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PubMed1984

The clinical features of homozygous alpha 2(I) collagen deficient osteogenesis imperfecta.

The detailed clinical features and progress of a child with homozygous alpha 2(I) collagen deficiency are described. Clinically, the disease presents as severe progressive Sillence type III osteogenesis imperfecta. The main biochemical defect is the synthesis of an abnormal pro alpha 2(I) chain which does not associate with pro alpha 1(I) chains and therefore is not incorporated into triple helical trimers of type I procollagen which can be used to assemble collagen fibres.

Child, Preschool↗
PubMed1994

An LI and ML motif in the cytoplasmic tail of the MHC-associated invariant chain mediate rapid internalization.

Invariant chain (Ii) is a transmembrane protein that associates with the MHC class II molecules in the endoplasmic reticulum. Two regions of the 30 residue cytoplasmic tail of Ii contain sorting information able to direct Ii to the endocytic pathway. The full-length cytoplasmic tail of Ii and the two tail regions were fused to neuraminidase (NA) forming chimeric proteins (INA). Ii is known to form trimers and when INA was transfected into COS cells it assembled as a tetramer like NA. The INA molecules were targeted to the endosomal pathway and cotransfection with Ii showed that both molecules appeared in the same vesicles. By labelling the INA fusion proteins with iodinated antibody it was found that molecules with either endocytosis signal were expressed at the plasma membrane and internalized rapidly. Point mutations revealed that an LI motif within the first region of the cytoplasmic tail and an ML motif in the second region were essential for efficient internalization. The region containing the LI motif is required for Ii to induce large endosomes but a functional LI internalization motif was not fundamental for this property. The cytoplasmic tail of Ii is essential for efficient targeting of the class II molecules to endosomes and the dual LI and ML motif may thus be responsible for directing these molecules to the endosomal pathway, possibly via the plasma membrane.

Amino Acid Sequence↗
PubMed1997

Assembly of membrane-containing bacteriophage PRD1 is dependent on GroEL and GroES.

Assembly of the broad-host-range bacteriophage PRD1 involves translocation of the virus-specific membrane to the inside of the icosahedral protein shell formed of trimeric coat proteins. The formation of PRD1 particles is, in addition to the virus-encoded assembly factors P10 and P17, dependent on GroEL/GroES chaperonins. The chaperonins assist in the folding of the capsid proteins P3 and P5 and in the assembly of viral membrane proteins.

Chaperonin 10↗
PubMed1991

Clathrin domains involved in recognition by assembly protein AP-2.

The domains on clathrin responsible for interaction with the plasma membrane-associated assembly protein AP-2 have been studied using a novel cage binding assay. AP-2 bound to pure clathrin cages but not to coat structures already containing AP that had been prepared by coassembly. Binding to preassembled cages also occurred in the presence of elevated Tris-HCl concentrations (greater than or equal to 200 mM) which block AP-2 interactions with free clathrin. AP-2 interactions with assembled cages could also be distinguished from AP-2 binding to clathrin trimers by sodium tripolyphosphate (NaPPPi), which binds to the alpha subunit of AP-2 (Beck, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4442-4447). At concentrations of 1-5 mM, NaPPPi blocked clathrin-triskelion binding; in contrast, interactions with cages persisted in the presence of 25 mM NaPPPi. To begin to identify the region(s) of the clathrin molecule important in recognition by AP-2, clathrin cages were proteolyzed to remove heavy chain terminal domains and portions of the distal leg as well as all of the light chains. AP-2 bound to these "clipped cages"; however, unlike the interaction with native cages, binding of AP-2 to clipped cages was sensitive to the lower concentrations of both Tris-HCl and NaPPPi which disrupt interactions of AP-2 with clathrin trimers. Reconstitution of the clipped cages with clathrin light chains did not restore resistance of AP-2 binding to Tris-HCl. We conclude that one binding site for AP-2 resides on the hub and/or proximal part of the clathrin triskelion whereas a second site is likely to involve the terminal domain and/or distal leg; the second site is manifested only in the assembled lattice structure. We suggest that these two distinct binding interactions may be mediated by the two unique large subunits within the AP-2 complex, acting sequentially during assembly.

Animals↗
PubMed2004

Exploratory studies to investigate a linked prebiotic origin of RNA and coded peptides.

