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PubMed2005

Three amino acids that are critical to formation and stability of the P22 tailspike trimer.

The P22 tailspike protein folds by forming a folding competent monomer species that forms a dimeric, then a non-native trimeric (protrimer) species by addition of folding competent monomers. We have found three residues, R549, R563, and D572, which play a critical role in both the stability of the native tailspike protein and assembly and maturation of the protrimer. King and colleagues reported previously that substitution of R563 to glutamine inhibited protrimer formation. We now show that the R549Q and R563K variants significantly delay the protrimer-to-trimer transition both in vivo and in vitro. Previously, variants that destabilize intermediates have shown wild-type chemical stability. Interestingly, both the R549Q and R563K variants destabilize the tailspike trimer in guanidine denaturation studies, indicating that they represent a new class of tailspike folding variants. R549Q has a midpoint of unfolding at 3.2M guanidine, compared to 5.6M for the wild-type tailspike protein, while R563K has a midpoint of unfolding of 1.8 M. R549Q and R563K also denature over a broader pH range than the wild-type tailspike protein and both proteins have increased sensitivity to pH during refolding, suggesting that both residues are involved in ionic interactions. Our model is that R563 and D572 interact to stabilize the adjacent turn, aiding the assembly of the dimer and protrimer species. We believe that the interaction between R563 and D572 is also critical following assembly of the protrimer to properly orient D572 in order to form a salt bridge with R549 during protrimer maturation.

Amino Acid Substitution↗
PubMed2000

Changes of matrilin forms during endochondral ossification. Molecular basis of oligomeric assembly.

To understand the molecular properties of matrilin-3, a newly discovered member of the novel extracellular matrix protein family, we cloned a MAT-3 cDNA from developing chicken sterna. Real time quantitative reverse-transcription polymerase chain reaction indicates that MAT-3 mRNA is mainly expressed in the proliferation zone of a growth plate. It is also expressed in the maturation zone, overlapping with that of the mature chondrocyte-abundant matrilin-1 mRNA. This suggests that matrilin-3 may self-assemble in the proliferation zone, in addition to its co-assembly with matrilin-1 during endochondral ossification. Transfection of a MAT-3 cDNA into COS-7 cells shows that MAT-3 predominantly forms a homotetramer but also a trimer and a dimer. Co-transfection of both MAT-3 and MAT-1 cDNAs results in three major matrilins as follows: (MAT-1)(3), (MAT-3)(4), and (MAT-1)(2)(MAT-3)(2). Thus matrilin-3 may assemble into both homotypic and heterotypic oligomers. Our analysis shows that the assembly of MAT-3 does not depend on the number of epidermal growth factor repeats within the molecule, but the presence of Cys(412) and Cys(414) within the coiled-coil domain, which form covalent disulfide linkage responsible for both homo-oligomerization of MAT-3 and hetero-oligomerization of MAT-3 and MAT-1. Our data suggest that the varying synthetic levels of matrilins in different zones of a growth plate may result in a change of matrilin oligomeric forms during endochondral ossification.

Animals↗
PubMed2004

Sequence-induced trimerization of phospholipase A2: structure of a trimeric isoform of PLA2 from common krait (Bungarus caeruleus) at 2.5 A resolution.

The venom of the common Indian krait (Bungarus caeruleus) contains about a dozen isoforms of phospholipase A2 (PLA2), which exist in different oligomeric forms as well as in complexes with low-molecular-weight ligands. The basic objective of multimerization and complexation is either to inactivate PLA2 in the venom for long-term storage, to generate a new PLA2 function or to make a more lethal assembly. The current isoform was isolated from the venom of B. caeruleus. Dynamic light-scattering studies indicated the presence of a stable trimeric association of this PLA2. Its primary sequence was determined by cDNA cloning. The purified protein was crystallized with 2.8 M NaCl as a precipitating agent using the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 80.9, b = 80.5, c = 57.1 A, beta = 90.3 degrees. The structure was refined to a final R factor of 0.198. This is a novel trimeric PLA2 structure in which the central pore formed by the association of three molecules is filled with water molecules. The interactions across the pore take place via multiple water bridges primarily to the side chains of Arg, Lys and Thr residues. Approximately 12% of the total solvent-accessible surface area is buried in the core of the trimer. The active sites of all three molecules are located on the surface and are fully exposed to the solvent, resulting in a highly potent enzymatic unit.

