Formation and turnover of NSF- and SNAP-containing "fusion" complexes occur on undocked, clathrin-coated vesicle-derived membranes

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle-derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.     

Determining protein-protein interactions by oxidative cross-linking of a glycine-glycine-histidine fusion protein

The Ni(II) complex of the tripeptide NH2-glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc)2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGH-ecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 A of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.     

alpha IIb beta 3 redistribution triggered by receptor cross-linking

Fibrinogen binding to alpha IIb beta 3 on adherent, spread platelets triggers active, cytoskeletally-directed redistribution of fibrinogen/alpha IIb beta 3 complexes on the platelet surface. Gold-conjugated fibrinogen, unlabeled, soluble fibrinogen, and individual fibrinogen molecules have been demonstrated to trigger receptor redistribution. Here we examine the respective roles of receptor cross-linking and ligand occupancy of receptors in initiating this movement. Monovalent, alpha IIb beta 3-binding fibrinogen fragments RGDS and HHLGGAKQAGDV did not trigger receptor redistribution, suggesting that ligand binding to a single receptor is an insufficient stimulus. Binding of monoclonal antibodies 10E5, AP2, and AP3 to the receptor did not trigger receptor movement. However, cross-linking these receptor-bound monoclonal antibodies by polyclonal anti-mouse IgG or by conjugation of the anti-receptor antibody to large colloidal gold particles triggered receptor redistribution identical in rate, pattern, and final distribution to that previously seen with fibrinogen binding. We conclude that receptor cross-linking provides the signal for initiation of fibrinogen/alpha IIb beta 3 complex redistribution on platelet surfaces.     

Close vicinity of Lhc b1 and Lhc b4 in Photosystem II--membrane fragments as verified by chemical cross-linking

The nearest neighbourhood of pigment-protein complexes within Photosystem II (PSII) membrane fragments has been studied by means of chemical cross-linking with o-phthalaldehyde (OPA) in conjunction with protein-chemical techniques. By means of OPA-induced cross-linking a major conjugate of about 60 kDa has been identified. This conjugate was shown to consist of two pigment-protein complexes of light-harvesting complex II (LHC II), Lhc b1 (CP27) and Lhc b4 (CP29) by means of SDS/PAGE in combination with an immunological analysis using mAbs directed against Lhc b4 and by matrix-assisted-laser-desorption-ionization mass spectrometry (MALDI-MS) and sequence analysis of peptides derived from a proteolytic digest of the conjugate. Domains of Lhc bl and Lhc b4 have been localized to a distance of not more than 5 A within LHC II. Our results are discussed in the light of recent models on the topography of the two subunits within the antenna system of Photosystem II.     

Formation of the amino acid-DNA complexes by hexavalent and trivalent chromium in vitro: importance of trivalent chromium and the phosphate group

We have recently shown that a substantial fraction of all Cr-DNA adducts in chromate-exposed cells are represented by ternary complexes involving amino acids or glutathione bridged by Cr-(III) to DNA. The tridentate amino acids such as cysteine, glutamic acid, and histidine were predominantly found cross-linked to DNA. The mechanism by which Cr can cross-link these amino acids to DNA has been modeled by reacting DNA and trivalent and hexavalent chromium with cysteine and histidine. The formation of a Cr(III)-amino acid binary complex was required before Cr(III) reacted with DNA to yield a ternary complex. Cr(III)-pretreated DNA did not bind cysteine or histidine even after prolonged incubations. Reduction of Cr(VI) in the presence of DNA gave rise to an extensive cross-linking of cysteine and histidine. Addition of DNA to Cr(VI) mixtures at the start of reduction or after the reduction was complete had little effect on the level of ternary complexes indicating that Cr(III)-amino acid binary complexes were DNA-attacking species. In order to identify DNA groups involved in the ternary complex formation, pre-formed Cr(III)-histidine complexes were reacted with nucleosides and nucleotide monophosphates followed by separation and analysis of the products. The incubation of the Cr(III)-histidine complexes with nucleotide monophosphates but not with nucleosides gave rise to ternary complexes that contained both histidine and Cr, showing the primary importance of the phosphate group in this reaction. All four DNA nucleotides were capable of the ternary complex formation with Cr(III) and histidine. No apparent base preference in the amino acid cross-linking was also found in the reaction of Cr(III)/cysteine and Cr(VI)/cysteine mixtures with oligonucleotides of base-specific composition.     

