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PubMed1992

A possible role for the conserved trimer interface of ferritin in iron incorporation.

Ferritin is a large protein, highly conserved among higher eukaryotes, which reversibly stores iron as a mineral of hydrated ferric oxide. Twenty-four polypeptides assemble to form a hollow coat with the mineral inside. Multiple steps occur in iron core formation. First, Fe2+ enters the protein. Then, several alternate paths may be followed which include oxidation at site(s) on the protein, oxidation on the core surface, and mineralization. Sequence variations occur among ferritin subunits which are classified as H or L; Fe2+ oxidation at sites on the protein appears to be H-subunit-specific or protein-specific. Other steps of ferritin core formation are likely to involve conserved sites in ferritins. Since incorporation of Fe2+ into the protein must precede any of the other steps in core formation, it may involve sites conserved among the various ferritin proteins. In this study, accessibility of Fe2+ to 1,10-phenanthroline, previously shown to be inaccessible to Fe2+ inside ferritin, was used to measure Fe2+ incorporation in two different ferritins under various conditions. Horse spleen ferritin (L/H = 10-20:1) and sheep spleen ferritin (L/H = 1:1.6) were compared. The results showed that iron incorporation measured as inaccessibility of Fe2+ to 1,10-phenanthroline increased with pH. The effect was the same for both proteins, indicating that a step in iron core formation common among ferritins was being measured. Conserved sites previously proposed for different steps in ferritin core formation are at the interfaces of pairs and trios of subunits. Dinitrophenol cross-links, which modify pairs of subunits and affect iron oxidation, had no effect on Fe2+ incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Trimerization domain of the collagen tail of acetylcholinesterase.

In the collagen-tailed forms of cholinesterases, each subunit of a specific triple helical collagen, ColQ, may be attached through a proline-rich domain (PRAD) situated in its N-terminal noncollagenous region, to tetramers of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). This heteromeric assembly ensures the functional anchoring of AChE in extracellulare matrices, for example, at the neuromuscular junction. In this study, we analyzed the influence of deletions in the noncollagenous C-terminal region of ColQ on its capacity to form a triple helix. We show that an 80-residue segment located downstream of the collagenous regions contains the trimerization domain, that it can form trimers without the collagenous regions, and that a pair of cysteines located at the N-boundary of this domain facilitates oligomerization, although it is not absolutely required. We further show that AChE subunits can associate with nonhelical collagen ColQ monomers, forming ColQ-associated tetramers (G4-Q), which are secreted or are anchored at the cell surface when the C-terminal domain of ColQ is replaced by a GPI-addition signal.

Acetylcholinesterase↗
PubMed1995

The crystal structure of bluetongue virus VP7.

Bluetongue virus (BTV), a representative of the orbivirus genus of the Reoviridae, is considerably larger (at 80 nm across), and structurally more complex, than any virus for which we have comprehensive structural information. Orbiviruses infect mammalian hosts through insect vectors and cause economically important diseases of domesticated animals. They possess a segmented double-stranded RNA genome within a capsid composed of four major types of polypeptide chains. An outer layer of VP2 and VP5 is removed as the virus enters the target cell, to leave an intact core within the cell. This core is 70 nm across and composed of 780 copies of VP7 (M(r) 38K) that, as trimers, form 260 'bristly' capsomeres clothing an inner scaffold constructed from VP3 (M(r) 103K). We report here the crystal structure of VP7 from BTV serotype 10, which reveals a molecular architecture not seen previously in viral structural proteins. Each subunit consists of two domains, one a beta-sandwich, the other a bundle of alpha-helices, and a short carboxy-terminal arm which might tie trimers together during capsid formation. A concentration of methionine residues at the core of the molecule could provide plasticity, relieving structural mismatches during assembly.

Amino Acid Sequence↗
PubMed2005

Vaccinia virus uracil DNA glycosylase interacts with the A20 protein to form a heterodimeric processivity factor for the viral DNA polymerase.

