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PubMed1996

Dimorphism of hepatitis B virus capsids is strongly influenced by the C-terminus of the capsid protein.

Hepatitis B virus (HBV) is an enveloped virus with an icosahedral capsid. Its homodimeric capsid protein ("core antigen") assembles into particles of two sizes, one with T = 3 icosahedral symmetry (90 dimers) and the other with T = 4 symmetry (120 dimers). We have investigated this assembly process in vitro, using a variety of purified, bacterially expressed, capsid proteins. All of our constructs lacked the predominantly basic C-terminal 34 amino acids of the full-length capsid protein (183 amino acids) and were further truncated to terminate at specific points between residues 138 and 149. While the smallest construct (138 residues) did not assemble into capsids, those terminating at residue 140, and beyond, assembled into mixtures of T = 3 and T = 4 particles. The two kinds of capsids could be separated on sucrose gradients and did not interconvert upon protracted storage. The proportion of T = 3 capsids, assayed by sucrose gradient fractionation, analytical ultracentrifugation, and cryoelectron microscopy, was found to increase systematically with larger deletions from the C-terminus. The variant terminating at residue 149 formed approximately 5% of T = 3 capsids, while the 140-residue protein produced approximately 85% of this isomorph. For the 147-residue capsid protein, the structures of both capsids were determined to 17 A resolution by three-dimensional reconstruction of cryoelectron micrographs. In these density maps, the boundaries of the constituent dimers can be clearly seen and the quaternary structures of the two capsids compared. The arrangement of dimers around their icosahedral five-fold axes is almost identical, whereas the quasi-six-fold arrangements of dimers are distinctly different.

Amino Acid Sequence↗

PubMed2002

Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT.

The biogenesis of the cytoskeletal proteins actin and tubulin involves interaction of nascent chains of each of the two proteins with the oligomeric protein prefoldin (PFD) and their subsequent transfer to the cytosolic chaperonin CCT (chaperonin containing TCP-1). Here we show by electron microscopy that eukaryotic PFD, which has a similar structure to its archaeal counterpart, interacts with unfolded actin along the tips of its projecting arms. In its PFD-bound state, actin seems to acquire a conformation similar to that adopted when it is bound to CCT. Three-dimensional reconstruction of the CCT:PFD complex based on cryoelectron microscopy reveals that PFD binds to each of the CCT rings in a unique conformation through two specific CCT subunits that are placed in a 1,4 arrangement. This defines the phasing of the CCT rings and suggests a handoff mechanism for PFD.

Actins↗

PubMed1994

Electron microscopy of cytochrome c oxidase crystals. Monomer-dimer relationship and cytochrome c binding site.

Cytochrome c oxidase was isolated from beef heart mitochondria by detergent extraction yielding two different crystal forms. Extraction with Triton detergents produced vesicular crystals with two-dimensional crystalline arrays of cytochrome c oxidase dimers while extraction with sodium deoxycholate produced crystalline sheets of cytochrome c oxidase monomers. The structures of both crystal forms were determined in two-dimensional projection along an axis normal to the plane of the membrane by cryoelectron microscopy of crystals embedded in vitreous ice (frozen-hydrated). The projection structures of unstained frozen hydrated monomers and of dimers are similar to the structures of the crystals in negative stain. The molecular outline of dimers can be approximated by a parallelogram 44 A by 82 A with an included angle of 80 degrees. Monomers are less regular consisting of two large domains with a smaller domain at one end and a total length of approximately 82 A. Comparison of the two structures reveals the orientation of cytochrome c oxidase monomers within dimers, an orientation which is different from earlier models of monomer-monomer interaction, and suggests a very close interaction between monomers when they associate to form dimers. The crystalline sheets of cytochrome c oxidase monomers bind tightly the small peripheral membrane protein substrate, cytochrome c, and this binding accentuates a tendency of these crystals to stack upon one another. Images of crystals of the cytochrome c oxidase/cytochrome c complex were analyzed by crosscorrelation analysis versus the monomer crystal image. Two types of two-layer crystals have been identified. Both types have one layer rotated by 180 degrees with respect to the other, but they differ in the shifts of origin along crystal axes of the two layers. Difference images formed by subtracting simulated multilayered crystal images (which have no bound cytochrome c) from the complex crystals (cytochrome c oxidase plus cytochrome c) contain one positive difference peak for each cytochrome oxidase monomer within a unit cell. Comparison of the difference peak loci among the different crystal forms is interpreted based upon a consensus cytochrome c binding site in the single layer cytochrome oxidase monomer crystal image.

