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PubMed2005

A coiled-coil interaction mediates cauliflower mosaic virus cell-to-cell movement.

The function of the virion-associated protein (VAP) of cauliflower mosaic virus (CaMV) has long been only poorly understood. VAP is associated with the virion but is dispensable for virus morphogenesis and replication. It mediates virus transmission by aphids through simultaneous interaction with both the aphid transmission factor and the virion. However, although insect transmission is not fundamental to CaMV survival, VAP is indispensable for spreading the virus infection within the host plant. We used a GST pull-down technique to demonstrate that VAP interacts with the viral movement protein through coiled-coil domains and surface plasmon resonance to measure the interaction kinetics. We mapped the movement protein coiled-coil to the C terminus of the protein and proved that it self-assembles as a trimer. Immunogold labeling/electron microscopy revealed that the VAP and viral movement protein colocalize on CaMV particles within plasmodesmata. These results highlight the multifunctional potential of the VAP protein conferred by its efficient coiled-coil interaction system and show a plant virus possessing a surface-exposed protein (VAP) mediating viral entry into host cells.

Amino Acid Sequence↗
PubMed2005

Crystal structure of Bacillus subtilis anti-TRAP protein, an antagonist of TRAP/RNA interaction.

In Bacillus subtilis the anti-TRAP protein (AT) is produced in response to the accumulation of uncharged tRNA(Trp). AT regulates expression of genes involved in tryptophan biosynthesis and transport by binding to the tryptophan-activated trp RNA-binding attenuation protein (TRAP) and preventing its interaction with several mRNAs. Here, we report the x-ray structure of AT at 2.8 angstroms resolution, showing that the protein subunits assemble into tight trimers. Four such trimers are further associated into a 12-subunit particle in which individual trimers are related by twofold and threefold symmetry axes. Twelve DnaJ-like, cysteine-rich zinc-binding domains form spikes on the surface of the dodecamer. Available data suggest several possible ways for AT to interact with the 11-subunit TRAP. Interaction between the two symmetry-mismatching molecules could be assisted by the flexible nature of AT zinc-binding domains.

Bacillus subtilis↗
PubMed1995

The type I collagen pro alpha 1(I) COOH-terminal propeptide N-linked oligosaccharide. Functional analysis by site-directed mutagenesis.

The C-propeptides of the pro alpha 1(I) and pro alpha 2(I) chains of type I collagen are each substituted with a single high-mannose N-linked oligosaccharide. Conservation of this motif among the fibrillar collagens has led to the proposal that the oligosaccharide has structural or functional importance, but a role in collagen biosynthesis has not been unambiguously defined. To examine directly the function of the pro alpha 1(I) C-propeptide N-linked oligosaccharide, the acceptor Asn residue was changed to Gln by site-directed mutagenesis. In transfected mouse Mov13 and 3T6 cells, unglycosylated mutant pro alpha 1(I) folded and assembled normally into trimeric molecules with pro alpha 2(I). In biosynthetic pulse-chase experiments mutant pro alpha 1(I) were secreted at the same rate as wild-type chains; however, following secretion, the chains were partitioned differently between the cell layer and medium, with a greater proportion of the mutant pro alpha 1(I) being released into the medium. This distribution difference was not eliminated by the inclusion of yeast mannan indicating that the high-mannose oligosaccharide itself was not binding to the matrix or the fibroblast surface after secretion. Subtle alterations in the tertiary structure of unglycosylated C-propeptides may have decreased their affinity for a cell-surface component. Further support for a small conformational change in the mutant C-propeptides came from experiments suggesting that unglycosylated pro alpha 1(I) chains were cleaved in vitro by the purified C-proteinase slightly less efficiently than wild-type chains. Mutant and normal pro alpha 1(I) were deposited with equal efficiency into the 3T6 cell accumulated matrix, thus the reduced cleavage by C-proteinase and altered distribution in the short pulse-chase experiments were not functionally significant in this in vitro extracellular matrix model system.

Amino Acid Sequence↗
PubMed2000

Characterization of the Saccharomyces cerevisiae high affinity copper transporter Ctr3.

Copper is an essential nutrient required for the activity of a number of enzymes with diverse biological roles. In the bakers' yeast Saccharomyces cerevisiae, copper is transported into cells by two high affinity copper transport proteins, Ctr1 and Ctr3. Although Ctr1 and Ctr3 are functionally redundant, they bear little homology at the amino acid sequence level. In this report, we characterize Ctr3 with respect to its localization, assembly, and post-transcriptional regulation. Ctr3 is an integral membrane protein that assembles as a trimer to form a competent copper uptake permease at the plasma membrane. Whereas the CTR1 and CTR3 genes are similarly regulated at the transcriptional level in response to copper, post-transcriptional regulation of these proteins is distinct. Unlike Ctr1, the Ctr3 transporter is neither regulated at the level of protein degradation nor endocytosis as a function of elevated copper levels. Our studies suggest that Ctr3 constitutes a fundamental module found in all eukaryotic high affinity copper transporters to date, which is sufficient for copper uptake but lacks elements for post-transcriptional regulation by copper.

