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PubMed2004

Conformational epitope mapping of OmpC, a major cell surface antigen from Salmonella typhi.

The outer membrane protein OmpC, a trimer made of 16 stranded beta-barrel monomers, is a major cell surface antigen from the human pathogen Salmonella typhi. The relative stability of the epitopes recognising a Salmonella specific MAb (referred as MPN5) and an Enterobacteria specific MAb (referred as P7D8) and the role of the trimeric organisation has been probed using gel electrophoresis and monoclonal antibodies. The assembly of the trimer and the stability of the beta-barrel are found to be important for epitope presentation. The Salmonella specific conformational epitope is found to be more stable than the Enterobacteria specific one. The important residues of the Salmonella specific (Asp 25 of loop 1, Asp 340 of loop 8, Lys 334 of loop 8, and Tyr 210 of loop 5) and the Enterobacteria specific (Asp 25 of loop 1, Tyr 210 of loop 5, and Lys 152 of loop 4) conformational epitope have been identified using monoclonal antibodies, chemical modification, and solid phase binding methods.

Antibodies, Monoclonal↗
PubMed2004

Differential impact of MexB mutations on substrate selectivity of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa.

The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a DeltamexB strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.

Anti-Bacterial Agents↗
PubMedDate not supplied

Deep-etch views of clathrin assemblies.

Clathrin assemblies were adsorbed to mica and freeze-dried by a new procedure that yields 3-D images with much topological detail. These permitted renewed inquiry into how clathrin trimers (i.e. "triskelions") assemble into polygonal coats or baskets. Freeze-drying revealed unsuspected differences in the relative shapes and dimensions of individual trimer building blocks, as compared with the completed polygonal networks, which indicate that the assembly scheme first proposed by Crowther and Peare (1) requires modification. Specifically, the freeze-etch images display the following new features: (1) Trimer arms possess terminal scroll-shaped hooks that can open or close and thus determine their lengths. (2) When extended, trimer arms are sufficiently long to pass around three sides of the final polygonal facet. Since current views hold that the arms pass around only two sides, the remaining length, including the terminal hook, must point into the basket interior. (3) Freeze-dried trimers display bends in their arms at specific loci that determine their final distribution in the completed baskets. (4) The completed struts of the final assemblies are uniformed in the calibre, cylindrical in profile, and travel directly between the vertices of each polygon, without any sign of the slew or width-variation that is predicted by the Crowther and Pearse model. Based on this direct comparison of promoter vs product, by a single technique that can image both, we offer a modified scheme for clathrin coat assembly, in which we predict that the individual arms in each clathrin triskelion emanate from its center in a slewed manner, but the final assembled struts of the basket need not be slewed. Attempts were made to capture assembly intermediates on mica to obtain support for the scheme, but these unfortunately yielded ambiguous images of incomplete polygons with blunt projections, rather than the expected "halo" of uncommitted trimer arms. These we interpret to be "dead ends" that failed to polymerize further because they included proteolyzed components. Further assembly experiments, avoiding such hazards, are indicated.

Aluminum Silicates↗
PubMed1993

Refined crystal structure of the catalytic domain of dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii at 2.6 A resolution.