Animals↗

PubMed2002

The cryptophycin-tubulin ring structure indicates two points of curvature in the tubulin dimer.

Cryptophycin-1 is the parent compound of a group of cyclic peptides with potent antineoplastic activity. Cryptophycins are thought to function by modulating the dynamic instability of spindle microtubules, and in vitro are known to bind in an equimolar ratio to the beta-tubulin subunit and to induce the formation of ring-like complexes. However, the detailed mechanisms whereby the cryptophycins interact with tubulin are not known. We have investigated the origin of the conformational changes in tubulin both biochemically and by electron microscopy and image analysis. Cryptophycin was found to protect both alpha- and beta-tubulin against proteolysis by trypsin, indicating conformational changes in specific regions of both subunits. The ring mass was determined to be approximately 0.81 MDa by sedimentation velocity combined with dynamic light scattering and by STEM, indicating a complex of eight alphabeta dimers. Statistical analysis of rings imaged by cryoelectron microscopy revealed 16-fold symmetry, corresponding to eight dimers. Computational averaging based on this symmetry yielded an image of a 24 nm diameter ring, at 2.6 nm resolution, that clearly distinguishes intradimer contacts from interdimer contacts, and allows discrimination of alpha-subunits from beta-subunits. Fitting of the tubulin dimer crystal structure into this projected density map indicates two points of curvature: a 13 degrees intradimer bend and a 32 degrees interdimer bend. We conclude that drug binding to one subunit (beta) results in two bends per dimer, affecting both subunits.

Animals↗

PubMed1996

Topography and immunogenicity of bluetongue virus VP7 epitopes.

The core of bluetongue virus (BTV) consists of ten dsRNA viral genome segments and five proteins, including two major (VP7 and VP3) and three minor (VP1, VP4 and VP6) components. The major core protein VP7 is believed to be an important structural constituent because it interacts, not only with the underlying core protein VP3, but also with two outer capsid proteins (VP2 and VP5). In this communication we summarise data on the mapping of at least six different epitopes of VP7 distributed along the molecule. Two of the six epitopes have not been mapped previously. The accessibility of these epitopes in intact virions and core particles was analysed using immunoelectron microscopy. The epitope located near the N-terminus of VP7 was accessible at the surface of intact virions and core particles. Epitopes in other parts of the VP7 molecule were detected weakly in core particles but not in intact virions. These results support the proposal that VP7 molecules are orientated with their N-terminus accessible on the surface of either the particle or at least one of the three different channels observed by cryoelectron microscopy in the outer capsid layer. Analysis of the immune response to BTV-infected or -immunised sheep and rabbits to three selected epitopes, which are located in different regions of the VP7 molecule, demonstrated that all of them were recognised by the animals tested. These results provided further molecular evidence suggesting that VP7 is indeed a major immunogenic antigen ideal for BTV antibody detection.

Animals↗

PubMed1998

Structure of a neutralizing antibody bound monovalently to human rhinovirus 2.

The structure of a complex between human rhinovirus 2 (HRV2) and the Fab fragment of neutralizing monoclonal antibody (MAb) 3B10 has been determined to 25-A resolution by cryoelectron microscopy and three-dimensional reconstruction techniques. The footprint of 3B10 on HRV2 is very similar to that of neutralizing MAb 8F5, which binds bivalently across the icosahedral twofold axis. However, the 3B10 Fab fragment (Fab-3B10) is bound in an orientation, inclined at approximately 45 degrees to the surface of the virus capsid, which is compatible only with monovalent binding of the antibody. The canyon around the fivefold axis is not directly obstructed by the bound Fab. The X-ray structures of a closely related HRV (HRV1A) and a Fab fragment were fitted to the density maps of the HRV2-Fab-3B10 complex obtained by cryoelectron microscope techniques. The footprint of 3B10 on the viral surface is largely on VP2 but also covers the VP3 loop centered on residue 3064 and the VP1 loop centered on residue 1267. MAb 3B10 can interact directly with VP2 residue 2164, the site of an escape mutation on VP2, and with VP1 residues 1264 to 1267, the site of a deletion escape mutation. Deletion of these residues shortens the VP1 loop, moving it away from the MAb binding site. All structural and biochemical evidence indicates that MAb 3B10 binds to a conformation epitope on HRV2.

