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PubMed1989

Identification of covalently linked trimeric and tetrameric D domains in crosslinked fibrin.

Following proteolytic conversion of fibrinogen to fibrin, clot assembly commences with formation of double-stranded fibrils that subsequently branch extensively in forming a three-dimensional network. Plasmin digests of fibrin clots that had first been covalently crosslinked by plasma transglutaminase (factor XIIIa) contained multimeric proteolytic fragments composed of crosslinked outer (D) domains of neighboring fibrin molecules. Two of these were larger than the well-known "D dimer" fragment and corresponded to D trimers and D tetramers, respectively. Whereas D dimers originate from crosslinked D domains at bimolecular junctions within two-stranded fibrils, D trimers and D tetramers evidently arise through crosslinking of contiguous D domains at trimolecular and tetramolecular junctions or at fibril branch points, respectively. Measurement of the widths of fibrils comprising trifunctional branches in thin fiber networks revealed tetramolecular branch points, which are formed by bifurcation of two double-stranded fibrils. In addition, another type of trifunctional structure, which we term the trimolecular branch point, was composed of three double-stranded fibrils. Crosslinking of D domains to form trimers may occur at this type of junction. These findings add to our understanding of the crosslinking arrangements that stabilize fibrin clot structure and the ways that fibrin molecules polymerize to form branches in the clot matrix.

Cross-Linking Reagents↗
PubMed1996

Structure, interactions and dynamics of PRD1 virus I. Coupling of subunit folding and capsid assembly.

Bacteriophage PRD1, which infects Escherichia coli and Salmonella typhimurium, consists of an icosahedral capsid enclosing a membrane-packaged double-stranded DNA genome. The viral shell has been investigated using time and temperature resolved Raman and ultraviolet-resonance Raman spectroscopy to reveal novel features of the capsid structure and its pathway of assembly from P3 subunits. Raman spectra show that the shell is thermostable to 50 degrees C, and disassembles between 50 and 70 C degrees with only a small change in P3 conformation. However, the products of thermal disassembly depend sensitively upon total protein concentration. Characterization by analytical ultracentrifugation indicates that below 8 mg/ml, the purified shell disassembles primarily into P3 trimers; at higher concentrations, larger multimers of P3 are formed. Guanidine hydrochloride (GuHCl) dissociation of the P3 shell yields similar results. Purified P3 trimers, isolated either by heat or GuHCl treatment, exhibit structure sensitivity between 30 and 50 degrees C. Thus, shell disassembly diminishes P3 thermostability. Both the lower temperature transition (30 degrees C to 50 degrees C) of the trimer and the higher temperature transition (50 degrees C to 70 degrees C) of the shell involve a conversion of approximately 5% of the P3 peptide backbone from alpha-helix to beta-strand. Deuterium exchange of the P3 peptide backbone reveals more rapid exchange in the shell than in the trimer, consistent with the observed non-specific polymerization of trimers at high concentration. Conversely, the exchange of indole 1NH groups shows that approximately 65% of tryptophan residues are protected against exchange in the assembled shell. The results suggest a mechanism for shell assembly in which the specific association of trimers into the correct shell architecture involves stabilization of a subunit alpha-helical domain and sequestering of selected side-chains from solvent access. We propose a capsid assembly model which couples P3 shell formation with the final step in folding of the P3 subunit.

Bacteriophages↗
PubMed1982

Maturation of the tail spike endorhamnosidase of Salmonella phage P22.

As part of a genetic analysis of the in vivo folding and subunit assembly of the P22 tail spike endorhamnosidase, we have studied the maturation of the newly synthesized 76,000-dalton polypeptide chains into thermostable tail spike oligomers. Four of 15 temperature-sensitive mutations in the structural gene for this protein result in electrophoretically distinct tail spikes. Cells mixedly infected with wild type and an electrophoretic variant produce two hybrid species, with mobilities intermediate between the parental species, indicating that the native tail spike is a trimer. Mature trimers are resistant to denaturation by sodium dodecyl sulfate (SDS): at room temperature the trimer migrates in an SDS gel as if it were not binding significant amounts of SDS, whereas the heat-denatured chain migrates as expected of an SDS-polypeptide complex. The mature trimer is also resistant to trypsin digestion. Lysates of infected cells contain SDS and trypsin-sensitive forms of the newly synthesized tail spike polypeptide chains. These are probably incompletely or incorrectly folded chains. SDS and trypsin resistance were used to measure the efficiency of in vivo folding and subunit assembly of the mature trimer from its polypeptide chains. This decreased from 90% at 27 degrees C to only 15% at 42 degrees C. These results are consistent with the existence or a labile intermediate or step in the folding or subunit assembly of the thermostable tail spike protein. We discuss the possibility that the achievement of certain structural features of mature proteins may entail difficulties in their folding pathways.