The vaccinia virus E9 protein, the catalytic subunit of the DNA polymerase holoenzyme, is inherently distributive under physiological conditions, although infected cells contain a highly processive form of the enzyme. The viral A20 protein was previously characterized as a stoichiometric component of the processivity factor, and an interaction between A20 and E9 was documented in vivo. A20 has been shown to interact with D4, the virally encoded uracil DNA glycosylase (UDG), by yeast-two hybrid and in vitro analysis. Here we confirm that UDG and A20 interact in vivo and show that temperature-sensitive viruses with lesions in the D4R gene show a profound defect in DNA synthesis at the non-permissive temperature. Moreover, cytoplasmic extracts prepared from these infections lack processive polymerase activity in vitro, implicating D4 in the assembly or activity of the processive polymerase. Upon overexpression of 3xFLAG-UDG, A20, and E9 in various combinations, we purified dimeric and trimeric UDG-A20 and UDG-A20-polymerase complexes, respectively. These complexes are stable in 750 mm NaCl and can be further purified by Mono Q chromatography. Notably, the trimeric complex displays robust processive polymerase activity, and the dimeric complex can confer processivity on purified E9. Consistent with previous reports that the catalytic activity of UDG is dispensable for virus replication in tissue culture, we find that the role of UDG role in the polymerase complex is not diminished by mutations targeting residues involved in uracil recognition or excision. Our cumulative data support the conclusion that A20 and UDG form a heterodimeric processivity factor that associates with E9 to comprise the processive polymerase holoenzyme.

Animals↗
PubMed1999

Solution assembly of the pseudo-high affinity and intermediate affinity interleukin-2 receptor complexes.

The high affinity interleukin-2 receptor is composed of three cell surface subunits, IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma. Functional forms of the IL-2 receptor exist, however, that enlist only two of the three subunits. On activated T-cells, the alpha- and beta-subunits combine as a preformed heterodimer (the pseudo-high affinity receptor) that serves to capture IL-2. On a subpopulation of natural killer cells, the beta- and gamma-subunits interact in a ligand-dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled-coil molecular recognition for the solution assembly of a heteromeric IL-2 receptor complex. In that study, although the receptor was functional, the coiled-coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled-coils to generate soluble forms of both the pseudo-high affinity and the intermediate affinity heterodimeric IL-2 receptors. The properties of these complexes were examined and their relevance to the physiological IL-2 receptor mechanism is discussed.

Amino Acid Sequence↗
PubMed1994

Fibritin encoded by bacteriophage T4 gene wac has a parallel triple-stranded alpha-helical coiled-coil structure.

The bacteriophage T4 late gene wac (whisker's antigen control) encodes a fibrous protein which forms a collar/whiskers complex. Whiskers function as a helper protein for the long tail fibres assembly and plays a role in regulating retraction of the long tail fibres in response to environmental conditions. In this work we show that expression of the cloned wac gene in Escherichia coli yields a protein oligomer of 53 nm length which we call fibritin, and which is able to complement gpwac T4 particles in vitro. CD spectroscopy of fibritin indicates a 90% alpha-helical content, and scanning calorimetry shows that the protein has several distinct domains. The analysis of the 486 amino acid sequence of fibritin reveals three structural components: a 408 amino acid region that contains 12 putative coiled-coil segments with a canonical heptad (a-b-c-d-e-f-g)n substructure where the "a" and "d" positions are preferentially occupied by apolar residues, and the N and C-terminal domains (47 and 29 amino acid residues, respectively) have no heptad substructure. The distribution of hydrophobic residues within heptads is more similar to a triple than to a double coiled-coil. The alpha-helical segments are separated by short "linker" regions, variable in length, that have a high proportion of glycine and proline residues. Each coiled-coil segment has, on the borders with linker regions, residues that are common to the N and C-terminal caps of the alpha-helices. Full-length and amino-terminally truncated fibritins can be reassembled in vitro after temperature-induced denaturation. Co-assembly of full-length fibritin and the N-terminal deletion mutant, as well as analytical centrifugation, indicates that the protein is a parallel triple-standard alpha-helical coiled-coil. Deletions of various N-terminal portions of fibritin did not block trimerisation but the mutant trimers are unable to bind to T4 particles. The last 18 C-terminal residues of fibritin are required for correct trimerisation of gpwac monomers in vivo. We propose that fibritin might serve as a convenient model for the investigation of folding and assembly mechanisms of alpha-fibrous proteins.

Amino Acid Sequence↗
PubMed2000

Structural hierarchy in erythrocruorin, the giant respiratory assemblage of annelids.

