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PubMed2006

Ring-opening polymerization of coordination complexes: silver(I) complexes with bis(amidopyridine) ligands derived from thiophene.

The thiophene-based bis(N-methylamido-pyridine) ligand SC4H2-2,5-{C(=O)N(Me)-4-C5H4N}2 reacts with silver(I) salts AgX to give 1 : 1 complexes, which are characterized in the solid state as the macrocyclic complexes [Ag(2){SC4H2-2,5-(CONMe-4-C5H4N)2}2][X]2, which have the cis conformation of the C(=O)N(Me) group, when X = CF3CO2, NO3, or CF3SO3 but as the polymeric complex [Ag(n){SC4H2-2,5-(CONMe-4-C5H4N)2}n][X]n, with the unusual trans conformation of the C(=O)N(Me) group, when X = PF6. The bis(amido-pyridine) ligand SC4H2-2,5-{C(=O)NHCH2-3-C5H4N}2 reacts with silver(I) trifluoroacetate to give the polymeric complex [Ag(n){SC4H2-2,5-(CONHCH2-3-C5H4N)2}n][X]n, X = CF3CO2. The macrocyclic complexes contain transannular argentophilic secondary bonds. The polymers self assemble into sheet structures through interchain C=O...Ag and S...Ag bonds in [Ag(n){SC4H2-2,5-(CONMe-4-C5H4N)2}n][PF6]n and through Ag...Ag, C=O...Ag and Ag...O(trifluoroacetate)...HN secondary bonds in [Ag(n){SC4H2-2,5-(CONHCH2-3-C5H4N)2}n][CF3CO2]n.

Journal Article↗

PubMed1994

In organello assembly of respiratory-chain complex I: primary structure of the 14.8 kDa subunit of Neurospora crassa complex I.

A cDNA encoding the 14.8 kDa subunit of complex I from Neurospora crassa was cloned and sequenced. The deduced primary structure of this subunit reveals a predominantly hydrophilic protein containing no obvious membrane-spanning domain. In agreement with this characteristic, we have localized the 14.8 kDa subunit in the peripheral arm of the enzyme. The 14.8 kDa subunit was found to be conserved in mammalian complex I. The conservation of this subunit in such distantly related organisms suggests that the 14.8 kDa subunit is an important component of complex I. We have used an in organello system to study the biosynthetic pathway of this subunit. The 14.8 kDa polypeptide could be efficiently imported into isolated mitochondria. Furthermore, a fraction of the in-vitro-imported subunit was found to assemble in complex I. This is the first time that assembly in complex I of an in-vitro-synthesized subunit is demonstrated.

Amino Acid Sequence↗

PubMed1996

Co-expression of the alpha subunit of human prolyl 4-hydroxylase with BiP polypeptide in insect cells leads to the formation of soluble and insoluble complexes. Soluble alpha-subunit-BiP complexes have no prolyl 4-hydroxylase activity.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the post-translational formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, their beta subunit being identical to protein disulphide isomerase (PDI). The function of the PDI-beta subunit in prolyl 4-hydroxylases is not fully understood, but it seems to be that of keeping the highly insoluble alpha subunits in solution. We report here that expression of the alpha subunit of human type I prolyl 4-hydroxylase in insect cells together with BiP polypeptide leads to the formation of both soluble and insoluble alpha-subunit-BiP complexes. Formation of the soluble complexes was evident from (1) a marked increase in the amount of the alpha subunit in the soluble fraction of the cell homogenates when expressed together with BiP, (2) immunoprecipitation experiments and (3) demonstration of the presence of some of the complexes by polyacrylamide gel electrophoresis under non-denaturing conditions. Formation of the insoluble complexes was suggested by an increase in the amount of BiP in the insoluble fraction when expressed together with the alpha subunit. Nevertheless the soluble alpha-subunit-BiP complexes had no prolyl 4-hydroxylase activity. This indicates that the function of the PDI-beta subunit in the prolyl 4-hydroxylase tetramer is not only that of keeping the alpha subunits in solution but appears to be more specific, probably that of keeping them in a catalytically active, non-aggregated conformation.