Computer calculation of multiple binding equilibrium isotherms: application to the binding of bivalent ligands to antibodies interacting with cell surface Fc-receptors

A method to calculate multiple binding equilibria by looking for a set of complexes satisfying the conservation principle among sets of concentrations of ligands, receptors satisfying the mass action equations is described. The method replaces complex analytical derivations of equations representing the interactions by the minimization of a single function. The method was implemented for use on microcomputers and applied to the calculation of the binding isotherms of the interactions between a bivalent ligand, a bivalent antibody and the cell surface Fc-receptor. The binding parameters were adjusted to experimental data obtained with P388D1 cells, a monoclonal antibody against DTPA-indium complexes and monovalent and bivalent DTPA-indium haptens. The binding of the antibody and of the haptens to P388D1 cells, as a function of antibody or hapten concentration, was satisfactorily represented using a model in which the antibody molecules bind co-operatively to the Fc-receptor in the presence of cross-linking bivalent hapten. The method can thus be used as a general tool for the numeric calculation of complex equilibrium involving simultaneous interactions of multiple receptors and ligands.     

Changes in integrin/adhesion molecule expression, but not in the T-cell receptor repertoire, in old mice infected with tuberculosis

The DNA binding and interstrand cross-linking properties of the dinuclear platinum complex [?cis-Pt(NH3)2Cl?2bpsu](NO3)2 (bpsu is 4,4'-dipyridyl sulfide) (II) and the mononuclear complex [cis-Pt(NH3)2Cl(4-methylpyridine)]NO3 (I) were compared with those of [?cis-Pt(NH3)2Cl?2H2N(CH2)4NH2](NO3)2 (III) in order to understand the mode of action of complexes I and II. Both compound I and compound II caused significantly different changes of conformation in poly(dG-dC) x poly(dG-dC) than compound III did. Studies of DNA binding, interstrand cross-linking and fluorescence assay suggest that compound I monofunctionally binds to DNA and compound II bifunctionally binds to DNA, that the dinuclear platinum complex II more efficiently interacts with DNA compared to its monomeric analog, and that platinum I and II complexes both interact with DNA in a non-intercalative mode. All the results indicate that the mode of action of the dinuclear complex II is different from that of the mononuclear complex I.     

Formation of a cleavasome: enhancer DNA-2 stabilizes an active conformation of NaeI dimer

Cleavage of DNA by NaeI-type restriction enzymes is stimulated by a DNA element with affinity for the activator site of the enzyme: a cleavage-enhancer DNA element. Measurements of the mobility of NaeI activity in comparison with protein standards on gel permeation columns and glycerol gradients demonstrated that NaeI, without enhancer, can form a 70,000 MW dimer. The dimer, however, is inactive: it could not cleave the "resistant" NaeI site in M13mp18 DNA in the absence of enhancer. In cleavage assays, enhancer stimulated either DNA nicking or DNA cleavage, depending upon NaeI concentration, and reduced the NaeI concentration required for the transition from nicking to cleavage activity. A gel mobility-shift assay of the interaction of NaeI with enhancer showed the formation of two complexes. Results using different sized DNAs and different percentage acrylamide gels for gel mobility-shift analysis implied that the two complexes were caused by NaeI monomer and dimer structures rather than one and two DNA binding. Dimer formation increased with the affinity of enhancer for NaeI. UV cross-linking "captured" the NaeI-enhancer complex; electrophoretic analysis of the cross-linked products showed NaeI dimer bound to enhancer. These results imply a model for cleavage enhancement in which enhancer binding stabilizes an active NaeI dimer conformation ("cleavasome") that cleaves both DNA strands before dissociating.     

Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments

Both the sec6/8 complex and septin filaments have been implicated in directing vesicles and proteins to sites of active membrane addition in yeast. The rat brain sec6/8 complex coimmunoprecipitates with a filament composed of four mammalian septins, suggesting an interaction between these complexes. One of the septins, CDC10, displays broad subcellular and tissue distributions and is found in postmitotic neurons as well as dividing cells. Electron microscopic studies showed that the purified rat brain septins form filaments of 8.25 nm in diameter; the lengths of the filaments are multiples of 25 nm. Glutaraldehyde-fixed rat brain sec6/8 complex adopts a conformation resembling the letter "T" or "Y". The sec6/8 and septin complexes likely play an important role in trafficking vesicles and organizing proteins at the plasma membrane of neurons.     

Experience in using ultrasonic and laser therapy in surgical diseases

Experience is presented gained with the use of ultratonotherapy, low-intensity laser radiation of infrared actions, and a number of drug electrophoreses in the treatment of 358 surgical patients over 1994-1996. A complex of combinations of different techniques common in physiotherapy has been designed with those of drug therapy during different phases of surgical disorders. Methods and complexes of the therapy treatments to be administered are submitted together with relevant indications. The proposed variants of complex physiotherapy make for earlier recovery, shorter treatment courses, longer remission.     

Direct analysis of sugar alcohol borate complexes in plant extracts by matrix-assisted laser desorption/ionization fourier transform mass spectrometry

Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI/FTMS), operating in the negative ion mode, is used to directly observe sugar alcohol borate complexes in a number of plant fractions. The method involves virtually no sample workup and, in the case of celery phloem sap, requires only 40 nL of sap to observe the borate complex. The isolation and characterization of such soluble borate complexes is important in understanding the distribution of boron in plants. The results show that the complexes are composed of two mannitol or sorbitol ligands (L) complexed to a single borate center (B). In some cases, the boron is complexed to non-alditol monosaccharides. Sustained off-resonance collision irradiation dissociation of the BL2- complex, where L is a mannitol, gives fragments that confirm the proposed structure. Complexes of larger oligosaccharides have also been successfully observed using MALDI/FTMS. Semiempirical molecular orbital calculations (AM1) of the mannitol BL2- complex show that the most favorable configuration is with carbons 3 and 4 of both mannitol residues complexed to the borate. This allows maximum interaction of the remaining hydroxyls with the borate center.     

Multivalent structure of an alphabetaT cell receptor

Whether there is one or multiple alphabetaT cell antigen receptor (TCR) recognition modules in a given TCR/CD3 complex is a long-standing controversy in immunology. We show that T cells from transgenic mice that coexpress comparable amounts of two distinct TCRbeta chains incorporate at least two alphabetaTCRs in a single TCR/CD3 complex. Evidence for bispecific alphabetaTCRs was obtained by immunoprecipitation and immunoblotting and confirmed on the surface of living cells both by fluorescence resonance energy transfer and comodulation assays by using antibodies specific for TCRbeta-variable regions. Such (alphabeta)2TCR/CD3 or higher-order complexes were evident in T cells studied either ex vivo or after expansion in vitro. T cell activation is thought by many, but not all, to require TCR cross-linking by its antigen/major histocompatibility complex ligand. The implications of a multivalent (alphabeta)2TCR/CD3 complex stoichiometry for the ordered docking of specific antigen/major histocompatibility complex, CD4, or CD8 coreceptors and additional TCRs are discussed.     

