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R M Burnett

Publications and source records attributed to R M Burnett.

At least 19 recordsLinked to original sources
PubMed2001

Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions.

BACKGROUND: The dsDNA bacteriophage PRD1 has a membrane inside its icosahedral capsid. While its large size (66 MDa) hinders the study of the complete virion at atomic resolution, a 1.65-A crystallographic structure of its major coat protein, P3, is available. Cryo-electron microscopy (cryo-EM) and three-dimensional reconstruction have shown the capsid at 20-28 A resolution. Striking architectural similarities between PRD1 and the mammalian adenovirus indicate a common ancestor. RESULTS: The P3 atomic structure has been fitted into improved cryo-EM reconstructions for three types of PRD1 particles: the wild-type virion, a packaging mutant without DNA, and a P3-shell lacking the membrane and the vertices. Establishing the absolute EM scale was crucial for an accurate match. The resulting "quasi-atomic" models of the capsid define the residues involved in the major P3 interactions, within the quasi-equivalent interfaces and with the membrane, and show how these are altered upon DNA packaging. CONCLUSIONS: The new cryo-EM reconstructions reveal the structure of the PRD1 vertex and the concentric packing of DNA. The capsid is essentially unchanged upon DNA packaging, with alterations limited to those P3 residues involved in membrane contacts. These are restricted to a few of the N termini along the icosahedral edges in the empty particle; DNA packaging leads to a 4-fold increase in the number of contacts, including almost all copies of the N terminus and the loop between the two beta barrels. Analysis of the P3 residues in each quasi-equivalent interface suggests two sites for minor proteins in the capsid edges, analogous to those in adenovirus.

Adenoviruses, Human↗
PubMed2001

Replication-defective vector based on a chimpanzee adenovirus.

An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.

Adenoviridae↗
PubMed2000

DARWIN: a program for docking flexible molecules.

A new program named "DARWIN" has been developed to perform docking calculations with proteins and other biological molecules. The program uses the Genetic Algorithm to optimize the molecule's conformation and orientation under the selective pressure of minimizing the potential energy of the complex. A unique feature of DARWIN is that it communicates with the molecular mechanics program CHARMM to make the energy calculations. A second important feature is its parallel interface, which allows simultaneous use of multiple stand-alone copies of CHARMM to rapidly evaluate large numbers of potential solutions. This permits an "accuracy first" approach to docking, which avoids many of the common assumptions and shortcuts often made to reduce computation time. The method was applied to three protein-carbohydrate complexes: the crystallographically determined structures of Concanavalin A and Fab Se155-4; and a model structure for Fab ME36.1. Conformations close to the crystal structures were obtained with this approach, but some "false positive" solutions were also selected. Many of these could be eliminated by introducing different methods for simulating solvent effects. An effective screening method for docking a database of compounds to a single target enzyme using DARWIN is also presented.

Algorithms↗
PubMed2000

Crystallization and preliminary X-ray analysis of receptor-binding protein P2 of bacteriophage PRD1.

Bacteriophage PRD1 has remarkable structural similarities to adenovirus, but is unusual in containing a membrane beneath its icosahedral capsid. Its monomeric receptor-binding protein, P2, is part of a complex at each capsid vertex and so is the functional equivalent of adenovirus fiber. P2 has been crystallized by the "hanging-drop" method of vapor diffusion and two different crystal forms were obtained. Macroseeding, used to increase the size of the initial small needles, gave rod-shaped crystals. These grew to a size of 0.08 x 0.08 x 0.50 mm(3) and diffracted to 2.6 A resolution. They have the orthorhombic space group P222(1), with unit cell dimensions a = 137.8 A, b = 46.5 A, c = 136.4 A. A few single crystals of a second form were grown without seeding under slightly different conditions. A parallelepiped crystal (0.10 x 0.10 x 0.35 mm(3)), with space group C222(1) and unit cell dimensions a = 182.3 A, b = 204.8 A, c = 133.3 A, diffracted to 3.5 A resolution. A rotation function for the second form revealed that four monomers of P2 are related by a noncrystallographic twofold axis. The structure of P2 will reveal how this arrangement relates to the trimeric adenovirus fiber.

Capsid↗
PubMed2000

Type-specific epitope locations revealed by X-ray crystallographic study of adenovirus type 5 hexon.

