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PubMed2005

New faces of the familiar clathrin lattice.

The clathrin triskelion self-assembles into a lattice that coats transport vesicles participating in several key membrane traffic pathways. A new model of a clathrin lattice at approximately 8 angstrom resolution, generated by Fotin et al. (Nature 2004;432:573) confirmed the basic structural features of clathrin that were defined over many years of biochemical and structural analysis. In addition, new structural features of the clathrin trimerization domain were modelled for the first time, and the predictions correlated well with previous biochemical studies. A second model, placing auxilin within the lattice suggested a possible lattice contact targeted during lattice disassembly (Fotin et al. Nature 2004;432:649). This contact predicts interactions of the newly modelled trimerization domain with a newly defined extension of the clathrin triskelion, the ankle domain. These aspects of the new models were emphasized in the published reports describing them and in recent commentary (Brodsky, Nature 2004;432:568). Also emerging from the new models is a better picture of how the clathrin structure is distributed throughout the lattice, allowing the first predictions of interacting molecular interfaces contributing to contacts in the assembled lattice. The focus of this interchange is to emphasize these additional features revealed by the recently published models from Fotin and colleagues.

Clathrin↗
PubMed2006

Structure of the Bet3-Tpc6B core of TRAPP: two Tpc6 paralogs form trimeric complexes with Bet3 and Mum2.

The transport protein particle (TRAPP) complexes are involved in the tethering process at different trafficking steps of vesicle transport. We here present the crystal structure of a human Bet3-Tpc6B heterodimer, which represents a core sub-complex in the assembly of TRAPP. We describe a conserved patch of Tpc6 with uncharged pockets, forming a putative interaction interface for an anchoring moiety at the Golgi. The structural and functional comparison of the two paralogs Tpc6A and Tpc6B, only found in some organisms, indicates redundancy and added complexity of TRAPP architecture and function. Both iso-complexes, Bet3-Tpc6A and Bet3-Tpc6B, are able to recruit Mum2, a further TRAPP subunit, and we identify the alpha1-alpha2 loop regions as a binding site for Mum2. Our study reveals similar stability of the iso-complexes and similar expression patterns of the tpc6 variants in different mouse organs. These findings raise the possibility that the Tpc6 paralogs might contribute to the formation of two distinct TRAPP complexes that differ in function.

Amino Acid Sequence↗
PubMed1992

Rotavirus VP6 modified for expression on the plasma membrane forms arrays and exhibits enhanced immunogenicity.

The major inner capsid protein of rotavirus is VP6, a 42-kDa polypeptide that forms the icosahedral surface of the rotavirus single-shelled particle. A chimeric form of VP6 (VP6sc) was constructed containing an upstream leader sequence derived from the influenza virus hemagglutinin and a downstream membrane-spanning (anchor) domain from a mouse immunoglobulin gene. When VP6sc was expressed in cells using a recombinant vaccinia virus, the protein was transported, glycosylated, and anchored in the plasma membrane as a trimer with the major domains of the protein orientated externally. Immunofluorescence and immunolabeling with colloidal gold indicated that VP6sc also localized in patches on the cell surface; electron microscopy revealed that the protein assembled into two-dimensional arrays which exhibited the same periodicity as the paracrystalline arrays formed by purified (viral) VP6. Mice inoculated with a recombinant vaccinia virus that expressed VP6sc produced rotavirus-specific antibodies at a titer 10 times higher than that achieved when wild-type, intracellular VP6 was delivered in the same way. Presentation at the cell surface therefore may represent a general method for enhancing the immunogenicity of rotavirus proteins.

Animals↗
PubMed2005

Expression and characterization of a baseplate protein for bacteriophage Mu, gp44.

The gene product of gene 44 of Mu phage (gp44) is an essential protein for baseplate assembly and has been designated as gpP, a traditional genetic assignment. The function of gp44 during the assembly or infection process is not known. In the present study, we purified the recombinant gp44 and characterized it by analytical ultracentrifugation and differential scanning microcalorimetry. The results indicate that gp44 forms a trimer comprising a complex consisting of the 42 kDa and 40 kDa subunits that had been cleaved in the C-terminal region. Thermodynamic analysis also suggested that the C-terminal region forms a flexible domain.

