Biomedical subjects

D I Stuart

Publications and source records attributed to D I Stuart.

At least 55 records · Page 3Linked to original sources

PubMed1999

Structure of the TRAIL-DR5 complex reveals mechanisms conferring specificity in apoptotic initiation.

TRAIL, an apoptosis inducing ligand, has at least four cell surface receptors including the death receptor DR5. Here we report the crystal structure at 2.2 A resolution of a complex between TRAIL and the extracellular region of DR5. TRAIL forms a central homotrimer around which three DR5 molecules bind. Radical differences in the surface charge of the ligand, together with variation in the alignment of the two receptor domains confer specificity between members of these ligand and receptor families. The existence of a switch mechanism allowing variation in receptor domain alignment may mean that it is possible to engineer receptors with multiple specificities by exploiting contact positions unique to individual receptor-ligand pairs.

Amino Acid Sequence↗

PubMed1999

The structure of a cypovirus and the functional organization of dsRNA viruses.

Cytoplasmic polyhedrosis virus (CPV) is unique among the double-stranded RNA viruses of the family Reoviridae in having a single capsid layer. Analysis by cryo-electron microscopy allows comparison of the single shelled CPV and orthoreovirus with the high resolution crystal structure of the inner shell of the bluetongue virus (BTV) core. This suggests that the novel arrangement identified in BTV, of 120 protein subunits in a so-called 'T=2' organization, is a characteristic of the Reoviridae and allows us to delineate structural similarities and differences between two subgroups of the family--the turreted and the smooth-core viruses. This in turn suggests a coherent picture of the structural organization of many dsRNA viruses.

Bluetongue virus↗

PubMed1999

ESPript: analysis of multiple sequence alignments in PostScript.

MOTIVATION: The program ESPript (Easy Sequencing in PostScript) allows the rapid visualization, via PostScript output, of sequences aligned with popular programs such as CLUSTAL-W or GCG PILEUP. It can read secondary structure files (such as that created by the program DSSP) to produce a synthesis of both sequence and structural information. RESULTS: ESPript can be run via a command file or a friendly html-based user interface. The program calculates an homology score by columns of residues and can sort this calculation by groups of sequences. It offers a palette of markers to highlight important regions in the alignment. ESPript can also paste information on residue conservation into coordinate files, for subsequent visualization with a graphics program. AVAILABILITY: ESPript can be accessed on its Web site at http://www.ipbs.fr/ESPript. Sources and helpfiles can be downloaded via anonymous ftp from ftp.ipbs.fr. A tar file is held in the directory pub/ESPript.

Amino Acid Sequence↗

PubMed1999

Crystallization and preliminary X-ray analysis of human rhinovirus serotype 2 (HRV2).

Human rhinoviruses, the major cause of mild recurrent infections of the upper respiratory tract, are small icosahedral particles. Over 100 different serotypes have been identified. The majority (91 serotypes) use intercellular adhesion molecule 1 as the cell-attachment site; ten serotypes (the minor group) bind to members of the low-density lipoprotein receptor. Three different crystal forms of the minor-group human rhinovirus serotype 2 (HRV2) were obtained by the hanging-drop vapour-diffusion technique using ammonium sulfate and sodium/potassium phosphate as precipitants. Monoclinic crystals, space group P2(1), diffracted at least to 2.8 A resolution, and two complete virus particles were located in the crystal asymmetric unit. A second type of crystals had a compact cubic like morphology and diffracted beyond 2.5 A resolution. These crystals belong to a primitive orthorhombic space group, with unit-cell parameters a = 309.3, b = 353.5, c = 759.6 A, and contain one virus particle in the asymmetric unit. A third type of crystals, with a prismatic shape and belonging to space group I222, was also obtained under similar crystallization conditions. These latter crystals, with unit-cell parameters a = 308.7, b = 352.2, c = 380.5 A, diffracted to high resolution (beyond 1.8 A) and contained 15 protomers per asymmetric unit; this requires that three perpendicular crystal twofold axes coincide with three of the viral particle's dyad axes.