Glycoside Hydrolases↗
PubMed1983

Negative dominance in gene lamB: random assembly of secreted subunits issued from different polysomes.

lamB is the structural gene for the lambda receptor, an oligomeric outer membrane protein from Escherichia coli K12 involved in phage lambda adsorption. We show that, under certain conditions, in a strain diploid for gene lamB, all the missense lamB mutations conferring lambda resistance that we have tested are dominant with respect to wild-type. We propose a model which allows a quantitative interpretation of the data. It is based on negative complementation at the level of oligomerisation. Wild-type and mutant subunits would assemble at random forming homo- and hetero-oligomers. Only wild-type homo-oligomers would be efficient for phage inactivation. For some classes of missense mutations the hetero-oligomers would have the capacity to bind, but not to inactivate the phage. The model confirms that active lambda receptor is a trimer and implies that for this secreted protein there is no preferential assembly of subunits originating from the same polysome.

Bacterial Outer Membrane Proteins↗
PubMed1995

Ethanol sensitivity of heteromeric NMDA receptors: effects of subunit assembly, glycine and NMDAR1 Mg(2+)-insensitive mutants.

In the current study, dimeric and trimeric combinations of N-methyl-D-aspartate (NMDA) receptor subunits were expressed in Xenopus oocytes and their sensitivity to ethanol was examined using conventional two electrode voltage clamp methods. In oocytes expressing the NR1/2A subunits, ethanol (25, 50 and 100 mM) inhibited NMDA (100 microM)/glycine (10 microM) induced currents by 21, 31 and 47%; respectively. NMDA-stimulated currents in oocytes expressing NR1/2B currents were inhibited by 13, 25 and 45% while NR1/NR2C currents were inhibited by 6, 11 and 24%. Ethanol inhibition of NMDA-stimulated currents in oocytes injected with NR1/2A/2B or NR1/2A/2C was not significantly different from that observed in NR1/2B or NR1/2C injected oocytes, respectively. With all receptor combinations, ethanol inhibition was rapid, reversible and not altered by pre-incubation. In the absence of ethanol, glycine enhanced NMDA-induced currents with an EC50 of 1.42 microM for the NR1/NR2A combination and 0.51 microM for the NR1/NR2C combination. Ethanol inhibited NMDA-induced currents at all glycine concentrations tested (1-100-microM) and did not significantly alter the EC50 value for glycine suggesting that ethanol does not compete for the glycine site on the NMDA receptor. Finally, three NR1 mutants which have been previously shown by others to possess either decreased Mg2+ sensitivity and Ca2+ permeability (N616Q and N616R) or reduced current amplitude (F609L) were tested for their ethanol sensitivity when expressed in combination with the NR2A subunit. Substitution of the wild-type NR1 with F609L did not alter the sensitivity of the receptor to ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Rapid intracellular assembly of tenascin hexabrachions suggests a novel cotranslational process.

Tenascin, an extracellular matrix protein that modulates cell adhesion, exists as a unique six-armed structure called a hexabrachion. The human hexabrachion is composed of six identical 320 kDa subunits and the structure is stabilized by inter-subunit disulfide bonds between amino-terminal segments. We have examined the biosynthesis of tenascin and its assembly into hexabrachions using pulsechase labeling of U-138 MG human glioma cells. Newly synthesized tenascin hexamers are secreted within 60 minutes of translation initiation. Intracellularly, as early as full length tenascin can be detected in pulse-labeled cell lysates, it is already in hexameric form. No precursors, such as monomers, dimers, or trimers, were identified that could be chased into hexamers. This lack of assembly intermediates suggests that nascent tenascin polypeptides associate prior to completion of translation. In contrast, fibronectin monomers in the same lysates are gradually formed into disulfide-bonded dimers. Although hexamer assembly is rapid, the rate-limiting step in secretion appears to be transport to the medial Golgi as endoglycosidase H-resistance was not detected until after a 30 minute chase. These results provide evidence for a novel co-translational mechanism of tenascin assembly which would be facilitated by its length and by the amino-terminal location of the assembly domain.