Amino Acid Sequence↗

PubMed2000

MBD2-MBD3 complex binds to hemi-methylated DNA and forms a complex containing DNMT1 at the replication foci in late S phase.

BACKGROUND: In vertebrates and plants, DNA methylation is one of the major mechanisms regulating gene expression. Recently, a family of methyl-CpG-binding proteins has been identified, and some members, such as MeCP2 and MBD2, were shown to mediate gene repression by recruiting histone deacetylase complexes to methylated genes. However, the function of another member of this family, MBD3, remained elusive. RESULTS: It was shown that MBD2 and MBD3 form homo- and hetero-dimers (or multimers) in vitro and in vivo. Significantly, the MBD2-MBD3 complex showed an affinity to hemi-methylated DNAs, a property that has never been reported with any member of the family proteins. MBD2 and MBD3 were co-localized with DNMT1 at replication foci in 293 cell nuclei at late S phase. Moreover, by a co-immunoprecipitation experiment, DNMT1 was shown to form a complex with MBD2 and MBD3. Finally, the abundance of MBD3 was highest in the late S phase when the DNMT1 is also most abundant, whereas the MBD2 level was largely constant throughout the cell cycle. CONCLUSIONS: The results suggest that MBD3 may play an important role in the S phase. We hypothesize that the MBD2-MBD3 complex recognizes hemi-methylated DNA concurrent with DNA replication and recruits histone deacetylase complexes, as well as DNMT1, to establish and/or maintain the transcriptionally repressed chromatin.

Chromatin↗

PubMed2003

Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro.

The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader/DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro, which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA.

Adenosine Triphosphate↗

PubMed2006

Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis.

The developing science called structural genomics has focused to date mainly on high-throughput expression of individual proteins, followed by their purification and structure determination. In contrast, the term structural biology is used to denote the determination of structures, often complexes of several macromolecules, that illuminate aspects of biological function. Here we bridge structural genomics to structural biology with a procedure for determining protein complexes of previously unknown function from any organism with a sequenced genome. From computational genomic analysis, we identify functionally linked proteins and verify their interaction in vitro by coexpression/copurification. We illustrate this procedure by the structural determination of a previously unknown complex between a PE and PPE protein from the Mycobacterium tuberculosis genome, members of protein families that constitute approximately 10% of the coding capacity of this genome. The predicted complex was readily expressed, purified, and crystallized, although we had previously failed in expressing individual PE and PPE proteins on their own. The reason for the failure is clear from the structure, which shows that the PE and PPE proteins mate along an extended apolar interface to form a four-alpha-helical bundle, where two of the alpha-helices are contributed by the PE protein and two by the PPE protein. Our entire procedure for the identification, characterization, and structural determination of protein complexes can be scaled to a genome-wide level.

Bacterial Proteins↗

PubMed1971

A complex between initiation factor IF2, guanosine triphosphate, and fMet-tRNA: an intermediate in initiation complex formation.

Evidence is presented that suggests the formation of a complex between polypeptide chain-initiation factor IF 2, GTP, and fMet-tRNA(f). This complex transfers both fMet-tRNA(f) and GTP to 30S ribosomal subunits in the presence of ApUpG and initiation factor IF 1. The resultant 30S initiation complex reacts with 50S subunits to form a 70S initiation complex. fMet-tRNA(f) in this 70S complex reacts with puromycin to form fMet-puromycin. These results suggest that [IF 2, GTP, fMet-tRNA(f)] is an intermediate in the initiation of protein synthesis in Escherichia coli.

Adenosine Monophosphate↗

PubMed1977

Theorems of Barth-Lefschetz type for complex subspaces of homogeneous complex manifolds.