Antibodies, Monoclonal↗

PubMed2004

Yaba-like disease virus protein Y144R, a member of the complement control protein family, is present on enveloped virions that are associated with virus-induced actin tails.

Yaba-like disease virus (YLDV) is a yatapoxvirus, a group of slow-growing poxviruses from primates. Analysis of the growth cycle of YLDV in tissue culture showed that maximum virus titres were reached 3 days post-infection and at this time only 3.3 % of infectious progeny was extracellular. The intracellular and extracellular virions have different buoyant densities and are separable on CsCl density gradients. They are also distinguishable by electron microscopy with the extracellular virions having an additional lipid envelope. In YLDV-infected cells, thick actin bundles with virions at their tips were seen protruding from the cell surface, despite the fact that YLDV lacks a protein comparable to Vaccinia virus A36R, which is required for VV-induced actin tail formation. In addition to these observations, the YLDV gene Y144R was characterized. This gene is predicted to encode a transmembrane protein containing three short consensus repeat (SCR) motifs common to members of the complement control protein family. Antibody generated against recombinant Y144R recognized products of 36, 41 and 48-55 kDa in YLDV-infected cells and purified extracellular enveloped virus (EEV) but not intracellular mature virus (IMV). Y144R protein is a glycoprotein with type I membrane topology that is synthesized early and late during infection. By immunoblot, indirect immunofluorescence and immuno-cryoelectron microscopy the Y144R protein was detected on the intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and EEV. This represents the first study of a YLDV IEV, CEV and EEV protein at the molecular level.

Actins↗

PubMed1997

Capsid assembly and DNA packaging in herpes simplex virus.

The genome of HSV-1 contains 80-85 open reading frames. Genetic and biochemical evidence suggests that at least 39 of these genes encode proteins that are components of the HSV-1 virion. The architecture of the HSV-1 virion consists of a trilaminar lipid envelope, an amorphous layer known as the tegument, a capsid shell, and a DNA-containing core. The capsid is an icosahedral shell whose major morphological features are 162 capsomers. It is composed of a major capsid protein called VP5 and three less abundant proteins, VP19C, VP23 and VP26. VP5 is the structural subunit of all 162 capsomers while VP19C and VP23 are located in the space between the capsomers. In addition to the structural proteins, capsid assembly involves participation of the HSV-1-encoded protease and the scaffolding protein, preVP22a. DNA packaging involves participation of DNA, empty capsids, and at least seven additional HSV-1-encoded proteins. Considerable advances have been made in understanding the structure of the capsid shell, largely as the result of applying cryoelectron microscopy techniques. Use of recombinant baculoviruses has allowed for a detailed analysis of the proteins required for capsid assembly. More recently, an in vitro system has been developed which has aided in defining the assembly pathway by identifying intermediates in the assembly of intact capsids. The in vitro system has identified a fragile roundish procapsid which matures into the polyhedral capsid in a transition similar to that undergone by bacteriophage proheads. This review is a summary of our present knowledge with respect to the structure and assembly of the HSV-1 capsid and what is known about the seven genes involved in DNA packaging. Copyright 1997 John Wiley & Sons Ltd.

Journal Article↗

PubMed1990

Method for forming two-dimensional paracrystals of biological filaments on lipid monolayers.

