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R C Stevens

Publications and source records attributed to R C Stevens.

At least 55 records · Page 3Linked to original sources
PubMed1998

Crystal structure of botulinum neurotoxin type A and implications for toxicity.

Botulinum neurotoxin type A (BoNT/A) is the potent disease agent in botulism, a potential biological weapon and an effective therapeutic drug for involuntary muscle disorders. The crystal structure of the entire 1,285 amino acid di-chain neurotoxin was determined at 3.3 A resolution. The structure reveals that the translocation domain contains a central pair of alpha-helices 105 A long and a approximately 50 residue loop or belt that wraps around the catalytic domain. This belt partially occludes a large channel leading to a buried, negative active site--a feature that calls for radically different inhibitor design strategies from those currently used. The fold of the translocation domain suggests a mechanism of pore formation different from other toxins. Lastly, the toxin appears as a hybrid of varied structural motifs and suggests a modular assembly of functional subunits to yield pathogenesis.

Binding Sites↗
PubMed1998

Biophysical characterization of the stability of the 150-kilodalton botulinum toxin, the nontoxic component, and the 900-kilodalton botulinum toxin complex species.

Botulinum neurotoxin serotype A is initially released from the bacterium Clostridium botulinum as a stable 900-kDa complex. The serotype A 900-kDa complex is one of the forms of the toxin being used as a therapeutic agent for the treatment of various neuromuscular disorders. Previous experiments have demonstrated that the 900-kDa complex form of the toxin protects the toxin from the harsh conditions of the gastrointestinal tract. To provide molecular level details of the stability and equilibrium of the 900-kDa complex, the nontoxic component, and the toxic (botulinum neurotoxin) component, the three species have been investigated with a series of biophysical techniques at the molecular level (dynamic light scattering, proteolysis, circular dichroism, pH incubations, and agglutination assays). These experiments were conducted under harsh conditions which mimic those found along the gastrointestinal tract. Separately, exposure to denaturing and proteolytic conditions degrades both the botulinum neurotoxin and the nontoxic component. In the 900-kDa complex, the botulinum neurotoxin is protected during exposure to the gastrointestinal environment and the nontoxic component is slightly modified. Surprisingly, the toxin protects the ability of the nontoxic component to agglutinate erythrocytes. Contrary to previous reports, the purified 900-kDa complex did not have agglutination ability until after exposure to the proteolytic conditions. These experiments provide new evidence and detail for the theory that the nontoxic component and the toxic component protect one another during exposure to harsh conditions, and a molecular model is presented for the passage of the toxin through the gastrointestinal tract.

Agglutination Tests↗
PubMed1997

The interplay between binding energy and catalysis in the evolution of a catalytic antibody.

Antibody catalysis provides an opportunity to examine the evolution of binding energy and its relation to catalytic function in a system that has many parallels with natural enzymes. Here we report such a study involving an antibody AZ-28 that catalyses an oxy-Cope rearrangement, a pericyclic reaction that belongs to a well studied and widely used class of reactions in organic chemistry. Immunization with transition state analogue 1 results in a germline-encoded antibody that catalyses the rearrangement of hexadiene 2 to aldehyde 3 with a rate approaching that of a related pericyclic reaction catalysed by the enzyme chorismate mutase. Affinity maturation gives antibody AZ-28, which has six amino acid substitutions, one of which results in a decrease in catalytic rate. To understand the relationship between binding and catalytic rate in this system we characterized a series of active-site mutants and determined the three-dimensional crystal structure of the complex of AZ-28 with the transition state analogue. This analysis indicates that the activation energy depends on a complex balance of several stereoelectronic effects which are controlled by an extensive network of binding interactions in the active site. Thus in this instance the combinatorial diversity of the immune system provided both an efficient catalyst for a reaction where no enzyme is known, as well as an opportunity to explore the mechanisms and evolution of biological catalysis.

Aldehydes↗
PubMed1997

Expression and purification of the Saccharomyces cerevisiae alpha-factor receptor (Ste2p), a 7-transmembrane-segment G protein-coupled receptor.