A major obstacle to the use of adenovirus as a vector for gene therapy is the host immune response to hexon, the major protein component of the icosahedral capsid. A solution lies in creating novel vectors with modified or chimeric hexons to evade the immune response to native hexon. The crystal structure of hexon from human adenovirus type 5 (ad5), the type primarily used for gene therapy, has been determined to facilitate the design of such molecules. As the 951-amino-acid (aa) ad5 hexon sequence is closely related to that of ad2 (967 aa; 86% aa identity), the ad5 structure was solved by molecular replacement with a model constructed from the known ad2 hexon. During refinement, greater than 25% of the sequence was reassigned, resulting in a relocation of two epitope regions, from buried positions in the ad2 model to external sites at the top of the ad5 molecule. The resultant model is in better agreement with crystallographic data, while maintaining the overall topology of ad2 hexon. This work suggests that all hexons have the same basic fold and that the ad5 hexon structure provides an accurate and representative model for designing new adenovirus vectors.

Adenoviruses, Human↗
PubMed1999

Viral evolution revealed by bacteriophage PRD1 and human adenovirus coat protein structures.

The unusual bacteriophage PRD1 features a membrane beneath its icosahedral protein coat. The crystal structure of the major coat protein, P3, at 1.85 A resolution reveals a molecule with three interlocking subunits, each with two eight-stranded viral jelly rolls normal to the viral capsid, and putative membrane-interacting regions. Surprisingly, the P3 molecule closely resembles hexon, the equivalent protein in human adenovirus. Both viruses also have similar overall architecture, with identical capsid lattices and attachment proteins at their vertices. Although these two dsDNA viruses infect hosts from very different kingdoms, their striking similarities, from major coat protein through capsid architecture, strongly suggest their evolutionary relationship.

Adenoviruses, Human↗
PubMed1998

Spherical viruses.

In the past two years, structural studies on spherical viruses have experienced a significant advance with the dramatic increase in the resolution attainable by cryo-electron microscopy and image reconstruction. X-ray crystallography, both alone and, increasingly, in combination with electron microscopy, continues to play a crucial role in elucidating how viruses function.

Animals↗
PubMed1997

The crystal structure of a Fab fragment to the melanoma-associated GD2 ganglioside.

The GD2 ganglioside is a cell-surface component that appears on the surface of metastatic melanoma cells and is a marker for the progression of the disease. The ME36.1 monoclonal antibody binds to the GD2 ganglioside and has shown potential as a therapeutic antibody. ME36.1 is a possible alternative therapy to radiation, which is often ineffective in late-stage melanoma. The crystal structure of the Fab fragment of ME36.1 has been determined using molecular replacement and refined to an R factor of 20.4% at 2.8 A resolution. The model has good geometry with root-mean-square deviations of 0.008 A from ideal bond lengths and 1.7 degrees from ideal bond angles. The crystal structure of the ME36.1 Fab shows that its complementarity determining region forms a groove-shaped binding site rather than the pocket-type observed in other sugar binding Fabs. Molecular modeling has placed a four-residue sugar, representative of GD2, in the antigen binding site. The GD2 sugar moiety is stabilized by a network of hydrogen bonds that define the specificity of ME36.1 toward its antigen.

Antibodies, Monoclonal↗
PubMed1994

The refined crystal structure of hexon, the major coat protein of adenovirus type 2, at 2.9 A resolution.

The crystal structure of hexon, the major coat protein from adenovirus type 2, has been refined at 2.9 A resolution. Hexon is a homo-trimer (molecular mass 3 x 109,077 Da) and crystallizes in the cubic space group P2(1)3, with a cell edge of 150.5 A. There are four molecules in the unit cell so that the crystallographic asymmetric unit contains one subunit of the trimer. The electron density in most regions is well-defined and 880 amino acid residues, of the 967 in this unusually long polypeptide chain, have been located and fitted. The N terminus (1 to 43) and three internal stretches (192 to 203, 270 to 291 and 444 to 453) are not defined, and a stretch (168 to 207) with unclear side-chain density is modelled as poly(Ala/Gly). The current refined model, consisting of 6943 non-hydrogen protein atoms and 85 water molecules, yields an R-factor of 19.9% for 18,176 reflections in the resolution range 5.0 to 2.9 A. The model has reasonable geometry with root-mean-square deviations from ideal bond lengths of 0.022 A and angle-related 1-3 distances of 0.056 A. The overall shape of the trimeric hexon molecule is unusual and may be divided into a pseudo-hexagonal base rich in beta-structure, and a triangular top formed from three long loops containing some secondary structure. The base contains two similar pedestal domains, P1 and P2, each of which is a flattened eight-stranded beta-barrel with the "jelly-roll greek key" topology characteristic of other viral coat proteins. P1 and P2 are related by an approximate 6-fold operation about the molecular 3-fold axis so that six barrels form the walls of the tubular hexon base. The hexon bases form close-packed p3 arrays on each facet of the icosahedral adenovirus virion. Unlike other viral capsids, the barrel axes are almost perpendicular to rather than parallel with the capsid surface. The hexon top, which consists of intimately interacting loops emerging from P1 and P2 in the base, has a triangular outline and so does not exhibit the pseudo-symmetry of the base. The structure of the hexon trimer shows how economically it meets the demands of its function as a stable protective viral coat, reveals the significance of the special features in its unusual amino acid sequence, and explains its biochemical and immunological properties. The molecule is hollow, with a large central cavity, and so has a high effective volume for its mass.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary X-ray analysis of human alpha-galactosidase A complex.