Crystallization↗

PubMed1999

Complementing crystallography: the role of cryo-electron microscopy in structural biology.

Dramatic improvements in experimental methods and computational techniques have revolutionized three-dimensional image reconstruction from electron micrographs (EM) of vitrified samples. Recent results include the first determination of a protein fold (for the core protein of the hepatitis B virus) by non-crystalline imaging techniques. These developments have generated interest within the crystallographic community and have led to a re-evaluation of the technique, particularly amongst those working in the field of virus structure or struggling with the phasing of large macromolecular assemblies. A simple discussion of the techniques of EM image reconstruction and its advantages and problems in terms familiar to crystallographers will hopefully allow an appreciation of the essential complementarity of the two techniques and the practical potentials for phasing applications.

Cryoelectron Microscopy↗

PubMed1999

The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site.

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.

Binding Sites↗

PubMed1999

Effects of N-butyldeoxynojirimycin and the Lec3.2.8.1 mutant phenotype on N-glycan processing in Chinese hamster ovary cells: application to glycoprotein crystallization.

Heterologous gene expression in either (1) the glycosylation-defective, mutant Chinese hamster ovary cell line, Lec3.2.8.1, or (2) the presence of the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin facilitates the trimming of N-linked glycans of glycoproteins to single N-acetylglucosamine (GlcNAc) residues with endoglycosidase H (endo H). Both approaches are somewhat inefficient, however, with as little as 12% of the total protein being rendered fully endo H-sensitive under these conditions. It is shown here that the combined effects of these approaches on the restriction of oligosaccharide processing are essentially additive, thereby allowing the production of glycoproteins that are essentially completely endo H-sensitive. The preparation of a soluble chimeric form of CD58, the ligand of the human T-cell surface recognition molecule CD2, illustrates the usefulness of the combined approach when expression levels are low or the deglycosylated protein is unstable at low pH. The endo H-treated chimera produced crystals of space group P3(1)21 or P3(2)21, and unit cell dimensions a = b = 116.4 A, c = 51.4 A alpha = beta = 90 degrees , gamma = 120 degrees , that diffract to a maximum resolution of 1.8 A.

1-Deoxynojirimycin↗

PubMed1999

A family of insertion mutations between codons 67 and 70 of human immunodeficiency virus type 1 reverse transcriptase confer multinucleoside analog resistance.

To investigate the occurrence of multinucleoside analog resistance during therapy failure, we surveyed the drug susceptibilities and genotypes of nearly 900 human immunodeficiency virus type 1 (HIV-1) samples. For 302 of these, the 50% inhibitory concentrations of at least four of the approved nucleoside analogs had fourfold-or-greater increases. Genotypic analysis of the reverse transcriptase (RT)-coding regions from these samples revealed complex mutational patterns, including the previously recognized codon 151 multidrug resistance cluster. Surprisingly, high-level multinucleoside resistance was associated with a diverse family of amino acid insertions in addition to "conventional" point mutations. These insertions were found between RT codons 67 and 70 and were commonly 69Ser-(Ser-Ser) or 69Ser-(Ser-Gly). Treatment history information showed that a common factor for the development of these variants was AZT (3'-azido-3'-deoxythymidine, zidovudine) therapy in combination with 2',3'-dideoxyinosine or 2',3'-dideoxycytidine, although treatment patterns varied considerably. Site-directed mutagenesis studies confirmed that 69Ser-(Ser-Ser) in an AZT resistance mutational background conferred simultaneous resistance to multiple nucleoside analogs. The insertions are located in the "fingers" domain of RT. Modelling the 69Ser-(Ser-Ser) insertion into the RT structure demonstrated the profound direct effect that this change is likely to have in the nucleoside triphosphate binding site of the enzyme. Our data highlight the increasing problem of HIV-1 multidrug resistance and underline the importance of continued resistance surveillance with appropriate, sufficiently versatile genotyping technology and phenotypic drug susceptibility analysis.

Anti-HIV Agents↗

PubMed1999

Virus crystallography.