A plasmid vector was developed that permitted high-level expression of a functional form of the Saccharomyces cerevisiae alpha-factor receptor (the STE2 gene product) tagged at its C-terminal end with an epitope (FLAG) and a His6 tract. When expressed in yeast from this plasmid, Ste2p was produced at a level at least 3-fold higher than that reported previously for any other 7-transmembrane-segment receptor expressed in the same cells. For purification, isolated cell membranes containing the overexpressed receptor were solubilized with detergent under specific conditions and subjected to immobilized metal affinity chromatography. Yields as high as 1 mg of nearly homogeneous (95%) receptor were routinely obtained even from relatively small scale preparations (60 g of frozen cell paste). The purified receptor was reconstituted into artificial phospholipid vesicles. Radioligand binding studies demonstrated that the purified receptor, in the reconstituted vesicles, bound its tridecapeptide ligand (alpha-factor) with a KD (155 nM) consistent with the affinity expected for this receptor in the absence of its associated G protein. Efficient restoration of ligand binding activity upon reconstitution required the addition of solubilized membranes prepared from a yeast strain lacking the receptor. Sufficient amounts of active material can be obtained by this procedure to allow physical studies of this receptor and other 7-transmembrane-segment receptors expressed in this system.

Amino Acid Sequence↗
PubMed1997

Structural insights into the evolution of an antibody combining site.

The crystal structures of a germline antibody Fab fragment and its complex with hapten have been solved at 2.1 A resolution. These structures are compared with the corresponding crystal structures of the affinity-matured antibody, 48G7, which has a 30,000 times higher affinity for hapten as a result of nine replacement somatic mutations. Significant changes in the configuration of the combining site occur upon binding of hapten to the germline antibody, whereas hapten binds to the mature antibody by a lock-and-key fit mechanism. The reorganization of the combining site that was nucleated by hapten binding is further optimized by somatic mutations that occur up to 15 from bound hapten. These results suggest that the binding potential of the primary antibody repertoire may be significantly expanded by the ability of germline antibodies to adopt more than one combining-site configuration, with both antigen binding and somatic mutation stabilizing the configuration with optimal hapten complementarity.

Antibodies, Catalytic↗
PubMed1997

Crystal structures of the free and liganded form of an esterolytic catalytic antibody.

The crystal structure of the esterase catalytic antibody 48G7 has been determined in the presence of hapten at 2.0 A resolution and in the absence of hapten at 2.7 A resolution. The root-mean-square difference between the two structures is 0.6 A for the variable domain and 0.7 A for the constant domain. Comparison of the active site shows that no significant changes occur upon hapten binding as main-chain and side-chain displacements are negligible. Complex formation occurs as hapten fits into a pre-formed pocket about 10 A deep. Although 151 water molecules were modeled into the 48G7-hapten structure, none are bound in the active site. Comparison of the 48G7 structures with those of other published ester hydrolysis antibodies illustrates an emerging theme used by esterolytic antibodies in binding their (nitro-)phenyl haptens and in hydrolysing their cognate esters and carbonates: hapten is bound with the aryl end buried deep in the binding pocket, and the phosphonate moiety is responsible for the majority of the binding energy to the antibody-hapten interaction.

Antibodies, Catalytic↗
PubMed1997

Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity.

The design, synthesis, and in vitro evaluation of the novel carbocycles as transition-state-based inhibitors of influenza neuraminidase (NA) are described. The double bond position in the carbocyclic analogues plays an important role in NA inhibition as demonstrated by the antiviral activity of 8 (IC50 = 6.3 microM) vs 9 (IC50 > 200 microM). Structure-activity studies of a series of carbocyclic analogues 6a-i identified the 3-pentyloxy moiety as an apparent optimal group at the C3 position with an IC50 value of 1 nM for NA inhibition. The X-ray crystallographic structure of 6h bound to NA revealed the presence of a large hydrophobic pocket in the region corresponding to the glycerol subsite of sialic acid. The high antiviral potency observed for 6h appears to be attributed to a highly favorable hydrophobic interaction in this pocket. The practical synthesis of 6 starting from (-)-quinic acid is also described.

Acetamides↗
PubMed1997

Electron density projection map of the botulinum neurotoxin 900-kilodalton complex by electron crystallography.