Inhibition of elastase by N-sulfonylaryl beta-lactams: anatomy of a stable acyl-enzyme complex

beta-Lactam inhibitors of transpeptidase enzymes involved in cell wall biosynthesis remain among the most important therapeutic agents in clinical use. beta-Lactams have more recently been developed as inhibitors of serine proteases including elastase. All therapeutically useful beta-lactam inhibitors operate via mechanisms resulting in the formation of hydrolytically stable acyl-enzyme complexes. Presently, it is difficult to predict which beta-lactams will form stable acyl-enzyme complexes with serine enzymes. Further, the factors that result in the seemingly special nature of beta-lactams versus other acylating agents are unclear-if indeed they exist. Here we present the 1.6 A resolution crystal structure of a stable acyl-enzyme complex formed between porcine pancreatic elastase and a representative monocyclic beta-lactam, which forms a simple acyl-enzyme. The structure shows that the ester carbonyl is not located within the oxyanion hole and the "hydrolytic" water is displaced. Combined with additional kinetic and mass spectrometric data, the structure allows the rationalization of the low degree of hydrolytic lability observed for the beta-lactam-derived acyl-enzyme complex.     

The herpes simplex virus type 1 origin binding protein. Specific recognition of phosphates and methyl groups defines the interacting surface for a monomeric DNA binding domain in the major groove of DNA

The UL9 gene of herpes simplex virus type 1 (HSV-1) encodes an origin binding protein (OBP). It is an ATP-dependent DNA helicase and a sequence-specific DNA-binding protein. The latter function is carried out by the C-terminal domain of OBP (DeltaOBP). We have now performed a quantitative analysis of the interaction between DeltaOBP and its recognition sequence, GTTCGCAC, in oriS. Initially optimal conditions for binding were carefully determined. We observed that complexes with different electrophoretic mobilities were formed. A cross-linking experiment demonstrated that nonspecific complexes containing 2 or more protein monomers per DNA molecule were formed at high protein concentrations. The specific complex formed at low concentrations of DeltaOBP had an electrophoretic mobility corresponding to a 1:1 complex. We then demonstrated that the methyl groups of thymine in the major groove were essential for high affinity binding. Changes in the minor groove had considerably smaller effects. Ethylation interference experiments indicated that specific contacts were made between OBP and three phosphates in the recognition sequence. Finally, these observations were used to present a model of the surface of DNA that interacts with DeltaOBP in a sequence-specific manner.     

Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.     

Expression and characterization of recombinant soluble peptide: I-A complexes associated with murine experimental autoimmune diseases

Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to beta-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.     

X-ray crystallographic studies of unique cross-linked lattices between four isomeric biantennary oligosaccharides and soybean agglutinin

Soybean agglutinin (SBA) (Glycine max) is a tetrameric GalNAc/Gal-specific lectin which forms unique cross-linked complexes with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal GalNAc or Gal residues [Gupta et al. (1994) Biochemistry 33, 7495-7504]. We recently reported the X-ray crystal structure of SBA cross-linked with a biantennary analog of the blood group I carbohydrate antigen [Dessen et al. (1995) Biochemistry 34, 4933-4942]. In order to determine the molecular basis of different carbohydrate-lectin cross-linked lattices, a comparison has been made of the X-ray crystallographic structures of SBA cross-linked with four isomeric analogs of the biantennary blood group I carbohydrate antigen. The four pentasaccharides possess the common structure of (beta-LacNAc)2Gal-beta-R, where R is -O(CH2)5COOCH3. The beta-LacNAc moieties in the four carbohydrates are linked to the 2,3-, 2,4-, 3,6-, and 2,6-positions of the core Gal residue(s), respectively. The structures of all four complexes have been refined to approximately 2.4-2.8 A. Noncovalent lattice formation in all four complexes is promoted uniquely by the bridging action of the two arms of each bivalent carbohydrate. Association between SBA tetramers involves binding of the terminal Gal residues of the pentasaccharides at identical sites in each monomer, with the sugar(s) cross-linking to a symmetry-related neighbor molecule. While the 2,4-, 3,6-, and 2,6-pentasaccharide complexes possess a common P6422 space group, their unit cell dimensions differ. The 2, 3-pentasaccharide cross-linked complex, on the other hand, possesses the space group I4122. Thus, all four complexes are crystallographically distinct. The four cross-linking carbohydrates are in similar conformations, possessing a pseudo-2-fold axis of symmetry which lies on a crystallographic 2-fold axis of symmetry in each lattice. In the case of the 3,6- and 2,6-pentasaccharides, the symmetry of their cross-linked lattices requires different rotamer orientations about their beta(1,6) glycosidic bonds. The results demonstrate that crystal packing interactions are the molecular basis for the formation of distinct cross-linked lattices between SBA and four isomeric pentasaccharides. The present findings are discussed in terms of lectins forming unique cross-linked complexes with glycoconjugate receptors in biological systems.     