Amino Acid Sequence↗
PubMed2006

Crystal structure of infectious bursal disease virus VP2 subviral particle at 2.6A resolution: implications in virion assembly and immunogenicity.

The structural protein VP2 of infectious bursal disease virus (IBDV) spontaneously forms a dodecahedral T=1 subviral particle (SVP), and is a primary immunogen of the virus. In this study, the structure of IBDV SVP was determined in a cubic crystal and refined to 2.6A resolution. It contains 20 independent VP2 subunits in a crystallographic asymmetric unit. Each subunit is folded mainly into a shell domain and a protrusion domain, both with the Swiss-roll topology, plus a small helical base domain. Three VP2 subunits constitute a tight trimer, which is the building block of IBDV (sub)viral particles. The structure revealed a calcium ion bound to three pairs of symmetry-related Asp31 and Asp174 to stabilize the VP2 trimer. Our results of treatment of SVP with EGTA, a Ca(2+)-chelating reagent, indicated that the metal-ion may be important not only in maintaining highly stable quaternary structure but also in regulating the swelling and dissociation of the icosahedral particles. A Ca(2+)-dependent assembly pathway was thus proposed, which involves further interactions between the trimers. The 20 independent subunits showed conformational variations, with the surface loops of the protrusion domain being the most diverse. These loops are targets of the neutralizing antibodies. Several common interactions between the surface loops were clearly observed, suggesting a possible major conformation of the immunogenic epitopes.

Amino Acid Sequence↗
PubMed1996

The role of prenylation in G-protein assembly and function.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) are vital components of numerous signal transduction pathways, including sensory and hormonal response systems. G-proteins transduce signals from heptahelical transmembrane receptors to downstream effectors. The localization of a G-protein to the plasma membrane, as well as its interaction with the appropriate receptor and effector, are essential for its function. In addition, the association of a G-protein's subunits to form its trimer is required for interaction with its receptor. The G-protein gamma subunits (G gamma) are subject to a set of carboxyl-terminal processing events that include prenylation of a cysteine, proteolysis, and methylation. Recent advances which elucidate the contributions that the post-translational modifications of the G gamma subunit have on the assembly, membrane association, and function of the G-protein trimer reveal that these modifications are required for important protein-protein, in addition to membrane-protein, interactions.

Animals↗
PubMed1984

Physicochemical characterization of the 68 000-dalton protein of bovine neurofilaments.

The 68 000-dalton protein from bovine neurofilaments was purified by a combination of chromatography on DEAE-cellulose and on hydroxylapatite in buffers containing 8 M urea. Although the separation of this protein from the other proteins of the neurofilament appeared to be hampered by a mixed association of the several components, a nearly homogeneous product was obtained for study. Sedimentation equilibrium experiments in buffers containing 8 M urea showed the molecule to be a monomer with a molecular weight of 70 600 +/- 2000. Circular dichroic spectra taken under the same conditions gave no evidence of residual alpha-helix. Molecular sieve chromatography in 8 M urea on controlled-pore glass showed that the molecule eluted at an unexpectedly small volume. The small elution volume did not depend significantly on protein concentration and is unlikely to be the result of intermolecular association. Rather, the monomer probably has a conformation more rigid or extended than a classical random coil. When dialyzed into 0.01 M tris(hydroxymethyl)aminomethane/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.1 mM dithioerythritol, pH 8.5, the protein does not assemble into filaments. Sedimentation velocity reveals that under these conditions it consists mainly of a 4.8S molecular species, containing few large particles; sedimentation equilibrium shows that it is composed of oligomers, the smallest present in significant concentration having a molecular weight approximately that of a trimer. Circular dichroism measurements lead to the interpretation that the molecule has refolded in this buffer into a structure that has approximately 55% alpha-helix. Assembly into filamentous particles resembling neurofilaments occurs when the protein is dialyzed against 0.1 M 2-(N-morpholino)ethane-sulfonic acid/0.1% beta-mercaptoethanol/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.17 M NaCl, pH 6.5. We suggest that the oligomeric species present in 0.01 M tris(hydroxymethyl)aminomethane may frequently be present in solubilized preparations of intermediate filaments and may represent an intermediate in the assembly process.