Bacteriophage mu↗
PubMed1987

Electron microscopy of nickel-containing methanogenic enzymes: methyl reductase and F420-reducing hydrogenase.

Methanogens catalyze the hydrogen-dependent eight-electron reduction of carbon dioxide to methane. Two of the key catalysts in the eight-electron reduction pathway are the nickel-containing enzymes F420-reducing hydrogenase and methyl reductase. In the present study, the structures of these archaebacterial enzymes from Methanobacterium thermoautotrophicum delta H have been determined by electron microscopy. By negative stain techniques, F420 hydrogenase was found to be a ring structure with a diameter of 15.7 nm and an inner channel 4 nm in diameter. Shadow-casting experiments demonstrated that the rings were 8.5 nm deep, indicating a holoenzyme molecular weight of 8.0 X 10(5). Methyl reductase appeared to be an oligomeric complex of dimensions 8.5 by 9 by 11 nm, with a central stain-penetrating region. The morphology and known subunit composition suggest a model in which the subunits are arranged as an eclipsed pair of open trimers. Methyl reductase was also found in the form of larger aggregates and in paracrystalline arrays derived from highly concentrated solutions. The extremely large size of F420 hydrogenase and the methyl reductase supramolecular assemblies may have relevance in vivo in the construction of multiprotein arrays that function in methane biogenesis.

Euryarchaeota↗
PubMed1977

[Quaternary structure of oligomeric immunoglobulin A forms from human blood serum].

A character of forces stabilyzing quaternary structure of dimer and more high molecular human immunoglobulin A oligomers is found to be different. Quaternary structure of IgA dimer is formed when joining subunits with disulfide bonds and is stabilized by non-covalent interactions between them. Disulfide bonds play a main part in the formation of trimers and tetramers. Dimer IgA reconstructs by 40% from subunits with intact interchain S--S bonds. The addition of exogenous J-chain does not significantly affect the process of dimer self-assembling from subunits with recovered and intact interchain disulfide bonds.

Chemical Phenomena↗
PubMed2006

Structure of the bacteriophage phi6 nucleocapsid suggests a mechanism for sequential RNA packaging.

Bacteriophage phi6 is an enveloped dsRNA virus with a segmented genome. Phi6 specifically packages one copy of each of its three genome segments into a preassembled polymerase complex. This leads to expansion of the polymerase complex, minus and plus strand RNA synthesis, and assembly of the nucleocapsid. The phi6 in vitro assembly and packaging system is a valuable model for dsRNA virus replication. The structure of the nucleocapsid at 7.5 A resolution presented here reveals the secondary structure of the two major capsid proteins. Asymmetric P1 dimers organize as an inner T = 1 shell, and P8 trimers organize as an outer T = 13 laevo shell. The organization of the P1 molecules in the unexpanded and expanded polymerase complex suggests that the expansion is accomplished by rigid body movements of the P1 monomers. This leads to exposure of new potential RNA binding surfaces to control the sequential packaging of the genome segments.

Bacteriophage phi 6↗
PubMed2002

Application of the anomeric samarium route for the convergent synthesis of the C-linked trisaccharide alpha-D-Man-(1-->3)-[alpha-D-Man-(1-->6)]-D-Man and the disaccharides alpha-D-Man-(1-->3)-D-Man and alpha-D-Man-(1-->6)-D-Man.

Studies are reported on the assembly of the branched C-trisaccharide, alpha-D-Man-(1-->3)-[alpha-D-Man-(1-->6)]-D-Man, representing the core region of the asparagine-linked oligosaccharides. The key step in this synthesis uses a SmI(2)-mediated coupling of two mannosylpyridyl sulfones to a C3,C6-diformyl branched monosaccharide unit, thereby assembling all three sugar units in one reaction and with complete stereocontrol at the two anomeric carbon centers. Subsequent tin hydride-based deoxygenation followed by a deprotection step produces the target C-trimer. In contrast to many of the other C-glycosylation methods, this approach employes intact carbohydrate units as C-glycosyl donors and acceptors, which in many instances parallels the well-studied O-glycosylation reactions. The synthesis of the C-disaccharides alpha-D-Man-(1-->3)-D-Man and alpha-D-Man-(1-->6)-D-Man is also described, they being necessary for the following conformational studies of all three carbohydrate analogues both in solution and bound to several mannose-binding proteins.