Dihydrolipoyl transacetylase (E2p) is both structurally and functionally the central enzyme of the pyruvate dehydrogenase multienzyme complex. The crystal structure of the catalytic domain, i.e. residues 382 to 637, of Azotobacter vinelandii E2p (E2pCD) was solved by multiple isomorphous replacement and refined by energy minimization procedures. The final model contains 2182 protein atoms and 37 ordered water molecules. The R-factor is 18.7% for 10,344 reflections between 10.0 and 2.6 A resolution. The root-mean-square shift deviation from the ideal values is 0.017 A for bond lengths and 3.3 degrees for bond angles. The N-terminal residues 382 to 394 are disordered and not visible in the electron density map, otherwise all residues have well-defined density. The catalytic domain forms an oligomer of 24 subunits, having octahedral 432 symmetry. In the E2pCD crystals, the 24 subunits are related by the crystallographic symmetry. The cubic arrangement of subunits gives rise to a large hollow cube with edges of 120 A. The faces of the cube have pores of diameter of 30 A. The true building block of the cube is the E2p trimer, eight of which occupy the corners of the cube. Two levels of intermolecular contacts can be distinguished: (1) the extensive interactions between 3-fold related subunits leading to a tightly associated trimer; and (2) the interactions along the 2-fold axis leading to the assembly of the trimers into the cubic 24-mer. Each subunit has a topology similar to chloramphenicol acetyltransferase (CAT) and comprises a central beta-sheet surrounded by five alpha-helices. The comparison of the two proteins indicates a large rotation of the N-terminal residues 395 to 426 of E2pCD, which reshapes the substrate binding site and extends the interaction between threefold related subunits. The catalytic centre consists of a 30 A long channel extending from the "inner" side of the trimer to the "outer" side, where inner and outer refer to the position in the 24-meric cubic core of the pyruvate dehydrogenase complex and correspond with CoA and lipoamide binding sites, respectively. The active site is formed by the residues with the lowest mobility as indicated by the atomic B-factors. Five proline residues surround the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗
PubMed2000

CD154 variant lacking tumor necrosis factor homologous domain inhibits cell surface expression of wild-type protein.

X-linked hyper-IgM (XHIM) syndrome is an immunological disorder resulting from mutations in the CD154 gene. Some mutations occur in splicing sites and result in transcripts encoding wild-type and mutant proteins. These mutants lack the tumor necrosis factor homologous (TNFH) domain and consequently fail to trimerize. Given that the TNFH domain is responsible for trimerization, one may predict that the TNFH mutant can not participate in the assembly of wild-type CD154. Thus, it was puzzling why these patients exhibit XHIM phenotype, presumably resulting from a lack of functional CD154. One possibility is that the TNFH mutant exhibits a dominant negative effect over the wild-type protein. To investigate this, we coexpressed the wild-type protein and a TNFH mutant and examined the biochemical and functional properties of the resulting CD154 products. We demonstrate that despite the lack of the TNFH domain, the TNFH mutant can associate with the wild-type protein. Furthermore, such an association compromises the ability of the wild-type protein to mature onto the cell surface. These results provide a mechanism for the defect of CD154 in XHIM patients producing both wild-type and TNFH variants and suggest that besides the TNFH domain, the stalk region participates in the assembly of CD154 trimers.

CD40 Ligand↗
PubMed2000

Functional dissection of the major structural protein of bluetongue virus: identification of key residues within VP7 essential for capsid assembly.

A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer. Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid. One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.

Amino Acid Sequence↗
PubMed2005

Molecular self-assemblies of a pi-conjugated redox-active bipyridinium cation with magnetic dimetallic oxalate-bridged trimeric clusters.

A series of molecular assemblies constructed from a pi-conjugated redox-active bipyridinium cation, 1,4-bis(4'-pyridyl-1'-pyridinio)phthalazine (Bpyph), and magnetic dimetallic oxalate-bridged trimeric clusters, has been synthesized and characterized by elemental analysis, IR, TGA, X-ray single-crystal diffraction and magnetic susceptibility studies. The molecular assemblies formulated as (Bpyph)2{M(II)(H2O)2[M(III)(C2O4)3]2}.12.5H2O [for M(III) = Fe; M(II) = Mn (1) or Co (2); for M(III) = Cr; M(II) = Mn (3) or Co4] are isostructural, their structures feature an alternative arrangement of Bpyph2+ cations and the linear trimeric oxalate complexes {M(II)(H2O)2[M(III)(C2O4)3]2} along all three crystallographic axes, in which the dimetallic trimers form two distinct homo-chiral helices along the b axis via intermolecular hydrogen bonding interactions. Within each trimeric cluster, the two M(III) sites have opposite chirality (Delta and Lambda). Studies on the magnetic properties reveal the presence of antiferromagnetic exchange interactions within the trimeric clusters for the Fe2M and ferromagnetic for the Cr2M series.

Journal Article↗
PubMed2006

Invariant chain transmembrane domain trimerization: a step in MHC class II assembly.