Animals↗
PubMed1995

The 6-kilodalton membrane protein of Semliki Forest virus is involved in the budding process.

Alphavirus genomes encode a small hydrophobic protein of 6 kDa (the 6K protein) that is expressed as part of a large polyprotein containing the sequences of the two virus transmembranal glycoproteins which form the spikes of the infectious particle. Although made in amounts equivalent to those of the glycoproteins, very little of the 6K protein is found in secreted infectious virions. The role of this protein in virus replication and structure has been studied by use of a variety of mutationally altered forms of 6K, which yield phenotypically distinct viruses. A complete deletion of the gene encoding the 6K protein (delta 6K) of Semliki Forest Virus (SFV) has been constructed from an SFV infectious cDNA and the transcribed RNA-produced progeny virus that closely resembled the normal virus (P. Liljeström, S. Lusa, D. Huylebroeck, and H. Garoff, J. Virol. 65:4107-4113, 1991). Further studies of this mutant have now been performed, and they show that growth of delta 6K has a strong dependency on its host cell, varying from 2 to 50% of the rate of formation of the wild-type SFV. Mammalian cells are much more defective than insect and avian cells in replication of the delta 6K mutant. This mutant is not defective in formation and transport of the glycoproteins or in production of nucleocapsids, which accumulate at the plasma cell membrane in infected BHK cells. The major defect, thus, is in the final assembly and budding of new virus. In BHK cells infected with the delta 6K strain, a relatively large fraction of the total infectious virus formed can be recovered by osmotic lysis of exhaustively washed cells. Infectious SFV totally lacking 6K is identical to wild-type SFV in the early stages of virus replication, i.e., binding and uptake. The particles themselves are more thermolabile than those of wild-type SFV, suggesting that the 6K protein may be a part of the structure of wild-type virus or that the slower budding leads to an altered configuration of the trimeric spikes. These data support other studies that implicate the 6K protein as an important but nonessential component in the assembly and budding of the alphavirus particle, perhaps by affecting the packing of the glycoproteins and their interactions with membrane lipid.

Animals↗
PubMed2002

Cutting edge: single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells.

We report in this work the expression and characterization of class I molecules expressed as single-chain trimers consisting of an antigenic peptide-spacer-beta(2)-microglobulin-spacer H chain. Our results indicate that these single-chain constructs assemble efficiently, maintain their covalent structure, and are unusually stable at the cell surface. Consequently, these constructs are at least 1000-fold less accessible to exogenous peptide than class I molecules loaded with endogenous peptides, and they are potent simulators of peptide-specific CTL and Abs. Our combined findings suggest that single-chain trimers may have applications as DNA vaccines against virus infection or tumors.

Animals↗
PubMed1989

Characterization of bacteriophage P22 tailspike mutant proteins with altered endorhamnosidase and capsid assembly activities.

The tailspike protein of Salmonella typhimurium phage P22 is a multifunctional homotrimer which is involved in the terminal reaction of phage assembly, the adsorption of the phage to susceptible cells, and the hydrolysis of the Salmonella O-antigen during the first steps of phage infection. The proteins made from 15 mutant tailspike structural genes carried on high level expression plasmids have been analyzed with respect to their in vivo stability, quaternary structure, capsid assembly activity, and enzymatic activity. Nine mutants synthesize tailspike proteins which fail to accumulate to any appreciable level in vivo, and thus these proteins are probably degraded. Four other altered proteins accumulate in vivo as soluble monomers. The remaining two altered proteins accumulate in vivo as stable trimers. Each of these two proteins is defective for at least one of the known functions of the tailspike protein. One is defective in the capsid assembly reaction and shows an unusual quaternary structural defect but is normal with respect to the enzymatic hydrolysis of O-antigen. The other is defective in the enzymatic hydrolysis of O-antigen but is normal with respect to its capsid assembly activity and quaternary structure. The known sequence changes which give rise to these altered proteins and those of previously identified mutants allow the description of possible functional and structural "domains" of this multifunctional protein.