Barth, Larsen, and others showed that complex submanifolds of complex projective space, CP(N), of small codimension strongly resemble CP(N) both homotopically and cohomologically. These results are generalized to yield analogous results for complex subspaces of arbitrary homogeneous complex manifolds. One very special corollary that gives the flavor of the results is:Corollary. Let A and B be complex submanifolds with B a connected submanifold of X, a simple Abelian variety, i.e., X has no proper sub-Abelian varieties. Then: [Formula: see text], where basepoints are suppressed for simplicity, and [Formula: see text]. Further, given any coherent sheaf on B, which may be assumed to be a connected local complete intersection, then: [Formula: see text] and given a coherent sheaf on X, then: [Formula: see text].

Journal Article↗

PubMed1979

Structural studies of of "active complex" of bleomycin: assignment of ligands to the ferrous ion in a ferrous-bleomycin-carbon monoxide complex.

Proton NMR studies at 360 MHz establish the binary Fe(II)-bleomycin complex to be paramagnetic with a spectrum covering 70 ppm. Addition of carbon monoxide generates a stable, diamagnetic Fe(II)-bleomycin-CO complex that is a putative structural analog of the "active" Fe(II)-bleomycin-O2 complex. The following six groups have been determined to be coordinated to the Fe(II) ion from analysis of the highly resolved 1H NMR spectra of this complex: CO, the primary and secondary amine nitrogens of the beta-aminoalanine moiety, the carbamoyl moiety on the 3-position of mannose, the pyrimidine N-1, and the imidazole N-1. The Fe(II)-bleomycin-CO complex binds to DNA, as shown by fluorescence quenching experiments, but Fe(II)-bleomycin-CO does not mediate thymine release. These results necessitate a major revision in the current model for metal coordination to bleomycin.

Bleomycin↗

PubMed1991

Bacterial chemotaxis signaling complexes: formation of a CheA/CheW complex enhances autophosphorylation and affinity for CheY.

We have demonstrated that a complex of the proteins CheA (CheAL and CheAS) and CheW can be isolated and constitutes a functional unit that responds to the signaling state of the chemoreceptors. The autophosphorylation rate of CheAL is much greater when CheAL and CheAS are complexed with CheW. Moreover, the presence of mutant chemoreceptors that cause cells to tumble increases this rate. At wild-type levels of expression, the isolated CheAL/CheAS/CheW complex accounts for about 10% of the total number of CheAL, CheAS, and CheW molecules and exists in a 1:1:1 stoichiometry. This complex is also required for CheAL/CheAS and CheW binding to the phosphorylation substrate, CheY. A separate interaction between CheY and another chemotaxis component, CheZ, was also detected. The CheY-CheZ interaction does not require participation of the CheAL/CheAS/CheW complex.

Adenosine Triphosphate↗

PubMed1991

Antigen-specific therapy of experimental allergic encephalomyelitis by soluble class II major histocompatibility complex-peptide complexes.

Experimental allergic encephalomyelitis is a T-cell-mediated, major histocompatibility complex (MHC) class II gene-linked autoimmune demyelinating disease of the central nervous system. To develop therapies that will specifically inactivate only the autoantigen-reactive T cells, mice were treated with soluble MHC class II molecules that had been complexed with encephalitogenic peptides. Intravenous injections of 300 micrograms of complexes consisting of encephalitogenic peptide 91-103 of myelin basic protein plus I-As protein on day 0, 4, and 7 were effective in preventing experimental allergic encephalomyelitis. Similarly, administration of 45 micrograms of I-As protein complexed to peptide 139-151 from proteolipoprotein on day 1, 4, and 7 prevented mortality and significantly reduced paralysis induced by immunization with the encephalitogenic proteolipoprotein peptide. Histological examination of sections of animal brains revealed that treatment with I-As protein plus myelin basic protein 91-103 peptide prevents the development of inflammatory lesions characteristic of experimental allergic encephalomyelitis. Thus, treatment with MHC-self-peptide complexes could serve as a highly specific therapeutic modality in treating autoimmune disease when the putative autoantigen and the MHC restricting elements are known.

Animals↗

PubMed1992

HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides.

