenged in transfected cells. In PtK2 cells, intact alpha-catenin and alpha-catenin1-670, harboring the beta-catenin- binding site, were directed to cell-cell contacts. In contrast, alpha-catenin697-906 fragments were recruited to cell-cell contacts, focal adhesions, and stress fibers. Our results imply that in vivo alpha-catenin, like vinculin, is tightly regulated in its ligand binding activity.

Animals↗
PubMed2001

Processing of Mycobacterium tuberculosis antigen 85B involves intraphagosomal formation of peptide-major histocompatibility complex II complexes and is inhibited by live bacilli that decrease phagosome maturation.

Mycobacterium tuberculosis (MTB) inhibits phagosomal maturation to promote its survival inside macrophages. Control of MTB infection requires CD4 T cell responses and major histocompatibility complex (MHC) class II (MHC-II) processing of MTB antigens (Ags). To investigate phagosomal processing of MTB Ags, phagosomes containing heat-killed (HK) or live MTB were purified from interferon-gamma (IFN-gamma)-activated macrophages by differential centrifugation and Percoll density gradient subcellular fractionation. Flow organellometry and Western blot analysis showed that MTB phagosomes acquired lysosome-associated membrane protein-1 (LAMP-1), MHC-II, and H2-DM. T hybridoma cells were used to detect MTB Ag 85B(241-256)-I-A(b) complexes in isolated phagosomes and other subcellular fractions. These complexes appeared initially (within 20 min) in phagosomes and subsequently (>20 min) on the plasma membrane, but never within late endocytic compartments. Macrophages processed HK MTB more rapidly and efficiently than live MTB; phagosomes containing live MTB expressed fewer Ag 85B(241-256)-I-A(b) complexes than phagosomes containing HK MTB. This is the first study of bacterial Ag processing to directly show that peptide-MHC-II complexes are formed within phagosomes and not after export of bacterial Ags from phagosomes to endocytic Ag processing compartments. Live MTB can alter phagosome maturation and decrease MHC-II Ag processing, providing a mechanism for MTB to evade immune surveillance and enhance its survival within the host.

Acyltransferases↗
PubMed2002

Structure of a complex of the human alpha/beta T cell receptor (TCR) HA1.7, influenza hemagglutinin peptide, and major histocompatibility complex class II molecule, HLA-DR4 (DRA*0101 and DRB1*0401): insight into TCR cross-restriction and alloreactivity.

The alpha/beta T cell receptor (TCR) HA1.7 specific for the hemagglutinin (HA) antigen peptide from influenza A virus is HLA-DR1 restricted but cross-reactive for the HA peptide presented by the allo-major histocompatibility complex (MHC) class II molecule HLA-DR4. We report here the structure of the HA1.7/DR4/HA complex, determined by X-ray crystallography at a resolution of 2.4 A. The overall structure of this complex is very similar to the previously reported structure of the HA1.7/DR1/HA complex. Amino acid sequence differences between DR1 and DR4, which are located deep in the peptide binding groove and out of reach for direct contact by the TCR, are able to indirectly influence the antigenicity of the pMHC surface by changing the conformation of HA peptide residues at position P5 and P6. Although TCR HA1.7 is cross-reactive for HA presented by DR1 and DR4 and tolerates these conformational differences, other HA-specific TCRs are sensitive to these changes. We also find a dependence of the width of the MHC class II peptide-binding groove on the sequence of the bound peptide by comparing the HA1.7/DR4/HA complex with the structure of DR4 presenting a collagen peptide. This structural study of TCR cross-reactivity emphasizes how MHC sequence differences can affect TCR binding indirectly by moving peptide atoms.

Alleles↗
PubMed2000

CAST: an iterative algorithm for the complexity analysis of sequence tracts. Complexity analysis of sequence tracts.