An introduction to the premise that RNA and genetically coded proteins should not be viewed as etiologically discrete entities in the origin of life is presented. This premise follows from the mutual interdependence of RNA and coded proteins in biology and the lack of prebiotically plausible constitutional self-assembly processes leading to either polymeric species. The RNA:coded peptides subsystem and its informational core, the genetic code, are then analysed retrosynthetically to suggest a (replicative) synthesis involving the intermediacy of aminoacyl-RNA trimers (cf. Scheme 5). A number of potential candidate aminoacyl-RNA trimers are identified (23-26; Scheme 6) and a chemical strategy to assess their validity is outlined. Experimental investigation of potential aminoacylation chemistry, nucleobase assembly and phosphate activation rules out three of the trimers but suggests that 26 is worthy of further investigation.

Evolution, Molecular↗
PubMed2002

Spectroscopic anatomy of molecular-imprinting of cyclodextrin. Evidence for preferential formation of ordered cyclodextrin assemblies.

The processes of molecular-imprinting of beta-cyclodextrin (beta-CyD) with cholesterol and stigmasterol (cross-linking agent = diisocyanate) have been analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. These templates enormously promote the formation of dimers and trimers of beta-CyD, which are only inefficiently formed in their absence. These ordered assemblies are the guest-binding sites, in which two or three beta-CyD molecules cooperate to bind large steroids. Ordered assemblies are also formed when 2,6-di-O-methyl-beta-cyclodextrin is used in place of beta-CyD. Direct spectroscopic evidence for molecular-imprinting effect has been obtained. Molecular imprinting of CyDs is potent for tailor-made preparation of synthetic receptors for nanometer-scaled guests.

Cholesterol↗
PubMed1996

Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity.

Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity. When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hybrid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions. Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154. Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.

Allosteric Regulation↗
PubMed1982

Characterization of a temperature-sensitive fiber mutant of type 5 adenovirus and effect of the mutation on virion assembly.

A temperature-sensitive, fiber-minus mutant of type 5 adenovirus, H5ts142, was biochemically and genetically characterized. Genetic studies revealed that H5ts142 was a member of one of the three apparent fiber complementation groups which were detected owing to intracistronic complementation. Recombination analyses showed that it occupied a unique locus at the right end of the adenovirus genetic map. At the nonpermissive temperature, the mutant made stable polypeptides, but they were not glycosylated like wild-type fiber polypeptides. Sedimentation studies of extracts of H5ts142-infected cells cultured and labeled at 39.5 degrees C indicated that a limited number of the fiber polypeptides made at the nonpermissive temperature could assemble into a form having a sedimentation value of 6S (i.e., similar to the trimeric wild-type fiber), but that this 6S structure was not immunologically reactive. When H5ts142-infected cells were shifted to the permissive temperature, 32 degrees C, fiber polypeptides synthesized at 39.5 degrees C were as capable of being assembled into virions as fibers synthesized in wild type-infected cells; de novo protein synthesis was not required to allow this virion assembly. In H5ts142-infected cells incubated at 39.5 degrees C, viral proteins accumulated and aggregated into particles having physical characteristics of empty capsids. These particles did not contain DNA or its associated core proteins. However, when the infected culture was shifted to 32 degrees C, DNA appeared to enter the empty particles and complete virions developed. The intermediate particles obtained had the morphology of adenoviruses, but they contained less than unit-length viral genomes as measured by their buoyant density in a CsCl density gradient and the size of their DNA as determined in both neutral and alkaline sucrose gradients. The reduced size of the intermediate particle DNA was demonstrated to be the result of incompletely packaged DNA molecules being fragmented during the preparative procedures. Hybridization of labeled DNA extracted from the intermediate particles to filters containing restriction fragments of the adenovirus genome indicated that the molecular left end of the viral genome preferentially entered these particles.

Adenoviruses, Human↗
PubMed1999

Clathrin self-assembly is mediated by a tandemly repeated superhelix.

Clathrin is a triskelion-shaped cytoplasmic protein that polymerizes into a polyhedral lattice on intracellular membranes to form protein-coated membrane vesicles. Lattice formation induces the sorting of membrane proteins during endocytosis and organelle biogenesis by interacting with membrane-associated adaptor molecules. The clathrin triskelion is a trimer of heavy-chain subunits (1,675 residues), each binding a single light-chain subunit, in the hub domain (residues 1,074-1,675). Light chains negatively modulate polymerization so that intracellular clathrin assembly is adaptor-dependent. Here we report the atomic structure, to 2.6 A resolution, of hub residues 1,210-1,516 involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association. The hub fragment folds into an elongated coil of alpha-helices, and alignment analyses reveal a 145-residue motif that is repeated seven times along the filamentous leg and appears in other proteins involved in vacuolar protein sorting. The resulting model provides a three-dimensional framework for understanding clathrin heavy-chain self-assembly, light-chain binding and trimerization.