A method is described for electron microscopic observation of two-dimensional paracrystals on unsupported lipid monolayers. The method uses a hydrophobic holey C-coated grid placed on a monolayer made positively charged by the inclusion of stearylamine (SA) and has been used to align scallop thin filaments and reconstituted actin/tropomyosin filaments to form paracrystals. The use of unsupported monolayers allows the paracrystals to be viewed in either negative stain or with cryoelectron microscopy. Those paracrystals in frozen hydrated specimens have better order than those with negative stain. It was found that varying the lipid composition between the less fluid distearolyphosphotidylcholine/SA and the more fluid egg yolk phosphotidylcholine/SA alters the size and order of the paracrystals, the more fluid system having smaller, more ordered paracrystalline domains. The advantage of the technique for studying actin/thin filaments is the ability to form large two-dimensional paracrystals under physiological conditions of [Mg2+] and pH.

Crystallization↗

PubMed1994

Polyethylene glycol modified phospholipids stabilize emulsions prepared from triacylglycerol.

A stable lipid-based carrier system containing a triacylglycerol core has been developed. This has been achieved by homogenization of corn oil (primarily triacylglycerol) in the presence of phosphatidylcholine (PC), preformed 100 nm liposomes prepared from PC/cholesterol (55: 45; mol:mol) and polyethylene glycol modified phosphatidylethanolamine (PEG2000-PE). The lipid/liposome mixtures were emulsified using a microfluidizer, and the resulting particles could, depending on the phospholipids used and the addition of cholesterol, be designed to exhibit a uniform mean particle size of less than 100 nm (as measured by quasielastic light scattering). The presence of an oil core within the emulsified lipid preparation was confirmed by freeze-fracture and cryoelectron microscopy. Stability of the resulting PEG2000-PE-coated triacylglycerol emulsion was determined by several techniques including (1) time dependent changes in light scatter determined by measuring changes in absorbance at 600 nm (decreases in absorbance are indicative of unstable oil emulsions), (2) column chromatography procedures evaluating the migration of incorporated lipids, specifically PEG2000-PE and triolein, after emulsions were incubated at 37 degrees C in the presence and absence of serum, and (3) in vivo plasma clearance data demonstrating that the lipid mixtures were maintained at ratios specified prior to i.v. administration. This emulsion technology has been used to prepare formulations of several water insoluble compounds, such as the drugs taxol and dibucaine and the lipophilic dye sudan IV.

Animals↗

PubMed1997

Cloning and some novel characteristics of mitochondrial Hsp70 from Chinese hamster cells.

The cDNA for Chinese hamster mitochondrial Hsp70 (mHsp70) was cloned and sequenced using a polymerase chain reaction probe based on conserved regions in the Hsp70 family of proteins. The encoded protein consists of 679 amino acids which includes a N-terminal mitochondrial targeting sequence of 46 amino acids. The mHsp70 protein contains several sequence signatures that are characteristics of prokaryotic and eukaryotic organellar Hsp70 homologs. In a phylogenetic tree based on Hsp70 sequences, it branches with the gram-negative proteobacteria, supporting the endosymbiotic origin of mitochondria from this group of prokaryotes. The mHsp70 cDNA was transcribed and translated in vitro and its import into isolated rat heart mitochondria was examined. The precursor mHsp70 was converted into a mature form of lower molecular mass (approximately 71 kDa) which became resistant to trypsin digestion. The import of mHsp70 into mitochondria was not observed in the presence of an uncoupler of energy metabolism or when the N-terminal presequence was lacking. The cDNA for mHsp70 was expressed in Escherichia coli and a polyclonal antibody to the purified recombinant protein was raised. The antibody shows no cross-reactivity to recombinant cytosolic Hsp70 protein and in 2-D gel blots it reacted specifically with the mHsp70 protein only. In immunofluorescence experiments, the antibody predominantly labeled mitochondria, and the observed labeling pattern was identical to that seen with a monoclonal antibody to the mitochondrial Hsp60 chaperonin. The affinity-purified antibody to mHsp70 was also employed to examine the subcellular distribution of the protein by cryoelectron microscopy and the immunogold-labeling technique. In these experiments, in addition to mitochondria, labeling with mitochondrial Hsp70 antibody was also observed on the plasma membrane and in unidentified cytoplasmic vesicles and granules. These studies raise the possibility that similar to the Hsp60 chaperonin and a number of other mitochondrial proteins, mHsp70 may have an extramitochondrial role.