Capsid↗
PubMed1996

Canine parvovirus capsid structure, analyzed at 2.9 A resolution.

The DNA-containing capsid of canine parvovirus (CPV) is analyzed following atomic refinement at 2.9 A resolution. The capsid contains 60 copies of the capsid protein related by icosahedral symmetry. The atomic model has been extended from the first residue (Gly37) of the unrefined 3.25 A structure towards the N terminus. The electron density shows that approximately 87% of the capsid proteins have N termini on the inside of the capsid, but for approximately 13%, the polypeptide starts on the outside and runs through one of the pores surrounding each 5-fold axis, explaining apparently conflicting antigenic data. Analysis of potential hydrogen bonds reveals approximately 50% more secondary structure than previously apparent. Most of the additional secondary structure are in the 71 and 221 residue-long loop insertions between beta-strands E and F and G and H, forming subunit-bridging sheets that likely add specificity to assembly interactions. Structural analysis of the extensive subunit interactions around the 3-fold axes shows that assembly is a multistep process with loops intertwining following initial contact. Estimated free energies of association suggest that the formation of 3 and 5-fold contacts likely takes precedence over 2-fold interactions. Energies for initial association into trimers or pentamers would be similar, but the intertwining of loops about the 3-fold axis adds an additional large activation barrier to dissociation. Analysis of the surfaces of the assembled capsid shows a surprising lack of basic amino acids that might have been expected to interact with the negatively charged phosphoribose backbone of the DNA. Instead, uncharged polar and van der Waal's interactions predominate in the packaging of single-stranded DNA into the capsid.

Amino Acid Sequence↗
PubMed2003

A heterologous coiled coil can substitute for helix I of the Sindbis virus capsid protein.

Alphavirus core assembly proceeds along an assembly pathway involving a dimeric assembly intermediate. Several regions of the alphavirus capsid protein have been implicated in promoting and stabilizing this dimerization, including a putative heptad repeat sequence named helix I. This sequence, which spans residues 38 to 55 of the Sindbis virus capsid protein, was implicated in stabilizing dimeric contacts initiated through the C-terminal two-thirds of the capsid protein and nucleic acid. The studies presented here demonstrate that helix I can be functionally replaced by the corresponding sequence of a related alphavirus, western equine encephalitis virus, and also by an unrelated sequence from the yeast transcription activator, GCN4, that was previously shown to form a dimeric coiled coil. Replacing helix I with the entire leucine zipper domain of GCN4 (residues 250 to 281) produced a virus with the wild-type phenotype as determined by plaque assay and one-step growth analysis. However, replacement of helix I with a GCN4 sequence that favored trimer formation produced a virus that exhibited approximately 40-fold reduction in virus replication compared to the wild-type Sindbis virus. Changing residues within the Sindbis virus helix I sequence to favor trimer formation also produced a virus with reduced replication. Peptides corresponding to helix I inhibited core-like particle assembly in vitro. On the basis of these studies, it is proposed that helix I favors capsid protein-capsid protein interactions through the formation of dimeric coiled-coil interactions and may stabilize assembly intermediates in the alphavirus nucleocapsid core assembly pathway.

Amino Acid Sequence↗
PubMed1984

Rate constants for actin polymerization in ATP determined using cross-linked actin trimers as nuclei.