Carbohydrate Conformation↗
PubMed2005

The design of coiled-coil structures and assemblies.

Protein design allows sequence-to-structure relationships in proteins to be examined and, potentially, new protein structures and functions to be made to order. To succeed, however, the protein-design process requires reliable rules that link protein sequence to structure?function. Although our present understanding of coiled-coil folding and assembly is not complete, through numerous bioinformatics and experimental studies there are now sufficient rules to allow confident design attempts of naturally observed and even novel coiled-coil motifs. This review summarizes the current design rules for coiled coils, and describes some of the key successful coiled-coil designs that have been created to date. The designs range from those for relatively straightforward, naturally observed structures-including parallel and antiparallel dimers, trimers and tetramers, all of which have been made as homomers and heteromers-to more exotic structures that expand the repertoire of Nature's coiled-coil structures. Examples in the second bracket include a probe that binds a cancer-associated coiled-coil protein; a tetramer with a right-handed supercoil; sticky-ended coiled coils that self-assemble to form fibers; coiled coils that switch conformational state; a three-component two-stranded coiled coil; and an antiparallel dimer that directs fragment complementation of larger proteins. Some of the more recent examples show an important development in the field; namely, new designs are being created with function as well as structure in mind. This will remain one of the key challenges in coiled-coil design in the next few years. Other challenges that lie ahead include the need to discover more rules for coiled-coil prediction and design, and to implement these in prediction and design algorithms. The considerable success of coiled-coil design so far bodes well for this, however. It is likely that these challenges will be met and surpassed.

Amino Acid Sequence↗
PubMed1986

Folding of carboxyl domain and assembly of procollagen I.

An early form of procollagen I was found in acetic acid extracts of radioactively labeled chick embryo skull bones. It resembled native procollagen I, but sedimented slightly faster, and its component chains were slightly underhydroxylated and were not disulfide-linked to each other, although its propeptides were internally disulfide-bonded. Pulse-chase experiments showed its conversion to disulfide-linked procollagen. As the same conversion occurred when proline hydroxylation was blocked by 2,2'-dipyridyl, we infer that the formation of this precursor from its component chains does not require collagen triple helix formation. We suggest that interaction between the folded carboxyl propeptides of individual pro-alpha (I) chains is an important step in the formation of this precursor and of procollagen I. Studies of the refolding and association of fully reduced and denatured carboxyl propeptides supported this concept. In the presence of glutathione the correct disulfide bonds could be reestablished, as judged by a mapping of some tryptic peptides. Individual carboxyl propeptides refolded first, and this occurred even in 2 M urea. Recognition between folded carboxyl propeptides occurred only when less than 0.5 M urea was present. The presence of the carboxyl telopeptides was important for trimeric reassembly. Individual propeptides also folded spontaneously during cell-free translation of pro-alpha (I) chains and were recognized by specific antibodies. We consider the role of carboxyl propeptides in the formation of procollagen I molecules and suggest a model of self-assembly, possibly facilitated by interactions with the luminal surface of the rough endoplasmic reticulum.

Animals↗
PubMed2001

Gamma subunit of the AP-1 adaptor complex binds clathrin: implications for cooperative binding in coated vesicle assembly.