Antiporters↗
PubMed2002

Passive acquisition of ligand by the MopII molbindin from Clostridium pasteurianum: structures of apo and oxyanion-bound forms.

MopII from Clostridium pasteurianum is a molbindin family member. These proteins may serve as intracellular storage facilities for molybdate, which they bind with high specificity. High resolution structures of MopII in a number of states, including the first structure of an apo-molbindin, together with calorimetric data, allow us to describe ligand binding and provide support for the proposed storage function of the protein. MopII assembles as a trimer of dimers and binds eight oxyanions at two types of binding sites located at intersubunit interfaces. Two type 1 sites are on the molecular 3-fold axis and three pairs of type 2 sites occur on the molecular 2-fold axes. The hexamer is largely unaffected by the binding of ligand. Molybdate is admitted to the otherwise inaccessible type 2 binding sites by the movement of the N-terminal residues of each protein chain. This contrasts with the structurally related molybdate-dependent transcriptional regulator ModE, which undergoes extensive conformational rearrangements on ligand binding. Despite similarities between the binding sites of ModE and the type 2 sites of MopII the molbindin has a significantly reduced ligand affinity, due, in part, to the high density of negative charges at the center of the hexamer. In the absence of ligand this effects the movement of an important lysine side chain, thereby partially inactivating the binding sites. The differences are consistent with a biological role in molybdate storage/buffering.

Amino Acid Sequence↗
PubMed1999

Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily.

N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) is a cytoplasmic bifunctional enzyme involved in the biosynthesis of the nucleotide-activated UDP-GlcNAc, which is an essential precursor for the biosynthetic pathways of peptidoglycan and other components in bacteria. The crystal structure of a truncated form of GlmU has been solved at 2.25 A resolution using the multiwavelength anomalous dispersion technique and its function tested with mutagenesis studies. The molecule is composed of two distinct domains connected by a long alpha-helical arm: (i) an N-terminal domain which resembles the dinucleotide-binding Rossmann fold; and (ii) a C-terminal domain which adopts a left-handed parallel beta-helix structure (LbetaH) as found in homologous bacterial acetyltransferases. Three GlmU molecules assemble into a trimeric arrangement with tightly packed parallel LbetaH domains, the long alpha-helical linkers being seated on top of the arrangement and the N-terminal domains projected away from the 3-fold axis. In addition, the 2.3 A resolution structure of the GlmU-UDP-GlcNAc complex reveals the structural bases required for the uridyltransferase activity. These structures exemplify a three-dimensional template for the development of new antibacterial agents and for studying other members of the large family of XDP-sugar bacterial pyrophosphorylases.

Acetyltransferases↗
PubMed1992

Porins and specific channels of bacterial outer membranes.

Porins and specific channels both produce water-filled pores that allow the transmembrane diffusion of small solutes, but the latter contain specific ligand-binding sites within the channels. Recent structural studies show that many or most of these proteins exist as beta-barrels with the beta-strands traversing the thickness of the outer membrane. The channels often have diameters in the range of 1 nm, and thus the penetration rates of solutes through porin channels are likely to be affected strongly by what appear to be minor differences in the size, shape, hydrophobicity or charge of the solute molecule. With the specific channels, the presence of binding sites can accelerate very significantly the diffusion of some ligands when they are present at low concentrations. Thus these simple channels can sometimes achieve a surprising degree of real or apparent specificity. Recent data tend to favour the idea that these proteins are first exported into the periplasm, and then inserted into the outer membrane. Although lipopolysaccharides seem to play a significant role in the final assembly of the trimeric porins, the details of the targeting process still remain to be elucidated.

Amino Acid Sequence↗
PubMed1993

Structure/function relationships in the pyruvate dehydrogenase complex from Azotobacter vinelandii. Role of the linker region between the binding and catalytic domain of the dihydrolipoyl transacetylase component.