Chemically cross-linked actin trimer, prepared from F-actin covalently cross-linked by N-N'-p-phenylenebismaleimide, was used to nucleate the assembly of actin polymers under a variety of ionic conditions at 25 degrees C. Gel-filtered G-actin (5% labeled with N-pyrenyl iodoacetamide) was prepared in buffers containing 5 mM Tris X HCl, 0.2 mM ATP, 0.05 mM MgCl2 or 0.1 mM CaCl2, 0.2 mM dithiothreitol, and 0.01% sodium azide, pH 7.5. Polymerization was initiated by adding salt to a solution of actin monomer (2-25 microM) mixed with actin trimer (0-24 nM). Pseudo-first-order polymerization kinetics were observed in all cases following a lag phase of 30-75 s. The length of the lag phase was independent of trimer concentration, monomer concentration, or preincubation of monomer or trimer in polymerizing buffer. A simple model is presented which explains all of the observed features of the lag phase. Lower limits for the sums of the elongation rate constants at the two filament ends, calculated assuming each trimer acts as a seed, were in the range 1.4-5.2 microM-1 s-1, and lower limits for the polymer dissociation rate constants (calculated from the elongation rate constants and the critical actin concentration) were 0.4-1.9 s-1, depending on ionic conditions. The observed rate constants were independent of actin concentration or trimer concentration under a given ionic condition. These polymerization rate constants are in good agreement with the values others have obtained by measuring the growth of actin filaments by electron microscopy.

Actins↗
PubMed2000

Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly.

The bacteriophage T4 DNA polymerase holoenzyme, consisting of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62), is loaded onto DNA in an ATP-dependent, multistep reaction. The trimeric, ring-shaped gp45 is loaded onto DNA such that the DNA passes through the center of the ring. gp43 binds to this complex, thereby forming a topological link with the DNA and increasing its processivity. Using stopped-flow fluorescence-resonance energy transfer, we have investigated opening and closing of the gp45 ring during the holoenzyme assembly process. Two amino acids that lie on opposite sides of the gp45 subunit interface, W91 and V162C labeled with coumarin, were used as the fluorescence donor and acceptor, respectively. Free in solution, gp45 has two closed subunit interfaces with W91 to V162-coumarin distances of 19 A and one open subunit interface with a W91 to V162C-coumarin distance of 40 A. Making the assumption that the distance across the two closed subunit interfaces is unchanged during the holoenzyme assembly process, we have found that the distance across the open subunit interface is first increased to greater than 45 A and is then decreased to 30 A during a 10-step assembly mechanism. The gp45 ring is not completely closed in the holoenzyme complex, consistent with previous evidence suggesting that the C-terminus of gp43 is inserted into the gp45 subunit interface. Unexpectedly, ATP-hydrolysis events are coupled to only a fraction of the total distance change, with conformational changes linked to binding DNA and gp43 coupled to the majority of the total distance change. Using the nonhydrolyzable ATP analogue ATP-gamma-S results in formation of a nonproductive gp45 x gp44/62 complex; however, adding an excess of ATP to this nonproductive complex results in rapid ATP/ATP-gamma-S exchange to yield a productive gp45 x gp44/62 complex within seconds.

Adenosine Diphosphate↗
PubMed2006

The structures of frataxin oligomers reveal the mechanism for the delivery and detoxification of iron.

Defects in the mitochondrial protein frataxin are responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1:40,000 children. Here, we present the crystal structures of the iron-free and iron-loaded frataxin trimers, and a single-particle electron microscopy reconstruction of a 24 subunit oligomer. The structures reveal fundamental aspects of the frataxin mechanism. The trimer has a central channel in which one atom of iron binds. Two conformations of the channel with different metal-binding affinities suggest that a gating mechanism controls whether the bound iron is delivered to other proteins or transferred to detoxification sites. The trimer constitutes the basic structural unit of the 24 subunit oligomer. The architecture of this oligomer and several features of the trimer structure demonstrate striking similarities to the iron-storage protein ferritin. The data reveal how stepwise assembly provides frataxin with the structural flexibility to perform two functions: metal delivery and detoxification.

Amino Acid Sequence↗
PubMed2000

TAP expression provides a general method for improving the recognition of malignant cells in vivo.

A major class of tumors lack expression of the transporters associated with antigen processing (TAP). These proteins are essential for delivery of antigenic peptides into the lumen of the endoplasmic reticulum (ER) and subsequent assembly with nascent major histocompatibility complex (MHC) class I, which results in cell surface presentation of the trimeric complex to cytolytic T lymphocytes. Cytolytic T lymphocytes are major effector cells in immunosurveillance against tumors. Here we have tested the hypothesis that TAP downregulation in tumors allows immunosubversion of this effector mechanism, by establishing a model system to examine the role of TAP in vivo in restoring antigen presentation, immune recognition, and effects on malignancy of the TAP-deficient small-cell lung carcinoma, CMT.64. To test the potential of providing exogenous TAP in cancer therapies, we constructed a vaccinia virus (VV) containing the TAP1 gene and examined whether VV-TAP1 could reduce tumors in mice. The results demonstrate that TAP should be considered for inclusion in cancer therapies, as it is likely to provide a general method for increasing immune responses against tumors regardless of the antigenic complement of the tumor or the MHC haplotypes of the host.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1972

Precursors of collagen secreted by cultured human fibroblasts.