Cell Adhesion Molecules, Neuronal↗
PubMed1998

Role of the pro-alpha2(I) COOH-terminal region in assembly of type I collagen: truncation of the last 10 amino acid residues of pro-alpha2(I) chain prevents assembly of type I collagen heterotrimer.

Procollagen (Type I) contains a noncollagenous COOH-terminal propeptide (C-propeptide) hypothesized to be important in directing chain association and alignment during assembly. We previously expressed human pro-alpha2(I) cDNA in rat liver epithelial cells, W8, that produce only pro-alpha1(I) trimer collagen (Lim et al. [1994] Matrix Biol. 14: 21-30). In the resulting cell lines, alpha2(I) assembled with alpha1(I) forming heterotrimers. Using this cell system, we investigated the importance of the COOH-terminal propeptide sequence of the pro-alpha2(I) chain for normal assembly of type I collagen. Full-length human pro-alpha2(I) cDNA was cloned into expression vectors with a premature stop signal eliminating the final 10 amino acids. No triple-helical molecules containing alpha2(I) were detected in transfected W8 cells, although pro-alpha2(I) mRNA was detected. Additional protein analysis demonstrated that these cells synthesize small amounts of truncated pro-alpha2(I) chains detected by immunoprecipitation with a pro-alpha2(I) antibody. In addition, since the human-rat collagen was less thermostable than normal intraspecies collagen, wild-type and C-terminal truncated mouse cDNAs were expressed in mouse D2 cells, which produced only type I trimers. Results from both systems were consistent, suggesting that the last 10 amino acid residues of the pro-alpha2(I) chain are important for formation of stable type I collagen.

Amino Acid Sequence↗
PubMed2006

A cooperative beads-on-a-string approach to exceptionally stable DNA triplexes.

A poly(dT) oligomer can serve as the string on which synthetic hairpins possessing poly(dA) and poly(dT) arms connected by a hydrophobic perylene diimide linker are assembled like beads on a string. The synthetic hairpins form head-to-head dimers and trimers, respectively, in the absence of the poly(dT) string and in the presence of a string with inverted polarity at mid-strand. However, they assemble in cooperative head-to-tail fashion on normal poly(dT) oligomers.

Circular Dichroism↗
PubMed1998

Organization of HIV-1 capsid proteins on a lipid monolayer.

In an in vitro system that mimics the assembly of immature human immunodeficiency virus (HIV) particles, ordered arrays of HIV-1 capsid (CA) proteins encoded by the viral gag gene have been obtained by incubation of histidine-tagged capsid proteins (His-HIVCA) beneath lipid monolayers containing the nickel-chelating lipid, 1,2-di-O-hexadecyl-sn-glycero-3-(1'-2"-R-hydroxy-3'-N-(5-amino-1- carboxypentyl)iminodiacetic acid)propyl ether. The membrane-bound His-HIVCA proteins formed small crystalline arrays of primitive (p1) unit cells with dimensions of a = 74.2 A, b = 126.2 A, gamma = 89.3 degrees. The image-analyzed two-dimensional projection of His-HIVCA assemblies shows a cage-like lattice, consisting of hexamer and trimer units, surrounding protein-free cage holes. The hexamer-coordinated cage holes of 26.3-A diameter are spaced at 74. 2-A intervals: these distances, and the hexamer-trimer arrangement, are consistent with previous, lower resolution studies on immature HIV-1 virus particles produced in vivo. Additionally, HIV-1 matrix protein trimer unit structures align to the His-HIVCA trimer units such that residues previously shown to interact with the HIV-1 gp120/gp41 envelope protein complex are oriented toward the hexamer cage holes. Our results form a bridge between results from conventional methods for the analysis of HIV particle structure.

Capsid↗
PubMedDate not supplied

Sequence-based modeling of Abeta42 soluble oligomers.