The 900-kDa botulinum neurotoxin complex serotype A has been crystallized by the lipid-layer two-dimensional crystallization technique. Based on the binding characteristics of the hemagglutinating portion of the complex, a number of ganglioside/ lipid mixtures were tested but only lactosyl ceramide/1-palmityl-2-oleoyl-sn-glycero-3-phosphocholine was found to crystallize the complex. The optimum lipid mixture contained 75 mass % lactosyl ceramide and 25 mass % 1-palmityl-2-oleoyl-sn-glycero-3-phosphocholine. Using protein concentrations from 5 to 500 micrograms/ml and pH and 5 acetate buffer, we have obtained crystals that diffract to better than 15 A when prepared in negative stain. A projection map with a resolution of 30 A was calculated with unit cell dimensions of a = b = 157 A and P3 symmetry. The complex is triangular in shape with six distinct lobes observed. Additionally, six smaller structures protrude from the triangular core.

Botulinum Toxins↗
PubMed1997

Recombinant expression and purification of the botulinum neurotoxin type A translocation domain.

Botulinum neurotoxin type A in its fully activated form exists as a dichain protein consisting of a 50-kDa light chain and a 100-kDa heavy chain linked by a disulfide bond (B. R. DasGupta and H. Sugiyama, Biochem. Biophys. Res. Commun. 48, 108-112, 1972). The protein can be further subdivided into three functional domains: a catalytic domain corresponding to the light chain, a translocation domain associated with the N-terminal half of the heavy chain, and a binding domain as the C-terminal half. To facilitate further structural and functional studies on the mechanism of toxin translocation, we report here the recombinant Escherichia coli expression and purification of the isolated translocation domain with a yield of 1 mg pure protein per 1 g cell paste. Circular dichroism, enzyme-linked immunosorbent assays, and preliminary crystallization experiments verify proper protein folding. This reagent should serve as a key tool in elucidating the mechanism of translocation and in determining how the catalytic domain, a large 50-kDa metalloprotease, is delivered to the cytosol.

Biological Transport↗
PubMed1997

Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for inherited neurodegenerative diseases.

Tyrosine hydroxylase (TyrOH) catalyzes the conversion of tyrosine to L-DOPA, the rate-limiting step in the biosynthesis of the catecholamines dopamine, adrenaline, and noradrenaline. TyrOH is highly homologous in terms of both protein sequence and catalytic mechanism to phenylalanine hydroxylase (PheOH) and tryptophan hydroxylase (TrpOH). The crystal structure of the catalytic and tetramerization domains of TyrOH reveals a novel alpha-helical basket holding the catalytic iron and a 40 A long anti-parallel coiled coil which forms the core of the tetramer. The catalytic iron is located 10 A below the enzyme surface in a 17 A deep active site pocket and is coordinated by the conserved residues His 331, His 336 and Glu 376. The structure provides a rationale for the effect of point mutations in TyrOH that cause L-DOPA responsive parkinsonism and Segawa's syndrome. The location of 112 different point mutations in PheOH that lead to phenylketonuria (PKU) are predicted based on the TyrOH structure.

Amino Acid Sequence↗
PubMed1997

Antibody mapping to domains of botulinum neurotoxin serotype A in the complexed and uncomplexed forms.

The domain organization of the botulinum neurotoxin serotype A was studied by using antibody mapping of 44 monoclonal single-chain variable fragments. The analysis was carried out on (i) the individual domains of botulinum neurotoxin holotoxin (binding, translocation, and catalytic), (ii) botulinum neurotoxin holotoxin, (iii) the botulinum neurotoxin holotoxin in complex with the nontoxic portion, and (iv) botulinum neurotoxin holotoxin and nontoxic portion of the complex recombined in vitro. All 44 antibodies mapped to individual domains of botulinum neurotoxin. Forty of the 44 single-chain variable fragments bound the botulinum neurotoxin holotoxin relative to the isolated domains, suggesting that 4 epitopes are covered when the individual domains are in the holotoxin form. Only 20 of the antibodies showed a positive reaction to the toxin while in complex with the nontoxic portion. All of the covered epitopes were mapped to the binding domain of botulinum neurotoxin, which suggested that the binding domain is in direct contact with the nontoxic portion in the complex. Based on the antibody mapping to the different domains of the botulinum neurotoxin holotoxin and the entire complex, a model of the botulinum neurotoxin complex is proposed.

Antibodies, Monoclonal↗
PubMed1996

Insights into antibody catalysis: structure of an oxygenation catalyst at 1.9-angstrom resolution.