Amino Acid Sequence↗

PubMed1993

Three-dimensional architecture of human alpha 2-macroglobulin transformed with methylamine.

A frozen-hydrated sample embedded in vitreous ice of human alpha 2-macroglobulin transformed by methylamine was imaged by cryoelectron microscopy and reconstructed in three dimensions. In the reconstruction, the cage-like architecture of this protease inhibitor is fully revealed with a clear visualization of two lozenge-shaped lateral walls connected by thin bridges. The shape and dimensions of the internal cavity normally containing the trapped protease(s) is described. The possible locations of the thiol ester sites and inter-subunit connections are also discussed.

Image Processing, Computer-Assisted↗

PubMed1994

Formation of two-dimensional arrays of annexin V on phosphatidylserine-containing liposomes.

Annexins are intracellular proteins which bind to membranes in a Ca(2+)-dependent manner and which have been proposed to play regulatory roles in different membrane processes. In the present study, the stoichiometry of the Ca(2+)-dependent binding of annexin V to phosphatidylserine molecules incorporated into liposomes was studied by fluorescence spectroscopy. The Ca(2+)-dependence of the binding was determined using liposomes made of dioleoylphosphatidylserine (PS) and dioleoylphosphatidylcholine (PC), with a PC/PS molar ratio ranging from 1 to 800. These liposomes were shown to be mostly unilamellar by cryoelectron microscopy. [Ca2+]1/2 concentrations required for half-maximal binding of annexin V range from 57 microM at PC/PS = 1 up to 96 mM at PC/PS = 800. Titration of accessible PS molecules showed that annexin V molecules bind equally well to liposomes of PC/PS ratio ranging from 1 to 400. The stoichiometry of the binding between annexin V and PS, determined at low PS content, is eight annexin V molecules per one PS molecule. We propose a novel model of the Ca(2+)-dependent interaction between annexin V and lipid membranes, based on the formation of two-dimensional arrays of annexin V molecules, stabilized by both protein-lipid and protein-protein interactions.

Annexin A5↗

PubMed1996

Conserved features in papillomavirus and polyomavirus capsids.

Capsids of papilloma and polyoma viruses (papovavirus family) are composed of 72 pentameric capsomeres arranged on a skewed icosahedral lattice (triangulation number of seven, T = 7). Cottontail rabbit papillomavirus (CRPV) was reported previously to be a T = 7laevo (left-handed) structure, whereas human wart virus, simian virus 40, and murine polyomavirus were shown to be T = 7dextro (right-handed). The CRPV structure determined by cryoelectron microscopy and image reconstruction was similar to previously determined structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1). CRPV capsids were observed in closed (compact) and open (swollen) forms. Both forms have star-shaped capsomeres, as do BPV-1 and HPV-1, but the open CRPV capsids are approximately 2 nm larger in radius. The lattice hands of all papillomaviruses examined in this study were found to be T = 7dextro. In the region of maximum contact, papillomavirus capsomeres interact in a manner similar to that found in polyomaviruses. Although papilloma and polyoma viruses have differences in capsid size (approximately 60 versus approximately 50 nm), capsomere morphology (11 to 12 nm star-shaped versus 8 nm barrel-shaped), and intercapsomere interactions (slightly different contacts between capsomeres), papovavirus capsids have a conserved, 72-pentamer, T = 7dextro structure. These features are conserved despite significant differences in amino acid sequences of the major capsid proteins. The conserved features may be a consequence of stable contacts that occur within capsomeres and flexible links that form among capsomeres.

Animals↗

PubMed1998

Formation of the gap junction intercellular channel requires a 30 degree rotation for interdigitating two apposing connexons.