The transmembrane (TM) domain of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) has long been implicated in both correct folding and function of the MHC class II complex. To function correctly, Ii must form a trimer, and the TM domain is one of the domains thought to stabilize the trimeric state. Specific mutations in the TM domain have been shown previously to disrupt MHC class II functions such as mature complex formation and antigen presentation, possibly due to disruption of Ii TM helix-helix interactions. Although this hypothesis has been reported several times in the literature, thus far no experimental measurements have been made to explore the relationship between TM domain structure and TM mutations that affect Ii function. We have applied biophysical and computational methods to study the folding and assembly of the Ii TM domain in isolation and find that the TM domain strongly self-associates. According to analytical ultracentrifugation analyses, the primary oligomeric state for this TM domain is a strongly associated trimer with a dissociation constant of approximately 120 nM in DPC micelles. We have also examined the effect of functionally important mutations of glutamine and threonine residues in the TM domain on its structure, providing results that now link the disruption of TM helix interactions to previously reported losses of Ii function.

Amino Acid Sequence↗
PubMed2003

Intermolecular interactions in a two-layered viral capsid that requires a complex symmetry mismatch.

The surface of the bluetongue virus core forms a T=13 quasiequivalent icosahedral protein shell with 260 trimers of a single gene product: VP7 protein. Underneath is a smooth layer, made up of VP3 protein, which appears to guide and nucleate the assembly of VP7 trimers. The contacts between the two shells are extensive but nonspecific, and construction of the T=13 icosahedral shell requires polymorphism in the association of the VP7 subunits, each of which has two domains that contribute to trimer formation. We used structural and relative sequence information to guide an investigation of how such a complex structure is achieved during virus assembly and what residues are required to form a stable capsid. Fifteen single or multiple site-specific substitution mutations were introduced into the helical domain of VP7, which is closely associated with the VP3 layer, and the effects on capsid assembly were analyzed. Our data show that both the position and the nature of single residues are critical for the attachment of VP7 to VP3 and that formation of a stable VP7 lattice is not the automatic consequence of trimer formation.

Amino Acid Sequence↗
PubMed1991

Assembly of adenovirus type 2 fiber synthesized in cell-free translation system.

Physicochemical and functional analyses of the translation products of fiber mRNA in rabbit reticulocyte lysate suggested that fiber polypeptide chains (monomers) were capable of self-assembling in vitro, forming trimeric fibers (trimers) without direct intervention of any other adenovirus-coded protein or cell nuclear matrix component. Kinetic studies showed that trimer formation occurred at a rate six times lower than that of fiber polypeptide synthesis. Fiber assembly was found to be relatively inefficient in vitro, with only 25-30% fiber polypeptides trimerized after 4-h translation reaction. The rate constant for fiber subunit assembly, extrapolated from the kinetic curves of trimer formation, was found to be in the order of magnitude of 10(5) M-1 s-1, with a t 1/2 of 1.3 h at 30 degrees C. A latence phase of approximately 40 min in the appearance of the first detectable trimers indicated that fiber assembly did not occur co-translationally, suggesting the existence of rate-limiting intermediate step(s) during assembly.

Adenoviruses, Human↗
PubMed2004

Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus.

Fusion of biological membranes is mediated by specific lipid-interacting proteins that induce the formation and expansion of an initial fusion pore. Here we report the crystal structure of the ectodomain of the Semliki Forest virus fusion glycoprotein E1 in its low-pH-induced trimeric form. E1 adopts a folded-back conformation that, in the final post-fusion form of the full-length protein, would bring the fusion peptide loop and the transmembrane anchor to the same end of a stable protein rod. The observed conformation of the fusion peptide loop is compatible with interactions only with the outer leaflet of the lipid bilayer. Crystal contacts between fusion peptide loops of adjacent E1 trimers, together with electron microscopy observations, suggest that in an early step of membrane fusion, an intermediate assembly of five trimers creates two opposing nipple-like deformations in the viral and target membranes, leading to formation of the fusion pore.

Amino Acid Sequence↗
PubMed2003

Assembly of TolC, a structurally unique and multifunctional outer membrane protein of Escherichia coli K-12.