MOTIVATION: Sensitive detection and masking of low-complexity regions in protein sequences. Filtered sequences can be used in sequence comparison without the risk of matching compositionally biased regions. The main advantage of the method over similar approaches is the selective masking of single residue types without affecting other, possibly important, regions. RESULTS: A novel algorithm for low-complexity region detection and selective masking. The algorithm is based on multiple-pass Smith-Waterman comparison of the query sequence against twenty homopolymers with infinite gap penalties. The output of the algorithm is both the masked query sequence for further analysis, e.g. database searches, as well as the regions of low complexity. The detection of low-complexity regions is highly specific for single residue types. It is shown that this approach is sufficient for masking database query sequences without generating false positives. The algorithm is benchmarked against widely available algorithms using the 210 genes of Plasmodium falciparum chromosome 2, a dataset known to contain a large number of low-complexity regions. AVAILABILITY: CAST (version 1.0) executable binaries are available to academic users free of charge under license. Web site entry point, server and additional material: http://www.ebi.ac.uk/research/cgg/services/cast/

Algorithms↗
PubMed2003

The effects of familiarity and complexity on appraisal of complex songs by cochlear implant recipients and normal hearing adults.

The purposes of this study were (a) to develop a test of complex song appraisal that would be suitable for use with adults who use a cochlear implant (assistive hearing device) and (b) to compare the appraisal ratings (liking) of complex songs by adults who use cochlear implants (n = 66) with a comparison group of adults with normal hearing (n = 36). The article describes the development of a computerized test for appraisal, with emphasis on its theoretical basis and the process for item selection of naturalistic stimuli. The appraisal test was administered to the 2 groups to determine the effects of prior song familiarity and subjective complexity on complex song appraisal. Comparison of the 2 groups indicates that the implant users rate 2 of 3 musical genres (country western, pop) as significantly more complex than do normal hearing adults, and give significantly less positive ratings to classical music than do normal hearing adults. Appraisal responses of implant recipients were examined in relation to hearing history, age, performance on speech perception and cognitive tests, and musical background.

Adult↗
PubMed1986

A joining-diversity-joining complex generated by inversion mechanism and a variable-diversity complex in the beta-chain gene of the human T-cell receptor.

We have analysed an inactive allele of the beta-chain gene of the T-cell receptor in a human T-cell line HPB-ALL. Comparison with germline sequences showed that HPB-ALL has a joining (J)-diversity (D)-J complex recombined in head-to-head configuration and a variable (V)-D complex in tail-to-tail configuration. These results demonstrate that the inversion mechanism functions in the beta-chain gene of the T-cell receptor. The presence of the V-D complex suggests that V-D recombination could occur prior to D-J recombination although there is no definite proof that the V-D complex is an intermediate to form the V-D-J complex.

Base Sequence↗
PubMed1976

Conformation and reactivity of DNA in the complex with protein. IV. Circular dichroism of poly-L-histidine model complexes with DNA polymers and specificity of the interaction.

The CD study of the DNA-poly-L-histidine complex at high degree of protonation revealed that complex formation is already observable at 2 M NaCl. The influence of salt together with 5 M urea suggests that in addition to electrostatic interactions probably hydrogen bonding may favour specific complexes. Affinity of protonated histidines to AT-rich regions is strongly supported by the complexes formed with (dA.dT)-containing polymers. The psi-type structure occurs with poly(dA-dT)-poly(dA-dT) while poly(dA)-poly(dT) is restricted to form a similar psi-state on interaction with highly protonated poly-L-histidine. Differences in the helix winding properties due to variation in the sequence is suggested as a possible factor in the formation of the psi-type complexes. The mechanism of interaction including hydrogen bonding of histidine side-chains with an AT pair at high degree of protonation and with GC-regions at lower degree of protonation in the polypeptide structure is discussed.

Binding Sites↗
PubMed2006

Structural basis of yeast aminoacyl-tRNA synthetase complex formation revealed by crystal structures of two binary sub-complexes.