Many annelids, including the earthworm Lumbricus terrestris, have giant cooperative respiratory proteins (molecular masses greater than 3.5 million Da) freely dissolved in the blood, rather than packaged in cells. These complexes, termed either erythrocruorins or hemoglobins, are assembled from many copies of both hemoglobin subunits and nonhemoglobin or "linker" subunits. In this paper, we present the crystal structure of Lumbricus erythrocruorin at 5.5-A resolution, which reveals a remarkable hierarchical organization of 144 oxygen-binding hemoglobin subunits and 36 nonhemoglobin linker subunits. The hemoglobin chains arrange in novel dodecameric substructures. Twelve trimeric linker complexes project triple-stranded helical coiled-coil "spokes" toward the center of the complex; interdigitation of these spokes appears crucial for stabilization. The resulting complex of linker chains forms a scaffold on which twelve hemoglobin dodecamers assemble. This structure specifies the unique, self-limited assemblage of a highly cooperative single molecule.

Animals↗
PubMed1994

Laminin chain assembly. Specific sequences at the C terminus of the long arm are required for the formation of specific double- and triple-stranded coiled-coil structures.

The assembly of laminin chains into double- and triple-stranded structures was studied with recombinant proteins derived from the C-terminal alpha-helical region of the long arm of laminin. Both affinity assays and structural studies demonstrated chain-specific assembly of the B1, B2, and A chains, while associations between homotypic and homologous chains could not be observed. These results suggest that chain selection is controlled by the C-terminal region. Deletion mapping in the B1e and B2e chains identified two sites important for dimer and trimer formation. These two sites were separated by 23 amino acids in the B1e chain, whereas they were adjacent in the B2e chain. The Ae and Am chains contained only one site for trimerization. Site-directed mutagenesis revealed that charged amino acid residues within these sites were essential for association. These results suggest that distinct sites within the C-terminal alpha-helical region of the laminin long arm are critical for the chain-specific assembly of these macro-molecules.

Amino Acid Sequence↗
PubMed2000

In vitro assembly of bacteriophage P4 procapsids from purified capsid and scaffolding proteins.

Bacteriophage P4 is a satellite virus of bacteriophage P2, which has acquired the ability to utilize the structural gene products of P2 to assemble its own capsid. The normal P2 capsid has a T = 7 icosahedral structure comprised of the gpN-derived capsid protein, whereas the capsid produced under the control of P4 has a smaller, T = 4 structure. The protein responsible for this size determination is the P4-coded gene product Sid, which forms an external scaffold on the P4 procapsid. Using an in vitro assembly system, we show that gpN and Sid can coassemble into procapsid-like particles, indistinguishable from those produced in vivo, in the absence of any other gene products. The fidelity of the assembly reaction is enhanced by the inclusion of PEG and has a pH optimum between 8.0 and 8.5. Analysis of the assembly properties of truncated versions of Sid and gpN suggests that the amino-terminal part of Sid is involved in gpN binding, while the carboxyl-terminal part forms trimeric Sid-Sid interactions, and that the first 31 amino acids of gpN are required for binding to Sid as well as for size determination.

Bacteriophages↗
PubMed2004

Light-harvesting macroring accommodating a tetrapodal ligand based on complementary and cooperative coordinations.

A porphyrin macroring, mimicking the light-harvesting complex of photosynthetic purple bacteria, was synthesized by self-assembling trisporphyrinatoZn(II) complexes with imidazolyl substituents at both molecular terminals. Very strong complementary coordination of imidazolyl to Zn(II) afforded exclusively the cyclic trimer of trisporphyrin under dilute conditions at 27 degrees C in CHCl3/MeOH = 9/1 (v/v). 1H NMR spectra of the macroring indicate the existence of two topological isomers, one symmetric and one asymmetric. Use of three noncoordinated porphyrinatoZn(II) sites allows a tetrapodal ligand to be incorporated into the cavity of the macroring. A Job plot and the clear bending behavior on UV-vis titration indicated the formation a 1:1 complex; the heterogeneity of two topological isomers was not observed, and both isomers similarly accommodated the tetrapodal ligand. The association constant obtained by curve fitting analysis was 8 x 108 M-1 in toluene. This large association constant reflects the cooperative nature of three coordination sites.

Ligands↗
PubMed1986

Interaction between proteins localized in membranes.

We present a conceptual framework for evaluating the effect on the self-association of proteins in membranes due to the presence of other proteins at high concentrations (excluded volume effect) and the high concentration and preoriented state of the reactive species. We have calculated the magnitude of such effects using plausible values for the concentrations of proteins in membranes, for the degree to which proteins may tilt and move vertically, and for their dimensions. Compared to the association of monomers tumbling freely in an experimentally realistic volume, we calculate that these factors alone can increase the likelihood of forming dimers 10(6)-fold and of forming trimers and higher oligomers many orders of magnitude greater. We discuss the implications of our calculations for experimental manipulations of membrane proteins, for biosynthetic assembly of multisubunit membrane proteins and formation of membrane lesions by assemblies of exogenous proteins, and for the activation of cellular events induced by the interaction of membrane receptors with themselves or with other membrane proteins.