TolC is a multifunctional outer membrane protein of Escherichia coli that folds into a novel alpha-beta-barrel conformation absent in the other model outer membrane proteins used in assembly studies. The data presented in this work show that the unique folded structure of TolC reflects a unique assembly pathway. During its assembly, the newly translocated nascent TolC monomers are released in the periplasm. Maturation of these nascent monomers, and possibly their oligomerization, in the periplasm precedes their insertion in the outer membrane. The completion of the assembly process is signaled by the development of a characteristic proteinase K-resistant fragment generated by cleavage at a single, periplasmically exposed, protease-sensitive site of the membrane-anchored trimer. None of the assembly steps of TolC is affected by known folding factors, such as SurA, Skp, and lipopolysaccharide, which have profound effects on the assembly of other model trimeric outer membrane proteins. Two assembly-defective TolC mutants were isolated and characterized. One of the mutants (TolC(I106N)) was defective in the folding of nascent monomers, while the other (TolC(S350F)) was impaired in steps involving trimerization and membrane insertion of folded monomers.

Bacterial Outer Membrane Proteins↗
PubMed1983

A hydrodynamic study of collagen fibrillogenesis by electric birefringence and quasielastic light scattering.

Neutral soluble collagen was extracted from lathyritic rat skin under proteolysis-inhibited conditions. Purified solutions were characterized by electric birefringence and heterodyne beat quasi-elastic light-scattering techniques under conditions where the monomeric form was stable (at 4 degrees C in 0.032 M phosphate buffer at pH 7.04). Solutions were then heated and the birefringence and light scattering followed during the fibrillogenesis reaction. The monomer presents a translational diffusion coefficient of 0.85 X 10(-7) cm2/s and a rotary diffusion coefficient of 1150 +/- 50 s-1; these values are consistent with a rodlike molecular model of 220 +/- 10 nm length and 4 +/- 1 nm diameter, substantially different from electron microscopic values of 290 and 1.5 nm, respectively. We propose that at pH 7.04 and relatively high ionic strength, the collagen monomer unit must exhibit substantial deviation from a completely rigid and extended rodlike structure. During the entire lag phase in a thermally induced fibrillogenesis reaction, the relaxation times for both translational and rotational motion remain virtually unchanged. The monomer polarity is also unchanged, as shown by reverse pulse birefringence data. No intermediate size soluble aggregates, such as dimers or trimers, have been detected between monomer and very large aggregates or fibrils during the process, although early multistep assembly products (dimers, trimers) could have been seen if present. These data suggest a model for fibrillogenesis emphasizing a monomer-related nucleation event, such as internal stiffening or conformational transition, followed by a rapid continuous growth up to large fibrils.

Animals↗
PubMed1997

Expression of the long arm sequence of mouse laminin alpha1, beta1, or gamma1 chain in COS1 cells and assembly of monkey-mouse hybrid laminin.

Mouse laminin alpha1, beta1, or gamma1 sequence covering truncated regions of the long arm was transiently expressed in monkey COS1 cells. Unlike natural laminins, in which only alpha beta gamma trimers are selectively assembled and disulfide-bonded at the long arm, a large fraction of mouse chains formed disulfide-bonded homopolymers. However, a small fraction of mouse beta1 (or gamma1) formed hybrid beta1gamma1 dimers with endogenous monkey gamma1 (or beta1). These hybrid beta1gamma1 dimers formed alpha1 beta1 gamma1 trimer with monkey alpha1. Mouse alpha1 also formed disulfide bonds with monkey beta1gamma1 dimer. Thus, a common mechanism is shared by laminin chains of different animal origins. Sequences in the E8 region at the C-terminal end of the long arm were crucial for this chain-selective assembly. When the C-terminal sequence of mouse beta1 long arm was extended beyond the alpha-loop, the hybrid trimer formation was diminished. This supported the model of altered chain arrangement around the alpha-loop.

Animals↗
PubMed2002

Expression, mutagenesis and kinetic analysis of recombinant K1E endosialidase to define the site of proteolytic processing and requirements for catalysis.