The x-ray crystal structures of the sulfide oxidase antibody 28B4 and of antibody 28B4 complexed with hapten have been solved at 2.2-angstrom and 1.9-angstrom resolution, respectively. To our knowledge, these structures are the highest resolution catalytic antibody structures to date and provide insight into the molecular mechanism of this antibody-catalyzed monooxygenation reaction. Specifically, the data suggest that entropic restriction plays a fundamental role in catalysis through the precise alignment of the thioether substrate and oxidant. The antibody active site also stabilizes developing charge on both sulfur and periodate in the transition state via cation-pi and electrostatic interactions, respectively. In addition to demonstrating that the active site of antibody 28B4 does indeed reflect the mechanistic information programmed in the aminophosphonic acid hapten, these high-resolution structures provide a basis for enhancing turnover rates through mutagenesis and improved hapten design.

Amino Acid Sequence↗
PubMed1996

Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance.

The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR). SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1. The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 x 10-12 M for GM1 to 1.88 x 10-10 M for asialo GM1. The picomolar values obtained by surface plasmon resonance are similar to Kd values determined with whole-cell binding assays. Both whole-cell assays and SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude. This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface. The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane. Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface.

Animals↗
PubMed1996

The immunological evolution of catalysis.

The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

Amino Acid Sequence↗
PubMed1996

A 'litmus test' for molecular recognition using artificial membranes.

BACKGROUND: Sensitive and selective molecular recognition is important throughout biology. Certain organisms and toxins use specific binding at the cell surface as a first step towards invasion. A new series of biomolecular materials, with novel optical and interfacial properties, have been designed to sense molecular recognition events. These polymers, the diacetylenic lipids, have previously been shown to undergo chromatic transitions in response to virus binding to the surface of the material. RESULTS: Gangliosides that specifically bind cholera toxin, heat-labile Escherichia coli enterotoxin and botulinum neurotoxin were incorporated into a matrix of diacetylenic lipids, 5-10% of which were derivatized with sialic acid. The lipids were self-assembled into Langmuir-Blodgett layers and polymerized with ultraviolet irradiation, yielding a polydiacetylene membrane with a characteristic blue color into which the ganglioside is non-covalently incorporated. When toxin is added, the polymerized membrane turns red. The response is specific and selective, and can be quantified by visible absorption spectrophotometry. CONCLUSIONS: Polydiacetylenic lipid membranes offer a general 'litmus test' for molecular recognition at the surface of a membrane. A concentration of 20 ppm of protein could be detected using polymerized thin films. The speed, sensitivity and simplicity of the design offers a new and general approach towards the direct colorimetric detection of a variety of different molecules.

Biosensing Techniques↗
PubMed1994

Determination of the absolute configuration of (+)-neopentyl-1-d alcohol by neutron and x-ray diffraction analysis.

The absolute configuration of (+)-neopentyl-1-d alcohol, prepared by the reduction of 2,2-dimethylpropanal-1-d by actively fermenting yeast, has been determined to be S by neutron diffraction. The neutron study was carried out on the phthalate half ester of neopentyl-1-d alcohol, crystallized as its strychnine salt. The absolute configuration of the (-)-strychninium cation was first determined by an x-ray anomalous dispersion study of its iodide salt. The chiral skeleton of strychnine then served as a reference from which the absolute configuration of the -O-CHD-C(CH3)3 group of neopentyl phthalate was determined. Difference Fourier maps calculated from the neutron data showed unambiguously that the -O-CHD-C(CH3)3 groups of both independent molecules in the unit cell had the S configuration. This work proves conclusively that the yeast system reduces aldehydes by delivering hydrogen to the re face of the carbonyl group. Crystallographic details: (-)-strychninium (+)-neopentyl-1-d phthalate, space group P2(1) (monoclinic), a = 18.564(6) A, b = 7.713(2) A, c = 23.361(8) A, beta = 94.18(4) degrees, V = 3336.0(5) A3, Z = 2 (T = 100 K). Final agreement factors are R(F) = 0.073 for 2768 reflections collected at room temperature (x-ray analysis) and R(F) = 0.144 for 960 reflections collected at 100 K (neutron analysis).

Crystallography, X-Ray↗