Cholesterol↗
PubMed2003

Alpha-helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily.

Alpha-helical coiled-coils are widely occurring protein oligomerization motifs. Here we show that most members of the collagen superfamily contain short, repeating heptad sequences typical of coiled coils. Such sequences are found at the N-terminal ends of the C-propeptide domains in all fibrillar procollagens. When fused C-terminal to a reporter molecule containing a collagen-like sequence that does not spontaneously trimerize, the C-propeptide heptad repeats induced trimerization. C-terminal heptad repeats were also found in the oligomerization domains of the multiplexins (collagens XV and XVIII). N-terminal heptad repeats are known to drive trimerization in transmembrane collagens, whereas fibril-associated collagens with interrupted triple helices, as well as collagens VII, XIII, XXIII, and XXV, were found to contain heptad repeats between collagen domains. Finally, heptad repeats were found in the von Willebrand factor A domains known to be involved in trimerization of collagen VI, as well as in collagen VII. These observations suggest that coiled-coil oligomerization domains are widely used in the assembly of collagens and collagen-like proteins.

Amino Acid Sequence↗
PubMed2003

Monomeric and dimeric byproducts are the principal functional elements of higher order P2X1 concatamers.

Heteromultimeric assembly of ion channel subunits generates high diversity in ion channel subtypes with distinct pharmacological and functional properties. To determine the subunit stoichiometry and order of ion channels, constructs with several concatenated subunits have been widely used in electrophysiological studies. Here we used primarily biochemical techniques to analyze the synthesis, assembly, and surface expression of P2X1 concatamers. We found that full-length concatamers consisting of two to six contiguous copies of the P2X1 subunit, although readily synthesized in Xenopus laevis oocytes, were entirely retained as aggregates in the endoplasmic reticulum. In contrast, minute levels of lower order byproducts, such as monomers and dimers, that were inherently formed with all the concatamers combined to form defined protein complexes equal in mass to the homotrimeric P2X1 receptor assembled from P2X1 monomers. Besides these complexes consisting of three monomers or one monomer plus one concatenated dimer, only small amounts of concatenated P2X1 trimers reached the plasma membrane. Complexes comprising more than three subunits were not observed in the plasma membrane. The byproduct complexes can account fully for the ATP-gated currents arising from expression of concatenated P2X1 subunits. These results strongly corroborate a trimeric architecture for P2X receptors yet indicate that formation of lower order by-products can be a pitfall of the concatamer approach.

Adenosine Triphosphate↗
PubMed2005

Human immunodeficiency virus (HIV) gp41 escape mutants: cross-resistance to peptide inhibitors of HIV fusion and altered receptor activation of gp120.

Human immunodeficiency virus (HIV) infects cells by fusing with cellular membranes. Fusion occurs when the envelope glycoprotein (Env) undergoes conformational changes while binding to cellular receptors. Fusogenic changes involve assembly of two heptad repeats in the ectodomain of the gp41 transmembrane subunit to form a six-helix bundle (6HB), consisting of a trimeric N heptad repeat (N-HR) coiled-coil core with three antiparallel C heptad repeats (C-HRs) that pack in the coiled-coil grooves. Peptides corresponding to the N-and C-HRs (N and C peptides, respectively) interfere with formation of the 6HB in a dominant-negative manner and are emerging as a new class of antiretroviral therapeutics for treating HIV infection. We generated an escape mutant virus with resistance to an N peptide and show that early resistance involved two mutations, one each in the N- and C-HRs. The mutations conferred resistance not only to the selecting N peptide but also to C peptides, as well as other types of N-peptide inhibitors. Moreover, the N-HR mutation altered sensitivity to soluble CD4. Biophysical studies suggest that the 6HB with the resistance mutations is more stable than the wild-type 6HB and the 6HB formed by inhibitor binding to either wild-type or mutant C-HR. These findings provide new insights into potential mechanisms of resistance to HIV peptide fusion inhibitors and dominant-negative inhibitors in general. The results are discussed in the context of current models of Env-mediated membrane fusion.

Amino Acid Sequence↗
PubMed2001

Molecular genetics of the HLA complex.