The role of the hinge region between the binding domain and the catalytic domain in dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii was addressed by deletion mutagenesis. Mutated dihydrolipoyl transacetylase proteins were constructed with a deletion of 11 amino acids in the hinge region between the binding domain and the N-terminal part of the catalytic domain of E2p [E2p(pAPE1)] and with a further deletion of 9 amino acids into the N-terminal sequence protruding from the globular structure of the catalytic domain [E2p(pAPE2)] and found to take part in the intratrimer interaction. Both proteins behaved as wild-type E2p with respect to catalytic activity and quaternary structure. The interaction of the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) with the mutated E2p proteins was studied. E2p(pAPE1) assembles to a trimeric pyruvate dehydrogenase complex (PDC) with 15% decreased complex activity. No difference in affinity towards the peripheral components was detected. Upon binding of E3, E2p(pAPE2) dissociates into trimers and monomers. At saturation, two dimers of E3 were bound/E2p monomer instead of one dimer/E2p chain in trimeric wild-type E2p or E2p (pAPE1). The monomeric E2p species was catalytically inactive. Upon binding of excess E1p, some monomer formation of the E2p mutant took place. E1p however can prevent monomerization by E3. It is concluded that E1p is bound between two different E2p chains in the trimer. The substrates CoA and acetyl-CoA also prevent monomerization because they are bound by amino acid residues of two different E2p chains. In the presence of CoA no difference in affinity with respect to E1p and E3 binding was observed. CoA (and acetylCoA) also prevent dissociation of the 24-subunit core structure of wild-type E2p when added before addition of E1p or E3. Therefore, it seems likely that in vivo A. vinelandii PDC is based on a 24-subunit E2p core, like Escherichia coli PDC. A functional difference between complexes based on a trimer or a 24-subunit core has not been observed. A role of the hinge region as a spacer to allow binding of E1p or E3 seems unlikely. The results are discussed on the basis of the three-dimensional structure of the catalytic domain.

Acetyltransferases↗
PubMed2004

Collectins: players of the innate immune system.

Collectins are a family of collagenous calcium-dependent defense lectins in animals. Their polypeptide chains consist of four regions: a cysteine-rich N-terminal domain, a collagen-like region, an alpha-helical coiled-coil neck domain and a C-terminal lectin or carbohydrate-recognition domain. These polypeptide chains form trimers that may assemble into larger oligomers. The best studied family members are the mannan-binding lectin, which is secreted into the blood by the liver, and the surfactant proteins A and D, which are secreted into the pulmonary alveolar and airway lining fluid. The collectins represent an important group of pattern recognition molecules, which bind to oligosaccharide structures and/or lipid moities on the surface of microorganisms. They bind preferentially to monosaccharide units of the mannose type, which present two vicinal hydroxyl groups in an equatorial position. High-affinity interactions between collectins and microorganisms depend, on the one hand, on the high density of the carbohydrate ligands on the microbial surface, and on the other, on the degree of oligomerization of the collectin. Apart from binding to microorganisms, the collectins can interact with receptors on host cells. Binding of collectins to microorganisms may facilitate microbial clearance through aggregation, complement activation, opsonization and activation of phagocytosis, and inhibition of microbial growth. In addition, the collectins can modulate inflammatory and allergic responses, affect apoptotic cell clearance and modulate the adaptive immune system.

Animals↗
PubMed1988

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes.

Bacterial Outer Membrane Proteins↗
PubMed2001

A C-Terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressed in vitro or in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms.While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585-610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585-610 of the ORF2 protein might be critical for capsid biogenesis.

Journal Article↗
PubMed1990

Iron uptake by human upper small intestine microvillous membrane vesicles. Indication for a facilitated transport mechanism mediated by a membrane iron-binding protein.

To investigate the hypothesis that iron absorption in man involves a carrier-mediated cellular uptake mechanism, influx velocity (Vo) of 59Fe3+ by isolated human microvillous membrane (MVM) vesicles of the upper small intestine was examined. Vo revealed saturation kinetics (Km = 315 nM; Vmax = 361 pmol Fe3+ x min-1 x mg protein-1) was temperature dependent and inhibited by pronase pretreatment of MVM. In the presence of an inwardly directed Na(+)-gradient a typical overshoot phenomenon with maximal uptake at 30-40 s was observed. The suggestion of an active, carrier-mediated uptake mechanism for iron was pursued by isolation of a 160-kD iron-binding protein from solubilized human MVM proteins. This glycoprotein was assembled as a trimer composed of 54-kD monomers. A monospecific antibody against the 54-kD subunit inhibited vesicular influx of Fe3+ into MVM by greater than 50%. Immunofluorescence and immunoblot analysis confirmed the localization of the protein in brush border plasma membranes. It was detectable in human intestinal mucosa and liver, but not in esophagus. These data indicate that the translocation of Fe3+ across human MVM represents a facilitated transport mechanism which is, at least in part, mediated by a membrane iron-binding protein.

Biological Transport↗
PubMed2002

[Creation of new supramolecular chemistry based on multiple interaction in aqueous solution].