The heterotetrameric AP-1 adaptor complex is involved in the assembly of clathrin-coated vesicles originating from the trans-Golgi network (TGN). The beta 1 subunit of AP-1 is known to contain a consensus clathrin binding sequence, LLNLD (the so-called clathrin box motif), in its hinge segment through which the beta chain interacts with the N-terminal domains of clathrin trimers. Here, we report that the hinge region of the gamma subunit of human and mouse AP-1 contains two copies of a new variant, LLDLL, of the clathrin box motif that also bind to the terminal domain of the clathrin heavy chain. High-affinity binding of the gamma hinge to clathrin trimers requires both LLDLL sequences to be present and the spacing between them to be maintained. We also identify an independent clathrin-binding site within the appendage domain of the gamma subunit that interacts with a region of clathrin other than the N-terminal domain. Clathrin polymerization is promoted by glutathione S-transferase (GST)-gamma hinge, but not by GST-gamma appendage. However, the hinge and appendage domains of gamma function in a cooperative manner to recruit and polymerize clathrin, suggesting that clathrin lattice assembly at the TGN involves multivalent binding of clathrin by the gamma and beta1 subunits of AP-1.

Adaptor Proteins, Vesicular Transport↗
PubMed2001

Molecular mass, stoichiometry, and assembly of 20 S particles.

N-Ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptor (neuronal SNARE) complexes form 20 S particles with a mass of 788 +/- 122 kDa as judged by scanning transmission electron microscopy. A single NSF hexamer and three alpha SNAP monomers reside within a 20 S particle as determined by quantitative amino acid analysis. In order to study the binding of alpha SNAP and NSF in solution, to define their binding domains, and to specify the role of oligomerization in their interaction, we fused domains of alpha SNAP and NSF to oligomerization modules derived from thrombospondin-1, a trimer, and cartilage oligomeric matrix protein, a pentamer, respectively. Binding studies with these fusion proteins reproduced the interaction of alpha SNAP and NSF N domains in the absence of the hexamerization domain of NSF (D2). Trimeric alpha SNAP (or its C-terminal half) is sufficient to recruit NSF even in the absence of SNARE complexes. Furthermore, pentameric NSF N domains are able to bind alpha SNAP in complex with SNAREs, whereas monomeric N domains do not. Our results demonstrate that the oligomerization of both NSF N domains and alpha SNAP provides a critical driving force for their interaction and the assembly of 20 S particles.

Amino Acid Sequence↗
PubMed2005

Cyclin H is targeted to the nucleus by C-terminal nuclear localization sequences.

Cdk-activating kinase (CAK) is a trimeric complex consisting of cdk7, cyclin H, and MAT1, which activates the cell-cycle-regulating cdks through T loop phosphorylation. In addition, other substrates of the CAK complex have been identified when CAK is assembled with the TFIIH core proteins, thereby regulating transcription and nucleotide excision repair. Little is known about the regulation of the CAK complex through cyclin H. In this study we further analyzed cyclin H regulation and identified two basic clusters in the C terminus of the protein as putative nuclear localization sequences (NLSs). Fusion constructs of full-length and truncated cyclin H sequences demonstrated the functionality of the NLSs. A peptide-binding assay revealed that at least one NLS interacts with the nuclear import receptors importin alpha/beta. Phosphorylation in the vicinity of the NLSs by cyclin C/cdk8 or protein kinase CK2, however, does not influence the nuclear translocation of cyclin H.

Amino Acid Sequence↗
PubMed2004

Six-helix bundle assembly and analysis of the central core of mumps virus fusion protein.

The fusion protein of enveloped viruses mediates the fusion between the viral and cellular membranes, allowing the penetration of the viral genomes into the host cell. Many of these proteins share a common fold comprising a central core trimer of anti-parallel coiled-coil heterodimers, which are formed by two discontinuous heptad repeat (HR) motifs located at the ectodomain of the fusion proteins. In this study, we constructed and purified the corresponding regions (HR1 and HR2) of mumps virus fusion protein that are predicted to form coiled coil. The HR1 and HR2 were expressed and purified separately or as a single chain connected by an amino acid linker (HR1-linker-HR2, named 2-Helix). Series of biochemical and biophysical analyses of the expressed proteins have shown that HR1 and HR2 of mumps virus fusion protein share the common features of other enveloped virus fusion proteins. CD spectral results show that HR1 forms an alpha-helical coil structure while HR2 exists as an unstructured monomer in PBS in nature. Mixtures of HR1 and HR2 could form a stable six-helix bundle, indicating the interaction of HR1 and HR2. The 2-Helix protein also shows characteristic properties of the 6-helix bundle. Therefore, mumps virus fusion protein has a common core architecture and its HR regions could be used as a drug target for virus fusion inhibitors.