Amino Acid Sequence↗
PubMed1982

Cyanobacterial phycobilisomes. Role of the linker polypeptides in the assembly of phycocyanin.

The phycocyanin-containing segments of the rod substructures of Anabaena variabilis phycobilisomes consist of complexes of phycocyanin with "linker" polypeptides of 27,000 and 32,500 daltons (Yu, M.-H., Glazer, A. N., and Williams, R. C. (1981) J. Biol. Chem. 256, 13130-13136). Complexes (alpha beta)3.27,000, (alpha beta)3.32,500, (alpha beta)6.27,000, [(alpha beta)6.32,500]n, (alpha beta)6.27,000 - (alpha beta)6.32,500 were prepared, where alpha beta represents a monomer of phycocyanin, and 27,000 and 32,500 represent the 27,000- and 32,500-dalton polypeptides, respectively. Tryptic digestion of (alpha beta)3.32,500 leads to a stable (alpha beta)3.28,000 complex which does not form higher aggregates. The 32,500 polypeptide is stable to trypsin in the [(alpha beta)6.32,500]n and (alpha beta)6.27,000 - [(alpha beta)6.32,500]n=1.2 aggregates. Upon trypsin treatment of all 27,000 still assembled into higher aggregates, (alpha beta)6.21,0900 and (alpha beta)6.21,000 - (alpha beta)6.32,500. The spectroscopic properties of phycocyanin-linker polypeptide complexes were not modified by the tryptic cleavages. These results show that the 32,500 polypeptide has two distinct functional domains, a 28,000 portion necessary to the stabilization of a trimeric phycocyanin complex and a 4,500 domain which links consecutive phycocyanin hexamers in the rod substructure. The 27,000 polypeptide likewise has two distinct functional domains: a 21,000 domain stabilizes a trimeric phycocyanin complex, a 6,000 domain is exposed in all of the assembly forms examined. From these and earlier studies, it is concluded that the 6,000 domain functions in the attachment of the rod substructures to the core of the phycobilisome.

Cyanobacteria↗
PubMed2002

The new MATH: homology suggests shared binding surfaces in meprin tetramers and TRAF trimers.

Although apparently functionally unrelated, intracellular TRAFs and extracellular meprins share a region with conserved meprin and traf homology, MATH(1). Both TRAFs and meprins require subunit assembly for function. By structural analysis of the sequences, we provide an explanation of how meprins, which form tetramers, and TRAF molecules, which form trimers, can share homology. Our analysis suggests it is highly likely that the same oligomerization surface is used. The analysis has implications for the widely distributed group of proteins containing MATH domains.

Amino Acid Sequence↗
PubMed1997

A single amino acid can switch the oligomerization state of the alpha-helical coiled-coil domain of cartilage matrix protein.

We have studied the oligomerization of an alpha-helical coiled-coil using as an example a peptide corresponding to the C-terminal domain of cartilage matrix protein. By replacing one arginine residue, which forms an interchain ionic interaction with a glutamic acid residue, with glutamine, we found that this peptide assembles into a homotetramer at neutral pH in contrast to the native molecule which forms homotrimers. At acidic and basic pH, however, we again observed the trimer conformation. Another arginine, which is probably involved in an intrachain salt bridge, has no effect on the assembly. Our data demonstrate that besides the specific distribution of hydrophobic residues, interchain ionic interactions can be crucial in modulating the association behavior of alpha-helical coiled-coil domains.

Amino Acid Sequence↗
PubMed1999

Crystal structure of the alpha appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly.

AP-2 adaptors regulate clathrin-bud formation at the cell surface by recruiting clathrin trimers to the plasma membrane and by selecting certain membrane proteins for inclusion within the developing clathrin-coat structure. These functions are performed by discrete subunits of the adaptor heterotetramer. The carboxyl-terminal appendage of the AP-2 alpha subunit appears to regulate the translocation of several endocytic accessory proteins to the bud site. We have determined the crystal structure of the alpha appendage at 1.4-A resolution by multiwavelength anomalous diffraction phasing. It is composed of two distinct structural modules, a beta-sandwich domain and a mixed alpha-beta platform domain. Structure-based mutagenesis shows that alterations to the molecular surface of a highly conserved region on the platform domain differentially affect associations of the appendage with amphiphysin, eps15, epsin, and AP180, revealing a common protein-binding interface.

Adaptor Proteins, Vesicular Transport↗
PubMed2003

Associations between light-harvesting complexes and Photosystem II from Marchantia polymorpha L. determined by two- and three-dimensional electron microscopy.