Abeta fibrils, which are central to the pathology of Alzheimer's disease, form a cross-beta-structure that contains likely parallel beta-sheets with a salt bridge between residues Asp23 and Lys28. Recent studies suggest that soluble oligomers of amyloid peptides have neurotoxic effects in cell cultures, raising the interest in studying the structures of these intermediate forms. Here, we present three models of possible soluble Abeta forms based on the sequences similarities, assumed to support local structural similarities, of the Abeta peptide with fragments of three proteins (adhesin, Semliki Forest virus capsid protein, and transthyretin). These three models share a similar structure in the C-terminal region composed of two beta-strands connected by a loop, which contain the Asp23-Lys28 salt bridge. This segment is also structurally well conserved in Abeta fibril forms. Differences between the three monomeric models occur in the N-terminal region and in the C-terminal tail. These three models might sample some of the most stable conformers of the soluble Abeta peptide within oligomeric assemblies, which were modeled here in the form of dimers, trimers, tetramers, and hexamers. The consistency of these models is discussed with respect to available experimental and theoretical data.

Adhesins, Escherichia coli↗
PubMed1993

Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: implications for TNF receptor activation.

The X-ray crystal structure of the complex of the extracellular domain of the human 55 kd tumor necrosis factor (TNF) receptor with human TNF beta has been determined at 2.85 A resolution. The complex has three receptor molecules bound symmetrically to one TNF beta trimer. The receptor fragment, a very elongated end to end assembly of four similar folding domains, binds in the groove between two adjacent TNF beta subunits. The structure of the complex defines the orientation of the ligand with respect to the cell membrane and provides a model for TNF receptor activation. The novel fold of the TNF receptor structure is likely to be representative of the nerve growth factor (NGF)/TNF receptor family as a whole.

Amino Acid Sequence↗
PubMed2004

The trimeric organisation of photosystem I is not necessary for the iron-stress induced CP43' protein to functionally associate with this reaction centre.

A mutant of Synechocystis PCC 6803 lacking the PsaL subunit of photosystem I (PSI) has been grown in iron-deficient media to induce the expression of the isiA gene, which encodes the chlorophyll a-binding protein CP43'. The purpose of this was to establish whether or not the formation of an 18-mer CP43'-PSI supercomplex reported for wild type Synechocystis cells [Nature 412 (2001) 743-745] was dependent on the trimeric conformation of the cyanobacterial PSI reaction centre. Structural characterisation by electron microscopy and single particle image analysis has revealed that the PsaL-mutant does not form trimers of PSI. However, despite this, CP43' was found to associate with the PSI monomer. The PSI monomer bound six or seven copies of CP43' along one edge of the PSI monomer and can be compared with one segment of the trimeric 18-mer CP43'-PSI supercomplex. We therefore conclude that the trimeric nature of cyanobacterial PSI is not required for the assembly of the CP43' antenna system under iron-deficient conditions.

Biopolymers↗
PubMed2004

Antigen binding and stability properties of non-covalently linked anti-CD22 single-chain Fv dimers.

By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V(H)-V(L) oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V(L)-0-V(H) scFv assembled to homogenous dimers, remained substantially more stable than the V(H)-5-V(L) diabody when incubated in human serum at 37 degrees C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V(L)-0-V(H) scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22(+) tumor cells.

Amino Acid Sequence↗
PubMed1982

A domain of clathrin that forms coats.

Triskelions, trimeric complexes of clathrin and associated light chains, are the assembly units of clathrin coats [Ungewickell, E. & Branton, D. (1981) Nature (London) 289, 420-422; Kirchhausen, T. & Harrison, S. C. (1981) Cell 23, 755-761]. We report here that triskelions whose outer arms have been removed by trypsin digestion retain the ability to be assembled into coats. These digested timers contain a 110,000 molecular weight domain of clathrin and lack intact light chains.

Animals↗
PubMed1996

Effects of assembly and mutations outside the active site on the functional pH dependence of Escherichia coli aspartate transcarbamylase.