Soluble precursors of collagen ("procollagens") in the culture medium of human diploid fibroblasts were characterized by molecular sieve chromatography on 6% agarose and by sodium dodecyl sulfate-acrylamide gel electrophoresis. Under the denaturing conditions for gels, the following molecular species were identified: a major species of three procollagen chains, a procollagen dimer, and free procollagen chains. Reduction with 2-mercaptoethanol completely dissociated the trimer and dimer, releasing procollagen alpha1 and procollagen alpha2 chains with molecular weights of about 120,000. Both pro alpha1 and pro alpha2 chains could be labeled with [(35)S]cystine. In addition, free procollagen chains with molecular weights of less than 120,000, but heavier than native alpha chains, were identified in gels. Radioactivity in all of the above molecules could be chased into native alpha chains extracted from extracellular fibers of the cell layers. The data indicate that: (i) collagen is secreted as a disulfidestabilized procollagen trimer with the composition, (pro alpha1)(2).pro alpha2; (ii) noncovalent interactions maintain the three-chain assembly during stepwise enzymatic excisions of N-terminal, cysteine-containing procellagen peptides; and (iii) after such excisions, native helical molecules, (alpha1)(2).alpha2, precipitate as fibers in the cell layers.

Carbon Isotopes↗
PubMed1997

Structure-function studies of the human immunodeficiency virus type 1 matrix protein, p17.

The human immunodeficiency virus type 1 (HIV-1) matrix protein, p17, plays important roles in both the early and late stages of the viral life cycle. Using our previously determined solution structure of p17, we have undertaken a rational mutagenesis program aimed at mapping structure-function relationships within the molecule. Amino acids hypothesized to be important for p17 function were mutated and examined for effect in an infectious proviral clone of HIV-1. In parallel, we analyzed by nuclear magnetic resonance spectroscopy the structure of recombinant p17 protein containing such substitutions. These analyses identified three classes of mutants that were defective in viral replication: (i) proteins containing substitutions at internal residues that grossly distorted the structure of recombinant p17 and prevented viral particle formation, (ii) mutations at putative p17 trimer interfaces that allowed correct folding of recombinant protein but produced virus that was defective in particle assembly, and (iii) substitution of basic residues in helix A that caused some relocation of virus assembly to intracellular locations and produced normally budded virions that were completely noninfectious.

Amino Acid Sequence↗
PubMed2001

Polyamines and cell migration.

Leeuwenhoek first described polyamines in 1677, but active investigation did not begin until the 1970's. When intracellular polyamine levels are reduced by inhibitors, mutation, or transfection, severe reductions occur in cell division, cell differentiation, and cell migration. These effects are not difficult to demonstrate and measure, and all can be prevented if supplemental exogenous polyamines are supplied. However, linking the overall effects to molecular events remains to be accomplished. In this review, we discuss work (mostly from the last 10 years) that relates to cell migration. Specifically, we have discussed the biology and biochemistry of the polyamines, their transport and regulation, the structure of the cytoskeleton and the mechanics of cell movement. We have also considered four specific processes that polyamines participate in that may affect cell migration significantly. These are: 1) the regulation of intracellular Ca++ concentration by voltage-gated K+ channels, 2) the maintenance of normal RhoA levels that, with Rac, regulate the assembly of actin stress fibers, focal adhesions, and contractility, 3) the formation of ATP-Mg++-polyamine trimers that enhance the phosphorylation activity of ATP toward enzymes in specific signaling pathways and, 4) alterations in the structure of RNA that change translation initiation sites and affect the expression of proteins.

Animals↗