Novel supramolecular chemistry of multinuclear zinc(II) complexes in aqueous solution has been created by utilizing multiple interactions with polyanions. We have established reliable and convenient synthetic methods of multidentate macrocyclic polyamines and their zinc(II) complexes and thereby undertook a focused investigation of four topics: 1) efficient inhibition of photo[2 + 2] cycloaddition of thymidine dimer by dimeric zinc(II) complexes; 2) selective extraction and transport of thymidine nucleotide derivatives from the aqueous phase to the organic phase by lipophilic zinc(II) complexes and selective recognition of thymidine nucleotides by ditopic zinc(II) complexes in aqueous solution; 3) supramolecular polyhedrons formed by self-assembly of a trimeric zinc(II) complex with cyanuric acid or trithiocyanuric acid; and 4) a selective fluorescent probe for lanthanide ions such as Y3+ and La3+ based on a double-functionalized ligand with carbamoyl and dansyl groups. This knowledge should afford new methodology for supramolecular chemistry in aqueous solution.

Anions↗
PubMed1996

Pericytes from microvessel fragment produce type IV collagen and multiple laminin isoforms.

In the microvascular system, pericytes are located at the abdominal side of capillary endothelial cells. To discover the role of pericytes in the microvascular system, we have analyzed the extracellular proteins secreted from pericytes isolated from microvessel fragments of rat epididymal fat pads and found that they synthesize substantial amounts of basement membrane components such as type IV collagen and laminins. Secretion of type IV collagen was markedly stimulated by ascorbic acid phosphate. Reducing and nonreducing sodium dodecyl sulfate gel electrophoresis showed that pericytes produce six laminin chains assembled into different trimeric isoforms. Two of them were similar to laminin variants produced by aortic and pulmonal endothelial cells but others were suggested to be novel variants.

Adipose Tissue↗
PubMed2000

Plasticity and steric strain in a parallel beta-helix: rational mutations in the P22 tailspike protein.

By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmonella phage P22 have been identified. Temperature-sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect thermal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel beta-helix. We modified tailspike by rational single amino acid replacements at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) mutations in a similar structural context as two of the four known su mutations, which have been suggested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the beta-helix core. The high-resolution crystal structure of mutant T326F proves that structural rearrangements only take place in the remarkably plastic lumen of the beta-helix, leaving the arrangement of the hydrogen-bonded backbone and thus the surface of the protein unaffected. This supports the notion that changes in the stability of an intermediate, in which the beta-helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mutants, on the other hand, appears to be more difficult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interactions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statistical analysis of the distribution of backbone dihedral angles in known three-dimensional protein structures that the adoption of straight phi/psi angles other than the most favorable ones is often caused by side-chain interactions. Proteins 2000;39:89-101.

Bacteriophage P22↗
PubMed1981

Assembly and processing of procollagen type III in chick embryo blood vessels.

The processing of [3H]proline-labeled procollagen III in excised chick embryo blood vessels was found to differ significantly from that of procollagen I in the same tissue. While first the amino propeptides and then the carboxyl propeptides were fairly rapidly cleaved from procollagen I, only the carboxyl propeptides were split off procollagen III, leaving pN-collagen III. This intermediate, which is only slowly converted to collagen III by loss of amino propeptides, was characterized by its sedimentation properties, isolation of the amino propeptide, and reaction with purified antibodies that are specific against bovine amino propeptide III. It is interchain disulfide-linked, both through the amino propeptide and the carboxyl ends of the collagen chains. The conversion of procollagen III to pN-collagen III either in blood vessels, or after isolation by a carboxyl procollagen peptidase obtained from chick tendon fibroblast cultures, is inhibited by 50 mM arginine. Underhydroxylated procollagen III was isolated from blood vessels treated with alpha, alpha'-dipyridyl. Its amino propeptides reacted with the above antibodies but were not linked to each other. In contrast, its carboxyl propeptides were interchain disulfide-bridged, supporting previous suggestions that the carboxyl propeptides play a role in the assembly of procollagen trimer.

Animals↗
PubMed1994

Export and trimerization pathways of maltoporin overlap in the inner membrane of Escherichia coli.

The production of functional porins involves multiple steps including: export of precursor polypeptides from the cytoplasm, assembly of monomers into trimers, and stabilization of trimers by association with lipopolysaccharide. In this report, a late export/assembly intermediate of the maltoporin (LamB) is found in the inner membrane of Escherichia coli using cell fractionation studies. This processed intermediate is transiently associated with a unique Triton X-100-insoluble subfraction of the inner membrane. The kinetics of appearance and solubility characteristics of this intermediate correspond to those of the metastable trimer form of LamB, suggesting that the export and assembly pathways overlap in the inner membrane prior to final localization in the bulk outer membrane.

Bacterial Outer Membrane Proteins↗
PubMed2004

A chimeric protein of simian immunodeficiency virus envelope glycoprotein gp140 and Escherichia coli aspartate transcarbamoylase.

The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R(2)) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C(6)R(6) ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.

Amino Acid Sequence↗