Electrostatics are central to the function and regulation of Escherichia coli aspartate transcarbamylase, and modeling has suggested that long range electrostatic effects are likely to be important (Glackin, M. P., McCarthy, M. P., Mallikarachchi, D., Matthew, J. B., and Allewell, N. M. (1989) Proteins Struct. Funct. Genet. 5, 66-77; Oberoi, H., Trikha, J., Yuan, X., and Allewell, N. M. (1995) Proteins Struct. Funct. Genet., in press). To investigate this possibility from an experimental standpoint, we have examined the effects both of assembly and of removing ionizable and polar side chains outside the active site (Glu-50, Tyr-165, and Tyr-240) on the pH dependence of the kinetic parameters of aspartate transcarbamylase. The holoenzyme (c6r6) assembles from three regulatory dimers (r2) and two catalytically active trimers (c3). pH dependences of the enzyme kinetic parameters suggest that the mechanisms of productive binding of L-Asp to the binary complexes of the catalytic subunit (c3) and holoenzyme (c6r6) with carbamyl phosphate are different. In contrast, the Michaelis complex appears similar for both c3 and c6r6, except for pK shifts of approximately 1 pH unit. Results also indicate that the catalytic mechanism of the holoenzyme does not involve reverse protonation, as has recently been proposed for the catalytic trimer (Turnbull, J. L., Waldrop, G. L., and Schachman, H. K. (1992) Biochemistry 31, 6562-6569). The tyrosines at positions 165 and 240 are part of a cluster of interactions that links the catalytic subunits in the T state (the cluc4 interface) and which is disrupted in the T --> R transition. The effects of mutating the two Tyr residues are quite different: Y240F has higher than wild-type activity and affinity over the entire pH range, while Y165F has activity and affinity an order of magnitude lower than wild-type. Removal of the regulatory subunits from Y165F increases activity and affinity and restores the pH dependence of the wild-type catalytic subunit. Like Y165F, E50A has low activity and affinity over the entire pH range. Linkage analysis indicates that there is long range energetic coupling among the active site, the ear subunit interfaces, and residue Y165. The substantial quantitative difference between Y165F and Y240F, both of which are at the c1:c4 interface about 14-16 A from the closest active site, demonstrates specific path dependence, as opposed to general distance dependence, of interactions between this interface and the active site.

Amino Acid Sequence↗
PubMed2004

Eukaryotic CTR copper uptake transporters require two faces of the third transmembrane domain for helix packing, oligomerization, and function.

Members of the copper uptake transporter (CTR) family from yeast, plants, and mammals including human are required for cellular uptake of the essential metal copper. Based on biochemical data, CTRs have three transmembrane domains and have been shown to oligomerize in the membrane. Among individual members of the family, there is little amino acid sequence identity, raising questions as to how these proteins adopt a common fold, oligomerize, and participate in copper transport. Using site-directed mutagenesis, tryptophan scanning, genetic complementation, subcellular localization, chemical cross-linking, and the yeast unfolded protein response, we demonstrated that at least half of the third transmembrane domain (TM3) plays a vital role in CTR structure and function. The results of our analysis showed that TM3 contains two functionally distinct faces. One face bears a highly conserved Gly-X-X-X-Gly (GG4) motif, which we showed to be essential for CTR oligomerization. Moreover, we showed that steric constraints reach past the GG4-motif itself including amino acid residues that are not conserved throughout the CTR family. A second face of TM3 contains three amino acid positions that, when mutated to tryptophan, cause predominantly abnormal localization but are still partially functional in growth complementation experiments. These mutations cluster on the face opposite to the GG4-bearing face of TM3 where they may mediate interactions with the remaining two transmembrane domains. Taken together, our data support TM3 as being buried within trimeric CTR where it plays an essential role in CTR assembly.

Amino Acid Motifs↗
PubMed2000

Structural and functional analysis of mutations in alkaptonuria.

Alkaptonuria (AKU), the prototypic inborn error of metabolism, was the first human disease to be interpreted as a Mendelian trait by Garrod and Bateson at the beginning of last century. AKU results from impaired function of homogentisate dioxygenase (HGO), an enzyme required for the catabolism of phenylalanine and tyrosine. With the novel 7 AKU and 22 fungal mutations reported here, a total of 84 mutations impairing this enzyme have been found in the HGO gene from humans and model organisms. Forty-three of these mutations result in single amino acid substitutions. This mutational information is analysed here in the context of the HGO structure and function using kinetic assays performed using purified AKU mutant enzymes and the crystal structure of human HGO. HGO is a topologically complex structure which assembles as a functional hexamer arranged as a dimer of trimers. We show how the intricate pattern of intra- and inter-subunit interactions and the extensive surfaces required for subunit folding and association of this oligomeric enzyme can be inactivated at multiple levels by single-residue substitutions. This explains, in part, the predominance of missense mutations (67%) in AKU.

Alkaptonuria↗