Assemblies of Photosystem II and light-harvesting proteins were purified from the liverwort Marchantia polymorpha and investigated by two- and three-dimensional transmission electron microscopy of negatively stained specimens. By single-particle analysis, it was determined that about 25% of the particles are rectangular or slightly S-shaped with dimensions of 285 A in length, 144 A in width, 84 A in height, while the membrane part is about 52 A thick. This structure reveals the same architecture as that of a Photosystem II-light-harvesting assembly from seed plants. An overlay of the projection structure of the liverwort's complex with a projection structure deduced from stained trimeric LHC II crystals from pea confirmed the locations of trimeric LHC II within the liverwort's complex. Remarkably tight associations of LHC II and other chlorophyll a/b binding proteins with the PS II core complex are observed. More than 50% of the Photosystem II particles from the liverwort carry one or two additional masses. These extra masses are found to consist of an additional LHC II trimer and probably a chlorophyll a/b binding protein. For the first time, a three-dimensional structure of such a large assembly is defined.

Journal Article↗
PubMed1996

Co-translational trimerization of the reovirus cell attachment protein.

The reovirus cell attachment protein, sigma1, is a trimer with a 'lollipop' structure. Recent findings indicate that the N-terminal fibrous tail and the C-terminal globular head each possess a distinct trimerization domain. The region responsible for N-terminal trimerization (formation of a triple alpha-helical coiled-coil) is located at the N-terminal one-third of sigma1. In this study, we investigated the temporality and ATP requirement of this trimerization event in the context of sigma1 biogenesis. In vitro co-synthesis of the full-length (FL) and a C-terminally truncated (d44) sigma1 protein revealed a preference for homotrimer over heterotrimer formation, suggesting that assembly at the N-terminus occurs co-translationally. This was corroborated by the observation that polysome-associated sigma1 chains were trimeric as well as monomeric. Truncated proteins (d234 and d294) with C-terminal deletions exceeding half the length of sigma1 were found to trimerize post-translationally. This trimerization did not require ATP since it proceeded normally in the presence of apyrase. In contrast, formation of stable FL sigma1 trimers was inhibited by apyrase treatment. Collectively, our data suggest that assembly of nascent sigma1 chains at the N-terminus is intrinsically ATP independent, and occurs co-translationally when the ribosomes have traversed past the midpoint of the mRNA.

Adenosine Triphosphate↗
PubMed1999

Interactions of the CCAAT-binding trimer NF-Y with nucleosomes.

NF-Y is a sequence-specific evolutionary conserved activator binding to CCAAT boxes with high affinity and specificity. It is a trimer formed by NF-YA and two putative histone-like subunits, NF-YB and NF-YC, showing similarity to histones H2B and H2A, respectively. We investigated the relationships between NF-Y and chromatin using an Artemia franciscana chromatin assembly system with plasmids containing the Major HistoCompatibility complex class II Ea promoter. The NF-Y trimer, but not single subunits, protects the Y box in the presence of reconstituted chromatin, and it can bind the target sequence during and after assembly. Using reconstitution assays with purified chicken histones, we show that NF-Y associates with preformed nucleosomes. Translational analysis of various Ea fragments of identical length in which the CCAAT box is at different positions indicated that the lateral fragment was slightly more prone to NF-Y binding. In competition experiments, NF-Y is able to prevent formation of nucleosomes significantly. These data support the idea that NF-Y is a gene-specific activator with a built-in capacity to interface with chromatin structures.

Animals↗
PubMed2002

Structure of an F-actin trimer disrupted by gelsolin and implications for the mechanism of severing.

Stable oligomers of filamentous actin were obtained by cross-linking F-actin with 1,4-N,N'-phenylenedimaleimide and depolymerization with excess segment-1 of gelsolin. Segment-1-bound and cross-linked actin oligomers containing either two or three actin subunits were purified and shown to nucleate actin assembly. Kinetic assembly data from mixtures of monomeric actin and the actin oligomers fit a nucleation model where cross-linked actin dimer or trimer reacts with an actin monomer to produce a competent nucleus for filament assembly. We report the three-dimensional structure of the segment-1-actin hexamer containing three actin subunits, each with a tightly bound ATP. Comparative analysis of this structure with twelve other actin structures provides an atomic level explanation for the preferential binding of ATP by the segment-1-complexed actin. Although the structure of segment-1-bound actin trimer is topologically similar to the helical model of F-actin (1), it has a distorted symmetry compared with that of the helical model. This distortion results from intercalation of segment-1 between actin protomers that increase the rise per subunit and rotate each of the actin subunits relative to their positions in F-actin. We also show that segment-1 of gelsolin is able to sever actin filaments, although the severing activity of segment-1 is significantly lower than full-length gelsolin.

Actins↗