Phosphatidylinositol 3-kinase is required for CD28 but not CD3 regulation of the TEC family tyrosine kinase EMT/ITK/TSK: functional and physical interaction of EMT with phosphatidylinositol 3-kinase

Ligation of the TCR or CD28 induces activation of phosphatidylinositol 3-kinase (PI3K), the TEC family protein tyrosine kinase, EMT/ITK/TSK (EMT), and the SRC family tyrosine kinase, LCK. LCK is required for the activation and phosphorylation of EMT induced by ligation of the TCR or CD28 placing LCK upstream of EMT in T cell signaling cascades. We report herein that inhibition of PI3K activity with the specific inhibitors LY294002 and wortmannin markedly decreased EMT activation induced by CD28 cross-linking but not by CD3 cross-linking. Further, inhibition of PI3K markedly decreased EMT in vitro autokinase activity induced by activated LCK. In contrast, PI3K inhibitors did not alter CD28 or CD3 cross-linking or LCK-induced EMT phosphorylation. Consistent with the requirement of PI3K activity for CD28 but not CD3-induced stimulation of the EMT in vitro autokinase activity, a small but significant portion of cellular EMT associates with PI3K following CD28 cross-linking but not following CD3 cross-linking. CD28-induced association of EMT with PI3K also requires functional expression of LCK. Fusion proteins containing the SRC homology 2 domain of EMT interact with PI3K or a PI3K-associated molecule in a tyrosine phosphorylation-dependent manner. Taken together, the data suggest that EMT is differentially regulated and recruited to different signaling complexes following ligation of CD28 or the TCR complex, perhaps contributing to the disparate roles that EMT appears to play downstream of CD28 and the TCR.     

The hepatic interleukin-6 receptor. Studies on its structure and regulation by phorbol 12-myristate 13-acetate-dexamethasone

Recombinant human 125I-interleukin-6 (IL-6) was cross-linked with the homobifunctional reagent disuccinimidyl suberate to human hepatoma cells (HepG2). Three recombinant human 125I-IL-6-containing complexes of apparent molecular masses of 100, 120, and 200 kDa were immunoprecipitated with specific antibodies to human IL-6 or to the 80-kDa IL-6 receptor subunit. We show by immunoprecipitation, peptide mapping, and by the use of a cleavable heterobifunctional cross-linker (Denny-Jaffe reagent) that different polypeptides are involved in the formation of the 100- and 120-kDa IL-6-containing complexes. The molecular compositions of the 100- and 120-kDa cross-linked complexes were identified. The 100-kDa complex consisted of one ligand and one IL-6 receptor subunit, glycoprotein 80 (gp80), whereas the 120-kDa complex was found to be composed of one ligand and a polypeptide which was immunoprecipitable with the monoclonal antibody AM64 directed against gp130. Exposure of HepG2 cells to phorbol 12-myristate 13-acetate (PMA) or PMA-dexamethasone led to an increase in the 80-kDa IL-6 receptor mRNA and functional receptor protein. Whereas treatment of HepG2 cells with PMA led to an increase in the formation of gp80.gp130.IL-6 complexes determined by cross-linking, no corresponding increase in high affinity binding sites was found. The existence of a third IL-6 receptor subunit present in limiting amounts on HepG2 cells is proposed to explain this discrepancy. Evidence is presented that the 80-kDa IL-6 receptor up-regulation by PMA-dexamethasone is caused by the depletion of protein kinase C since the protein kinase C inhibitor staurosporine mimics the effect of PMA-dexamethasone.