Human alpha-galactosidase A (alpha-D-galactoside galactohydrolase; EC 3.2.1.22), the glycosylated lysosomal enzyme deficient in Fabry disease, has been crystallized as a complex with the inhibitor N-6-aminohexanoyl-alpha-D-galactopyranosylamine. The "hanging drop" method of vapor diffusion was used to grow crystals from solutions containing 50 mM sodium phosphate (pH 4.0 to 4.5), 120 to 170 mM ZnCl2 and 8 to 10% polyethylene glycol 3350. X-ray diffraction data collected from these crystals indicate that the crystals belong to the orthorhombic space group C222(1) with cell dimensions of a = 93.8 A, b = 141.1 A and c = 184.4 A. The crystals diffract to a resolution of 3 A and native data have been collected to 3.5 A resolution. Assuming one dimer per asymmetric unit with a total molecular mass of 110 kDa (with oligosaccharide chains), the Matthews' coefficient is Vm = 2.77 A3/dalton corresponding to a solvent content of 55% (v/v). The self-rotation function reveals that a non-crystallographic 2-fold axis relates the subunits of each dimer.

Crystallization↗
PubMed1994

Sequence and structural analysis of murine adenovirus type 1 hexon.

The genomic region encoding the major capsid protein (hexon) of murine adenovirus type 1 (MAV-1) has been isolated and sequenced. The sequence predicts a 908 residue MAV-1 hexon protein and is flanked by a portion of the upstream pVI gene and the downstream endoproteinase gene. The order of these genes and their location in the middle of the genome are the same as those found in other adenoviruses sequenced to date. Multiple sequence alignment with the other five known hexon protein sequences reveals an overall residue identity of 51% and residue conservation of 66%. In comparison with human adenovirus type 2 (Ad2), MAV-1 hexon has major deletions between residues 141 to 170, 270 to 284 and 446 to 455. Since these regions in the Ad2 hexon are partially exposed on the outer surface of the virion, they may represent type-specific antigenic determinants. The MAV-1 hexon sequence has been modelled using the known three-dimensional structure of the Ad2 hexon. The variable regions in which the mutations, deletions and insertions occur are located in the l1 and l2 loops of the molecule that form the protruding hexon towers on the external surface of the virion.

Amino Acid Sequence↗
PubMed1993

Crystallization of the major coat protein of PRD1, a bacteriophage with an internal membrane.

The major multimeric coat protein, P3, of the bacterial virus PRD1 has been crystallized by vapor diffusion from polyethylene glycol 4000. The PRD1-P3 crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 121.6 A, b = 123.2 A, c = 128.6 A and diffract to 3.0 A resolution. Density measurements show that there is one trimer (3 x 43.1 kDa) per asymmetric unit and a high solvent content of 67%. A self-rotation function calculation shows a pronounced peak indicating a non-crystallographic threefold axis. This indicates that the major viral capsomer is a trimer and allows the viral T-number to be postulated.

Bacteriophages↗
PubMed1993

Difference imaging of adenovirus: bridging the resolution gap between X-ray crystallography and electron microscopy.

While X-ray crystallography provides atomic resolution structures of proteins and small viruses, electron microscopy provides complementary structural information on the organization of larger assemblies at lower resolution. A novel combination of these two techniques has bridged this resolution gap and revealed the various structural components forming the capsid of human type 2 adenovirus. An image reconstruction of the intact virus, derived from cryo-electron micrographs, was deconvolved with an approximate contrast transfer function to mitigate microscope distortions. A model capsid was calculated from 240 copies of the crystallographic structure of the major capsid protein and filtered to the correct resolution. Subtraction of the calculated capsid from the corrected reconstruction gave a three-dimensional difference map revealing the minor proteins that stabilize the virion. Elongated density penetrating the hexon capsid at the facet edges was ascribed to polypeptide IIIa, a component required for virion assembly. Density on the inner surface of the capsid, connecting the ring of peripentonal hexons, was assigned as polypeptide VI, a component that binds DNA. Identification of the regions of hexon that contact the penton base suggests a structural mechanism for previously proposed events during cell entry.

Adenoviruses, Human↗
PubMed1991

Image reconstruction reveals the complex molecular organization of adenovirus.