Intercellular communication via gap junction membrane channels cannot occur until two apposing hemichannels (connexons) meet and dock to form a sealed cell-cell conduit. In particular, an important question is how does the structure at the extracellular surface influence the molecular recognition of the two connexons. In this study, cryoelectron microscopy and computer modeling provide evidence that the formation of the gap junction intercellular channel requires a 30 degree rotation between hemichannels for proper docking. With this amount of rotation, the peaks (protrusions) on one connexon fit into the valleys of the apposed connexon in the 3-D model, which would make for an ionically tight interface necessary for a functional cell-cell channel. Docking appears to be governed by a "lock and key" mechanism via a simple interdigitation of the six protrusions from each connexon. This interdigitation increases significantly the contact surface area and potential number of hydrogen bonds or hydrophobic interactions and/or other attractive interactions. Having a larger surface area than if the surfaces were flat would explain the biochemical requirements for conditions characterized previously for splitting of channels into hemichannels. The docked connexons were computationally fitted into two gap junction structures, which further confirmed the interdigitated manner of docking.

Cell Communication↗

PubMed1998

Nucleotide-dependent structural changes in dimeric NCD molecules complexed to microtubules.

Complexes consisting of motor domains of the kinesin-like protein ncd bound to reassembled brain microtubules were visualised using cryoelectron microscopy and helical image reconstruction. Different nucleotide-associated states of a dimeric construct (NDelta295-700) of ncd were analysed to reveal ADP-containing, AMP.PNP-containing and empty (rigor) conformations. In these three states, each thought to mimic a different stage in ATP turnover, the double-headed motors attach to the microtubules by one head only, with the free head tethered in relatively fixed positions. The three structures differ both in the way the attached heads interact with tubulin and in the position of the tethered heads. In the strongly binding rigor and AMP.PNP (ATP-like) states, the attached head makes close contact with both subunits of a tubulin heterodimer. In the weakly bound ADP state, the contact made by the attached head with the monomer closer to the plus end appears to be more loose. Also, in the ATP-like state, the free head tilts nearer to the plus end than in the other two states. The data argue against model mechanisms in which a conformational change in the bound head guides the free head closer to its next binding site; on the contrary, the transition from ADP-filled via rigor to the AMP.PNP (ATP-like) state of the bound head produces a small motion of the free head in the counter-productive direction. However, the observation that the tethered head points towards the minus end, in all three states, is consistent with the idea that the relative arrangement of the heads in a dimer is a major determinant of directionality.

Adenosine Diphosphate↗

PubMedDate not supplied

Structure of correctly self-assembled bluetongue virus-like particles.

Bluetongue virus-like particles (VLPs), synthesized by coexpression of VP2, VP3, VP5, and VP7 using recombinant baculoviruses, have been examined by cryoelectron microscopy and image analysis. The 3-D reconstruction of these VLPs reveals an icosahedral structure 86 nm in diameter with essentially the same features as for the native Bluetongue virus (BTV) particle. The VLP is thus shown to contain the four constituent proteins as the native virus particle, with each of the protein positions highly occupied. Since the BTV core-like particle formed by coexpression of VP3 and VP7 lacks five VP7 trimers around each of the five-fold axes, it appears that the presence of the outer capsid proteins VP2 and VP5 is necessary for the adhesion of these VP7 trimers around the five-fold axes. The observed spontaneous formation of complete VLP in the absence of the BTV nonstructural proteins implies that the nonstructural proteins are not necessary for the formation of the double-shelled viral capsid. However, the nonstructural proteins may be involved in different aspects of genome replication and packaging.

Baculoviridae↗

PubMedDate not supplied

Three-dimensional reconstruction of Androctonus australis hemocyanin labeled with a monoclonal Fab fragment.

An immunocomplex formed by the 4 x 6 meric hemocyanin of the scorpion Androctonus australis with the monoclonal antibody L104 was studied by cryoelectron microscopy and subjected to three-dimensional (3D) reconstruction. The reconstructed particle reflects the structure of the immunocomplex in its hydrated state and is devoid of the flattening that was previously observed with a negatively stained preparation. A 3D fitting of the X-ray data of the Panulirus interruptus hemocyanin and of the Fab R19.9 to the reconstruction volume allowed the first quantitative measurement of the structural parameters of the antigen, and the localization of the epitope at the surface of subunit Aa6. The independent alignment of the Fabs and the hexamers provides a direct verification for the accuracy of the fit and allows the building of the most detailed model of a cheliceratan 4 x 6meric complex and its attached monoclonal antibodies.

Animals↗