The two subunits of the human class I histocompatibility antigen (HLA)-A2 have been expressed at high levels (20-30 mg/liter) as insoluble aggregates in bacterial cells. The aggregates were dissolved in 8 M urea and then refolded to form an HLA-A2-peptide complex by removal of urea in the presence of an antigenic peptide. Two peptides from the matrix protein and nucleoprotein of influenza virus are known to bind to HLA-A2, and both support the refolding of the recombinant HLA-A2 molecule. An additional peptide, a nonamer from the gp120 envelope protein of human immunodeficiency virus type 1, also supported refolding. Yields of purified recombinant HLA-A2 are 10-15%. In the absence of an HLA-A2-restricted peptide, a stable HLA-A2 complex was not formed. Monoclonal antibodies known to bind to native HLA-A2 also bound to the recombinant HLA-A2-peptide complex. Three purified HLA-A2-peptide complexes refolded from bacterially produced protein aggregates crystallize under the identical conditions as HLA-A2 purified from human lymphoblastoid cells. Crystals of the recombinant HLA-A2 molecule in complex with the influenza matrix nonamer peptide, Mp(58-66), diffract to greater than 1.5-A resolution.

Amino Acid Sequence↗

PubMed1993

The t-complex polypeptide 1 complex is a chaperonin for tubulin and actin in vivo.

A role in folding newly translated cytoskeletal proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide 1 (TCP1). In this study, we investigated tubulin and actin biogenesis in Chinese hamster ovary (CHO) cells. When extracts of pulse-labeled cells were analyzed by anion-exchange and size-exclusion chromatography, newly synthesized alpha-tubulin, beta-tubulin, and actin were observed to enter a large molecular mass complex (approximately 900 kDa). These proteins were released from this complex capable, in the case of tubulin, of forming heterodimers. The large molecular mass complexes coeluted with TCP1 and could be immunoprecipitated by using an anti-TCP1 antibody. These findings demonstrate that there is a cytosolic pathway for folding tubulin and actin in vivo that involves the TCP1 complex.

Actins↗

PubMed1993

Stimulation of T cells by antigenic peptide complexed with isolated chains of major histocompatibility complex class II molecules.

Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins with one alpha and one beta polypeptide chain of similar molecular size. In this report, we describe the binding of an acetylated N-terminal peptide of myelin basic protein, [Ala4]MBP-(1-14), to purified individual alpha and beta chains of murine I-Ak molecules. Purified complexes of isolated single chains and antigenic peptide bind to cloned T cells restricted by I-Ak and [Ala4]MBP-(1-14) tetradecapeptide. The binding is blocked by alpha/beta anti-T-cell receptor (TCR) monoclonal antibody. Cell triggering as measured by an increase in extracellular acidification rate is observed when cloned T cells are exposed to purified complexes of isolated chains and antigenic peptide. This increase in the extracellular acidification rate is antigen specific and MHC-restricted, as chains alone or irrelevant chain-peptide complexes do not trigger an increase in the metabolic acidification rate. These results together demonstrate that in vitro cloned T cells are triggered by complexes of specific antigenic peptides and isolated individual chains of their cognate MHC proteins.

Amino Acid Sequence↗

PubMed1996

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides.

Amino Acid Sequence↗

PubMed1995

Hormonal modulation of major histocompatibility complex class I gene expression involves an enhancer A-binding complex consisting of Fra-2 and the p50 subunit of NF-kappa B.

Hydrocortisone decreases major histocompatibility complex (MHC) class I gene expression in rat thyroid cells and counteracts increases induced by interferons. Using FRTL-5 cells transfected with class I promoter-reporter gene chimeras, we show that hydrocortisone action is transcriptional and mediated by an element located between 180 and 170 base pairs upstream of the start of transcription. Gel shift assays reveal that hydrocortisone causes the decrease of a specific protein-DNA complex; this same complex, referred to as Mod-1, is increased by interferon. Oligonucleotide competition assays reveal that the Mod-1 complex is associated with enhancer A of the class I gene, -180 to -170 base pairs (5'-GGGGAGTCCCC-3'), immediately upstream of the interferon response element. Antibodies to fra-2, a fos family member, and to the p50, but not the p65, subunit of NF-kappa B supershift the Mod-1 complex. We suggest that hydrocortisone decreases MHC class I gene expression by reducing the formation of Mod-1, which contains both p50 and fra-2; interferon reverses the hydrocortisone effect and increases Mod-1 formation. These observations are relevant to the molecular basis of hydrocortisone therapy in autoimmune thyroid disease and to the actions of interferon to exacerbate or induce autoimmune disease.