Virus crystallography can provide atomic resolution structures for intact isometric virus particles and components thereof. The methodology is illustrated by reference to a particularly complex example, the core of the bluetongue virus (700 A).

Bluetongue virus↗

PubMed1998

Determination of the affinity and kinetic constants for the interaction between the human virus echovirus 11 and its cellular receptor, CD55.

The biochemical properties of the molecular interactions mediating viral-cell recognition are poorly characterized. In this study, we use surface plasmon resonance to study the affinity and kinetics of the interaction of echovirus 11 with its cellular receptor decay-accelerating factor (CD55). As reported for interactions between cell-cell recognition molecules, the interaction has a low affinity (KD approximately 3.0 microM) as a result of a very fast dissociation rate constant (kon approximately 10(5) M-1.s-1, koff approximately 0.3 s-1). This contrasts with the interaction of soluble ICAM-1 (sICAM-1, CD54) with human rhinovirus 3 which has been reported to have a similar affinity but 10(2)-10(3)-fold slower kinetics (Casasnovas, J. M., and Springer, T. A. (1995) J. Biol. Chem. 270, 13216-13224). The extracellular portion of decay-accelerating factor comprises four short consensus repeat domains (domains 1-4) and a mucin-like stalk. By comparison of the binding affinity for echovirus 11 of various fragments of decay-accelerating factor, we are able to conclude that short consensus repeat domain 3 contributes approximately 80% of the binding energy.

Biosensing Techniques↗

PubMed1998

Crystal structures of HIV-1 reverse transcriptase in complex with carboxanilide derivatives.

The carboxanilides are nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT), of potential clinical importance. The compounds differ in potency and in their retention of potency in the face of drug resistance mutations. Whereas UC-84, the prototype compound, only weakly inhibits many RTs bearing single point resistance mutations, inhibition by UC-781 is little affected. It has been proposed that UC-38 and UC-781 may form quaternary complexes with RT at a site other than the known binding pocket of other NNIs. X-ray crystal structures of four HIV-1 RT-carboxanilide complexes (UC-10, UC-38, UC-84, and UC-781) reported here reveal that all four inhibitors bind in the usual NNI site, forming binary 1:1 complexes with RT in the absence of substrates with the amide/thioamide bond in cis conformations. For all four complexes the anilide rings of the inhibitors overlap aromatic rings of many other NNIs bound to RT. In contrast, the second rings of UC-10, UC-84, and UC-781 do not bind in equivalent positions to those of other "two-ring" NNIs such as alpha-APA or HEPT derivatives. The binding modes most closely resemble that of the structurally dissimilar NNI, Cl-TIBO, with a common hydrogen bond between each carboxanilide NH- group and the main-chain carbonyl oxygen of Lys101. The binding modes differ slightly between the UC-10/UC-781 and UC-38/UC-84 pairs of compounds, apparently related to the shorter isopropylmethanoyl substituents of the anilide rings of UC-38/UC-84, which draws these rings closer to residues Tyr181 and Tyr188. This in turn explains the differences in the effect of mutated residues on the binding of these compounds.

Anilides↗

PubMed1998

The atomic structure of the bluetongue virus core.

The structure of the core particle of bluetongue virus has been determined by X-ray crystallography at a resolution approaching 3.5 A. This transcriptionally active compartment, 700 A in diameter, represents the largest molecular structure determined in such detail. The atomic structure indicates how approximately 1,000 protein components self-assemble, using both the classical mechanism of quasi-equivalent contacts, which are achieved through triangulation, and a different method, which we term geometrical quasi-equivalence.

Amino Acid Sequence↗

PubMed1998

Continuous and discontinuous changes in the unit cell of HIV-1 reverse transcriptase crystals on dehydration.

A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a high-resolution limit of 3.7 A. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2 A. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

Crystallization↗

PubMed1998

3'-Azido-3'-deoxythymidine drug resistance mutations in HIV-1 reverse transcriptase can induce long range conformational changes.