The Human Leukocyte Antigen (HLA) system can be defined as a set of glycoproteins which take up peptides intracellulary and become ligands for immune receptors once they are expressed on the cell membrane. Functional HLA molecules are trimers consisting of a heavy chain and a light chain which bind a peptide. The production of HLA molecules in the endoplasmic reticulum, their assembly and their trafficking to the cell membrane have been studied in great detail. A salient feature of HLA molecules is their polymorphism which is essentially a tremendous amount of variation in the amino acid sequence of mainly the heavy chain between different HLA types. Polymorphic chains of HLA molecules are encoded in the human major histocompatibility complex (MHC) on the short arm of chromosome 6. A map of the nucleotide sequence of the human MHC has been established. The polymorphism of HLA molecules, i.e. the HLA type, is intimately associated with the nature of the peptides bound. In principle, HLA molecules can bind a variety of different peptides. However, peptides with distinct biochemical features are preferentially bound according to the HLA-type. HLA-typing is applied clinically in transplantation and disease association. Recently, recombinant HLA molecules have been used as a tool to define the T cell receptor repertoire.

Alleles↗
PubMed2006

Directed self-assembly of gold-tipped CdSe nanorods.

Gold-tipped CdSe rods (nanodumbbells) were solubilized in an aqueous phase and self-assembled in a head-to-tail manner using biotin disulfide and avidin. The disulfide end of the biotin molecule attaches to the gold tip of the nanodumbbell, and the biotin end of the molecule is able to conjugate to an avidin protein. The avidin can strongly conjugate up to four biotin molecules. Changing the ratios of biotin to nanodumbbells leads to the formation of dimers, trimers, and flowerlike structures. To further improve the distribution of chain lengths, a separation method based upon weight was applied using a concentration gradient. The gold tips provide effective anchor points for constructing complex nanorod structures by self-assembly.

Journal Article↗
PubMed2001

Detection and binding properties of GABA(A) receptor assembly intermediates.

Density gradient centrifugation of native and recombinant gamma-aminobutyric acid, type A (GABA(A)) receptors was used to detect assembly intermediates. No such intermediates could be identified in extracts from adult rat brain or from human embryonic kidney (HEK) 293 cells transfected with alpha(1), beta(3), and gamma(2) subunits and cultured at 37 degrees C. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts from the brain of 8-10-day-old rats and from alpha(1)beta(3)gamma(2) transfected HEK cells cultured at 25 degrees C. In both systems, alpha(1), beta(3), and gamma(2) subunits could be identified in subunit dimers, indicating that different subunit dimers are formed during GABA(A) receptor assembly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated alpha(1) and beta(3) or alpha(1) and gamma(2) subunits and co-immunoprecipitation with subunit-specific antibodies indicated that even subunits containing no transmembrane domain can assemble with each other. Whereas alpha(1)gamma(2), alpha(1)Ngamma(2), alpha(1)gamma(2)N, and alpha(1)Ngamma(2)N, combinations exhibited specific [(3)H]Ro 15-1788 binding, specific [(3)H]muscimol binding could only be found in alpha(1)beta(3) and alpha(1)beta(3)N, but not in alpha(1)Nbeta(3) or alpha(1)Nbeta(3)N combinations. This seems to indicate that a full-length alpha(1) subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.

Animals↗
PubMed2001

Rotavirus assembly - interaction of surface protein VP7 with middle layer protein VP6.

The interaction between the rotavirus proteins viral protein 6 (VP6) and VP7 was examined in several exogenous protein expression systems. These proteins associated in the absence of other rotaviral proteins as demonstrated by a coimmunoprecipitation assay. Deletion analysis of VP7 indicated that truncations of either the mature amino or carboxyl terminus disrupted the proper folding of the protein and were not able to coimmunoprecipitate VP6. Truncation analysis of VP6 indicated that trimerization of VP6 was necessary, but not sufficient, for VP7 binding. MAb mapping and coimmunoprecipitation interference assays indicate that the VP6 amino acid residues between 271 and 342 are required for VP7 interaction. The interaction of VP6 and VP7 was also examined by the assembly of soluble VP7 onto baculovirus-expressed virus-like particles containing VP2 and VP6. Abrogation of this binding by preincubation of the particles with VP6 MAbs mapped to this same domain of VP6, validated our coimmunoprecipitation results. VP6 IgA MAbs that have been shown to be protective in vivo, but not a nonprotective IgA MAb, can interfere with VP7 binding to VP6. This suggests that these IgA MAbs may protect against rotavirus infection by blocking rotavirus assembly.

Animals↗