Animals↗

PubMed1995

Internalization of vitronectin-thrombin-antithrombin complex by endothelial cells leads to deposition of the complex into the subendothelial matrix.

Internalization of the ternary vitronectin-thrombin-antithrombin (VN-TAT) complex by human umbilical vein endothelial cells was investigated. Radiolabeled VN-TAT was bound to the cell surface at 4 degrees C, and internalization was initiated by increasing the temperature to 37 degrees C. After 30 min about half of the VN-TAT complex disappeared from the cell surface and accumulated in the subendothelial matrix. Translocation of VN-TAT complex from the luminal to the basolateral side was confirmed by electron microscopic evaluation of cross-sections of endothelial cells incubated with gold-conjugated VN-TAT complex. Furthermore, cells cultured in VN-TAT deficient serum, incubated with purified VN-TAT, and subsequently assayed for fluorescent staining using a monoclonal antibody directed against thrombin-modified antithrombin and a polyclonal antibody against vitronectin showed co-localization of both antibodies in punctates. Punctates were randomly distributed in both the xy and xz plane of endothelial cells as evidenced by confocal laser scanning microscopy. Trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis showed that VN-TAT was not degraded during translocation and inhibition of the microfilament system reduced release of VN-TAT to the matrix, indicating that transcytosis was responsible for translocation. These findings emphasize that VN-TAT complex is taken up by endothelial cells, not only leading to the removal of inactivated thrombin from the circulation but also to deposition of VN into the subendothelial matrix.

Antithrombin III↗

PubMed1997

Analysis of a yeast SNARE complex reveals remarkable similarity to the neuronal SNARE complex and a novel function for the C terminus of the SNAP-25 homolog, Sec9.

SNARE proteins represent a family of related proteins that are thought to have a central role in vesicle targeting and fusion in all eukaryotic cells. The binding properties of the neuronal proteins synaptobrevin 1 (VAMP1), syntaxin 1, SNAP-25, and soluble N-ethylmaleimide-sensitive factor attachment protein (alpha-SNAP), have been extensively studied. We report here the first biochemical characterization of a nonneuronal SNARE complex using recombinant forms of the yeast exocytic SNARE proteins Snc1, Sso1, and Sec9 and the yeast alpha-SNAP homolog, Sec17. Despite the low level of sequence identity, the association properties of the yeast and neuronal complexes are remarkably similar. The most striking difference we have found between the yeast and neuronal proteins is that individually neither of the target membrane SNAREs (t-SNAREs), Sso1 nor Sec9, show any detectable binding to the synaptobrevin homolog, Snc1. However, as a hetero-oligomeric complex, Sec9 and Sso1 show strong binding to Snc1. The clear dependence on the Sso1-Sec9 complex for t-SNARE function suggests that regulating the formation of this complex may be a key step in determining the site of vesicle fusion. In addition, we have used this in vitro assay to examine the biochemical effects of several mutations in Sec9 that result in pronounced growth defects in vivo. As expected, a temperature-sensitive mutation in the region most highly conserved between Sec9 and SNAP-25 is severely diminished in its ability to bind Sso1 and Snc1 in vitro. In contrast, a temperature-sensitive mutation near the C terminus of Sec9 shows no defect in SNARE binding in vitro. Similarly, a deletion of the C-terminal 17 residues, which is lethal in vivo, also binds Sso1 and Snc1 normally in vitro. Interestingly, we find that these same two C-terminal mutants, but not mutants that show SNARE assembly defects in vitro, act as potent dominant negative alleles when expressed behind a strong regulated promoter. Taken together these results suggest that the C-terminal domain of Sec9 is specifically required for a novel interaction that is required at a step following SNARE assembly.

Amino Acid Sequence↗