HIV reverse transcriptase (RT) is one of the main targets for the action of anti-AIDS drugs. Many of these drugs [e.g., 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI)] are analogues of the nucleoside substrates used by the HIV RT. One of the main problems in anti-HIV therapy is the selection of a mutant virus with reduced drug sensitivity. Drug resistance in HIV is generated for nucleoside analogue inhibitors by mutations in HIV RT. However, most of these mutations are situated some distance from the polymerase active site, giving rise to questions concerning the mechanism of resistance. To understand the possible structural bases for this, the crystal structures of AZT- and ddI-resistant RTs have been determined. For the ddI-resistant RT with a mutation at residue 74, no significant conformational changes were observed for the p66 subunit. In contrast, for the AZT-resistant RT (RTMC) bearing four mutations, two of these (at 215 and 219) give rise to a conformational change that propagates to the active site aspartate residues. Thus, these drug resistance mutations produce an effect at the RT polymerase site mediated simply by the protein. It is likely that such long-range effects could represent a common mechanism for generating drug resistance in other systems.

Binding Sites↗

PubMed1998

Crystallization and preliminary diffraction studies of the extracellular region of human p58 killer cell inhibitory receptor (KIR2).

Molecules of the human killer cell inhibitory receptor (KIR) family, which belong to the immunoglobulin superfamily (IgSF), are expressed on the surface of natural killer (NK) cells and some subsets of T cells. These receptors function to mediate the inhibition or activation of cytotoxic activity by recognizing HLA class I molecules on the target cell. The extracellular region of a p58 KIR specific for HLA-Cw1,3,7 (KIR2) has been overproduced in Escherichia coli and purified. The recombinant KIR2 has been crystallized in 9-10% poly(ethylene glycol) methyl ether (average Mr = 8000), 50mM HEPES, 8% ethylene glycol, 0.5% octyl-beta-glucoside, pH 7.5, at 294 K using the sitting-drop vapour-diffusion method. Preliminary X-ray diffraction studies reveal the space group to be hexagonal (P6122 or P6522) with lattice constants a = b = 95.3, c = 130.8 A. A native data set (3 A resolution) has been collected at the Photon Factory (lambda = 1.0 A).

Crystallization↗

PubMed1998

Production, crystallization, and preliminary X-ray analysis of the human MHC class Ib molecule HLA-E.

HLA-E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA-E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA-E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA-E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA-E forms a stable complex with beta2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 A with space group P3(1)21 and unit cell dimensions a = 182.2 A, b = 182.2 A, c = 88.4 A.

Amino Acid Sequence↗

PubMed1998

Structural studies of orbivirus particles.

We are using crystallographic methods to investigate the structure of AHSV and BTV. Our initial approach was to investigate the structure of the major protein component of the viral core, VP7(T13). This trimeric protein has been studied in several crystal forms from both orbiviruses and reveals a structure made up of conserved domains, capable of conformational changes and possessing a cleavage site. Further crystallographic analyses of native particles have provided a picture of the VP7(T13) and VP3(T2) layers of the BTV core. The VP7(T13) layer consists of 260 trimers arranged rather symmetrically and possessing very similar structures, thereby following the rules of quasi equivalence. The VP3(T2) layer is thin and contains 120 copies of 100 kDa protein arranged as 60 approximate dimers. This type of icosahedral construction has not been observed before and appears to contain a genome which is highly ordered. We anticipate that all of these features will be common to AHSV.

African Horse Sickness Virus↗

PubMed1998

Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E.

The crystal structure of the nonclassical human class lb MHC molecule HLA-E has been determined in complex with a prototypic ligand, the nonamer peptide (VMAPRTVLL), derived from the highly conserved residues 3-11 of the human MHC class la leader sequence. The mode of peptide binding retains some of the standard features observed in MHC class la complexes, but novel features imply that HLA-E has evolved to mediate specific binding to a tightly defined set of almost identical hydrophobic peptides from the highly conserved class l leader sequences. These molecular adaptations make HLA-E a rigorous checkpoint at the cell surface reporting on the integrity of the antigen processing pathway to CD94/NKG2 receptor-bearing natural killer cells.

Crystallography↗