Catalytically active, recombinant fusion proteins of bacteriophage E endosialidase were expressed and purified from Escherichia coli. Constructs with different fusion partners added to the amino terminus of the endosialidase were enzymatically active. A post-translational proteolytic cleavage was shown to occur between serine 706 and aspartate 707 to generate the 76 kDa mature enzyme from the 90 kDa translation product. Endosialidase truncated at the C-terminus from aspartate 707 was observed to have the same 76 kDa molecular weight as wild-type enzyme using denaturing SDS-PAGE but, under native PAGE conditions, was not observed to form the approximately 250 kDa trimeric wild-type enzyme, implying that the C-terminus of the enzyme may be required for correct assembly of active trimer, rather than as part of the active site as has been previously suggested. Mutagenesis of aspartate 138 to alanine greatly reduced enzyme activity whereas conversion of other selected aspartate residues to alanine had less effect, consistent with similarities between the structure and cata-lytic mechanism of bacteriophage E endosialidase and those of exosialidases.

Alanine↗
PubMed1981

Characterization of two temperature-sensitive mutants of type 5 adenovirus with mutations in the 100,000-dalton protein gene.

Complementation analysis assigned the mutations of strains H5ts115 and H5ts116, two hexon-minus mutants, to the 100,000-dalton (100K) protein gene. Heterotypic marker rescue (i.e., type 5 adenovirus [Ad5] temperature-sensitive mutants DNA X EcoRI restriction fragments of Ad2 DNA) confirmed the results of previous marker rescue mapping studies, and the heterotypic recombinants yielded unique hybrid (Ad5-Ad2) 100K proteins which were intermediate in size between Ad5 and Ad2 proteins and appeared to be as functionally active as the wild-type 100K protein. Phenotypic characterization of these mutants showed that both the hexon polypeptides and the 100K polypeptides were unstable at the nonpermissive temperature, whereas fiber and penton were not degraded, and that the 100K protein made at 39.5 degrees C could not be utilized after a shift to the permissive temperature (32 degrees C). The role of the 100K protein in the assembly of the hexon trimer was also examined by in vitro protein synthesis. Normally, hexon polypeptides synthesized during an in vitro reaction are assembled into immunoreactive hexons. However, this assembly was inhibited by preincubation of the cell extract with anti-100K immunoglobulin G; neither anti-fiber immunoglobulin G nor normal rabbit immunoglobulin G inhibited hexon assembly. It is postulated that an interaction between the 100K protein and hexon polypeptides is required for effective assembly of hexon trimers.

Adenoviruses, Human↗
PubMed1999

A role for the transmembrane domain in the trimerization of the MHC class II-associated invariant chain.

MHC class II and invariant chain (Ii) associate early in biosynthesis to form a nonameric complex. Ii first assembles into a trimer and then associates with three class II alphabeta heterodimers. Although the membrane-proximal region of the Ii luminal domain is structurally disordered, the C-terminal segment of the luminal domain is largely alpha-helical and contains a major interaction site for the Ii trimer. In this study, we show that the Ii transmembrane domain plays an important role in the formation of Ii trimers. The Ii transmembrane domain contains an unusual patch of hydrophilic residues near the luminal interface. Substitution of these polar residues with nonpolar amino acids resulted in a decrease in the efficiency of Ii trimerization and subsequent class II association. Moreover, N-terminal fragments of Ii were found to trimerize independently of the luminal alpha-helical domain. Progressive C-terminal truncations mapped a homotypic association site to the first 80 aa of Ii. Together, these results implicate the Ii transmembrane domain as a site of trimer interaction that can play an important role in the initiation of trimer formation.

Amino Acid Sequence↗
PubMed1989

Existence and purification of porin heterotrimers of Escherichia coli K12 OmpC, OmpF, and PhoE proteins.

Porin is a trimeric membrane protein that functions as a diffusion pore in the outer membrane of Escherichia coli. We report the existence and purification of porin heterotrimers between the ompC, ompF, and phoE porin gene products. Separation was achieved using a high resolution anion exchange column. The amount of each heterotrimer species present depended on the level of expression of the subunits and was consistent with random mixing of trimer subunits. A strong effect of bacterial lipopolysaccharide on the chromatography of porin was also detected. These results imply that assembly of porin trimers occurs between subunits synthesized on different polysomes and that subunit contacts between the porin subunits occur in conserved regions of the primary sequence.

Bacterial Outer Membrane Proteins↗