Amino Acid Sequence↗
PubMed2003

Plants lacking the main light-harvesting complex retain photosystem II macro-organization.

Photosystem II (PSII) is a key component of photosynthesis, the process of converting sunlight into the chemical energy of life. In plant cells, it forms a unique oligomeric macrostructure in membranes of the chloroplasts. Several light-harvesting antenna complexes are organized precisely in the PSII macrostructure-the major trimeric complexes (LHCII) that bind 70% of PSII chlorophyll and three minor monomeric complexes-which together form PSII supercomplexes. The antenna complexes are essential for collecting sunlight and regulating photosynthesis, but the relationship between these functions and their molecular architecture is unresolved. Here we report that antisense Arabidopsis plants lacking the proteins that form LHCII trimers have PSII supercomplexes with almost identical abundance and structure to those found in wild-type plants. The place of LHCII is taken by a normally minor and monomeric complex, CP26, which is synthesized in large amounts and organized into trimers. Trimerization is clearly not a specific attribute of LHCII. Our results highlight the importance of the PSII macrostructure: in the absence of one of its main components, another protein is recruited to allow it to assemble and function.

Arabidopsis↗
PubMed2006

The 2.6-Angstrom structure of infectious bursal disease virus-derived T=1 particles reveals new stabilizing elements of the virus capsid.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 A resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.

Capsid↗
PubMed1997

Theoretical investigation of IL-6 multiprotein receptor assembly.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates cell growth, differentiation, and cellular functions in many cell lineages. Recently, evidences for the formation of an active hexameric complex with an IL-6:IL-6R alpha:gp130 stoichiometry of 2:2:2 have been obtained by different experimental approaches. Analysis of the electrostatic potential complementarity between IL-6 and its receptors has been used, in this study, to guide the assembly of homology-based 3D models of the components. The results strongly support a mechanism whereby the active cytokine (IL-6: IL-6R alpha) associates with the signal transducing gp130 protein, and the trimeric complex formed further dimerizes to form the hexameric species. Furthermore, computational simulations of the multiprotein complexes provide a rationalization of data from mutation experiments and highlight some key protein-protein interactions which have not yet been the subject of mutagenesis studies.

Amino Acid Sequence↗
PubMed2002

Forced subunit assembly in alpha1beta2gamma2 GABAA receptors. Insight into the absolute arrangement.

The major isoform of the gamma-aminobutyric acid type A (GABA(A)) receptor is thought to be composed of 2alpha(1), 2beta(2), and 1gamma(2) subunit(s), which surround the ion pore. Definite evidence for the subunit arrangement is lacking. We show here that GABA(A) receptor subunits can be concatenated to a trimer that can be functionally expressed upon combination with a dimer. Many combinations did not result in the functional expression. In contrast, four different combinations of triple subunits with dual subunit constructs, all resulting in the identical pentameric receptor gamma(2)beta(2)alpha(1)beta(2)alpha(1), could be successfully expressed in Xenopus oocytes. We characterized the functional properties of these receptors in respect to agonist, competitive antagonist, and diazepam sensitivity. All properties were similar to those of wild type alpha(1)beta(2)gamma(2) GABA(A) receptors. Thus, together with information on the crystal structure of the homologous acetylcholine-binding protein (Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K., (2001) Nature 411, 269-276, we provide evidence for an arrangement gamma(2)beta(2)alpha(1)beta(2)alpha(1), counterclockwise when viewed from the synaptic cleft. Forced subunit assembly will also allow receptors containing different subunit isoforms or mutant subunits to be expressed, each in a desired position. The methods established here should be applicable to the entire ion channel family comprising nicotinic acetylcholine, glycine, and 5HT(3) receptors.

Animals↗