The three-dimensional structure of adenovirus has been determined by image reconstruction from cryo-electron micrographs. Comparison with the high resolution X-ray crystal structure of hexon, the major capsid protein, enabled an unusually detailed interpretation of the density map and confirmed the validity of the reconstruction. The hexon packing in the capsid shows more extensive intermolecular interfaces between facets than previously proposed. The reconstruction provides the first three-dimensional visualization of the vertex proteins, including the penton base and its associated protruding fiber. Three minor capsid proteins that stabilize and modulate capsomer interactions are revealed. One of these components stabilizes the group-of-nine hexons in the center of each facet and the other two bridge hexons in adjacent facets. The strategic positions of these proteins highlight the importance of cementing proteins in stabilizing a complex assembly.

Adenoviruses, Human↗
PubMed1991

Distribution and complementarity of hydropathy in multisubunit proteins.

A survey of 40 multisubunit proteins and 2 protein-protein complexes was performed to assay quantitatively the distribution of hydropathy among the exterior surface, interior, contact surface, and noncontact exterior surface of the isolated subunits. We suggest a useful way to present this distribution by using a "hydropathy level diagram." Additionally, we have devised a function called "hydropathy complementarity" to quantitate the degree to which interacting surfaces have matching hydropathy distributions. Our survey revealed the following patterns: (1) The difference in hydropathy between the interior and exterior of subunits is a fairly invariant quantity. (2) On average, the hydropathy of the contact surface is higher than that of the exterior surface, but is not greater than that of the protein as a whole. There was variation, however, among the proteins. In some instances, the contact surface was more hydrophilic than the noncontact exterior, and in a few cases the contact surface was as hydrophobic as the protein interior. (3) The average interface manifests significant hydropathy complementarity, signifying that proteins interact by placing hydrophobic centers of one surface against hydrophobic centers of the other surface, and by similarly matching hydrophilic centers. As a measure of recognition and specificity, hydropathy complementarity could be a useful tool for predicting correct docking of interacting proteins. We suggest that high hydropathy complementarity is associated with static inflexible interactions. (4) We have found that some subunits that bind predominantly through hydrophilic forces, such as hydrogen bonds, ionic pairs, and water and metal bridges, are involved in dynamic quaternary organization and allostery.

Animals↗
PubMed1989

Adenovirus polypeptide IX revealed as capsid cement by difference images from electron microscopy and crystallography.

Particles of adenovirus type 2 (ad2), when disassembled, consistently yield groups-of-nine (GON) hexons, which are the major virion shell component. The location of a minor component (6%) of the GON has been determined using a novel combination of electron microscopy and X-ray crystallography. The Brookhaven Scanning Transmission Electron Microscope (STEM) was used to estimate the distribution of protein in the GON to a resolution of 15-18 A. The relative hexon positions then were determined to within 1 A using a model of the hexon derived from the X-ray crystal structure to search the STEM image. The difference image between the STEM image and a model hexon group reveals individual monomers of polypeptide IX extending along the hexon--hexon interfaces. The distribution confirms our earlier proposal that four trimers of polypeptide IX are embedded in the large cavities in the upper surface of the GON to cement hexons into a highly-stable assembly.

Adenoviruses, Human↗
PubMed1989

The adenovirus type 40 hexon: sequence, predicted structure and relationship to other adenovirus hexons.

The gene encoding the major capsid protein (hexon) of human adenovirus type 40 (Ad40) has been isolated and sequenced. Comparison of the predicted amino acid sequence of the Ad40 hexon with the corresponding polypeptide of the human enteric adenovirus, Ad41, reveals an overall identity of 88%. The majority of the changes in sequence are located in two areas, amino acids 131 to 287 and 390 to 425. Regions in the hexon protein that vary between Ad40 and Ad41 (subgroup F) were the same regions that varied between Ad2 and Ad5 (subgroup C) suggesting that these areas of the protein represent type-specific antigenic determinants. Other areas were conserved within members of a subgroup but varied between subgroups. Fitting of the Ad40 hexon sequence to the known three-dimensional structure of the Ad2 hexon demonstrates that the variable regions are located in the 1(1), 1(2) and 1(4) loops that form the surface of the virion. Of major significance is the absence in Ad40 of the highly acidic region present in both Ad2 and Ad5. In Ad2 this region stretches down into the D-strand of the beta-barrel forming the P1 domain. Molecular modelling indicates that the amino acids in Ad40 which correspond to the acidic region of Ad2 can also be accommodated in the eight-stranded beta-barrel, thereby maintaining the integrity of the barrel. Since the acidic region is also absent from the hexon of Ad41, the sequence of amino acids that replaces the acidic residues may be responsible for some of the distinctive biological properties of the subgroup F adenoviruses.

Adenoviruses, Human↗