The yeast aminoacyl-tRNA synthetase (aaRS) complex is formed by the methionyl- and glutamyl-tRNA synthetases (MetRS and GluRS, respectively) and the tRNA aminoacylation cofactor Arc1p. It is considered an evolutionary intermediate between prokaryotic aaRS and the multi- aaRS complex found in higher eukaryotes. While a wealth of structural information is available on the enzymatic domains of single aaRS, insight into complex formation between eukaryotic aaRS and associated protein cofactors is missing. Here we report crystal structures of the binary complexes between the interacting domains of Arc1p and MetRS as well as those of Arc1p and GluRS at resolutions of 2.2 and 2.05 A, respectively. The data provide a complete structural model for ternary complex formation between the interacting domains of MetRS, GluRS and Arc1p. The structures reveal that all three domains adopt a glutathione S-transferase (GST)-like fold and that simultaneous interaction of Arc1p with GluRS and MetRS is mediated by the use of a novel interface in addition to a classical GST dimerization interaction. The results demonstrate a novel role for this fold as a heteromerization domain specific to eukaryotic aaRS, associated proteins and protein translation elongation factors.

Amino Acid Sequence↗
PubMed1981

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. II. The multi-domain structure of actin-myosin S1 complex.

A three-dimensional image of the "rigor" complex of actin and chymotryptic myosin subfragment-1 (S1) was reconstituted from electron micrographs of specimens embedded in unbroken and unbacked stain sheets of uranyl acetate over the holes of perforated carbon films to an effective resolution of 20 A radially and 26 A axially. The morphological unit of actin-S1 complex consists of at least three domains and myosin S1 shows a multi-domain submolecular structure. Possible ways to assign actin to one or more of these three domains are discussed. Two candidates for the shape of S1 molecule are also shown. Both candidates have a complex "embryo"-like shape. The solid model of the actin-S1 complex appears to be far less polar than that shown in the original electron micrographs or the projected density map of the reconstituted image shown by Toyoshima and Wakabayashi (1). The conspicuous polarity of the arrowhead pattern is related to the projected image of the spiral shape of the main part of the S1 molecule, which is almost at right angles to the helix axis. As one way to reconcile the non-tilted configuration of S1 in the rigor complex with sliding theory, the possibility of a pivoting mechanism (rotation of S1 head in the horizontal plane normal to the helix axis rather than in the vertical plane parallel to the helix axis) is discussed.

Actins↗
PubMed2001

Modeling multi-component protein-DNA complexes: the role of bending and dimerization in the complex of p53 dimers with DNA.

We used molecular modeling to study the optimal conformation of the complex between two p53 DNA-binding domain monomers and a 12 base-pair target DNA sequence. The complex was constructed using experimental data on the monomer binding conformation and a new approach to deform the target DNA sequence. Combined with an internal/helicoidal coordinate model of DNA, this approach enables us to bend the target sequence in a controlled way while respecting the contacts formed with each p53 monomer. The results show that the dimeric complex favors DNA bending towards the major groove at the dimer junction by a value close to experimental findings. In contrast to inferences from earlier models, the calculation of key contributions to the free energy of the complexes indicates a determinant role for DNA in the formation of the complex with the dimer of the p53 DNA-binding domains.

Binding Sites↗
PubMed1987

Paramagnetic complexes of manganese(II), iron(III), and gadolinium(III) as contrast agents for magnetic resonance imaging. The influence of stability constants on the biodistribution of radioactive aminopolycarboxylate complexes.

Paramagnetic complexes of manganese(II), iron(III), and gadolinium(III) with many ligands appear to undergo ligand substitution in vivo, producing biodistribution data similar to the hydrated metal ions. To identify ligands likely to be valuable in the preparation of paramagnetic contrast agents, a series of aminopolycarboxylate complexes with stability constants increasing in the order iminodiacetic acid (IDA) less than nitrilotriacetic acid (NTA) less than EDTA less than CDTA less than or equal to DTPA was prepared with 54Mn(II), 59Fe(III), and 153Gd(III) at both tracer and carrier levels. Biodistribution studies in mice suggested that complexes remained unchanged in vivo if their stability constants (K1) were approximately greater than 10(16) for Mn(II) and Gd(III) and greater than 10(22) for Fe(III) complexes at tracer levels. Metal complexes with added carrier appeared to be effectively more stable in vivo, possibly due to dissociation and saturation of metal-binding sites. To avoid the accumulation of metal ions in tissues, new paramagnetic contrast agents containing these metal ions will require stability constants equal to or greater than those identified here.

Animals↗