Biomedical subjects

R C Stevens

Publications and source records attributed to R C Stevens.

At least 37 records · Page 2Linked to original sources

PubMed2000

Combining structural genomics and enzymology: completing the picture in metabolic pathways and enzyme active sites.

An important goal of structural genomics is to complete the structural analysis of all the enzymes in metabolic pathways and to understand the structural similarities and differences. A preliminary glimpse of this type of analysis was achieved before structural genomics efforts with the glycolytic pathway and efforts are underway for many other pathways, including that of catecholamine metabolism. Structural enzymology necessitates a complete structural characterization, even for highly homologous proteins (greater than 80% sequence homology), as every active site has distinct structural features and it is these active site differences that distinguish one enzyme from another. Short cuts with homology modeling cannot be taken with our current knowledge base. Each enzyme structure in a pathway needs to be determined, including structures containing bound substrates, cofactors, products and transition state analogs, in order to obtain a complete structural and functional understanding of pathway-related enzymes.

Binding Sites↗

PubMed2000

Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 A resolution.

Botulinum neurotoxin serotype B is a zinc protease that disrupts neurotransmitter release by cleaving synaptobrevin-II (Sb2), one of three SNARE proteins involved in neuronal synaptic vesicle fusion. The three-dimensional crystal structure of the apo botulinum neurotoxin serotype B catalytic domain (BoNT/B-LC) has been determined to 2.2 A resolution, and the complex of cleaved Sb2 with the catalytic domain (Sb2-BoNT/B-LC) has been determined to 2.0 A resolution. A comparison of the holotoxin catalytic domain and the isolated BoNT/B-LC structure shows a rearrangement of three active site loops. This rearrangement exposes the BoNT/B active site. The Sb2-BoNT/B-LC structure illustrates two distinct binding regions, which explains the specificity of each botulinum neurotoxin for its synaptic vesicle protein. This observation provides an explanation for the proposed cooperativity between binding of full-length substrate and catalysis and suggest a mechanism of synaptobrevin proteolysis employed by the clostridial neurotoxins.

Amino Acid Sequence↗

PubMed2000

Automation of X-ray crystallography.

Structure-based biological discovery is entering a new era with the development of industrialized macromolecular structure determination pipelines. Intense, highly focused X-rays from integrated synchrotron radiation beam lines combined with significant advances in protein expression, purification, and micro-crystallization automation allow for the full streamlining of the traditionally tedious and time consuming process of determining the three dimensional structures of macromolecules.

Automation↗

PubMed1999

A structural view of evolutionary divergence.

Two directed evolution experiments on p-nitrobenzyl esterase yielded one enzyme with a 100-fold increased activity in aqueous-organic solvents and another with a 17 degrees C increase in thermostability. Structures of the wild type and its organophilic and thermophilic counterparts are presented at resolutions of 1.5 A, 1.6 A, and 2.0 A, respectively. These structures identify groups of interacting mutations and demonstrate how directed evolution can traverse complex fitness landscapes. Early-generation mutations stabilize flexible loops not visible in the wild-type structure and set the stage for further beneficial mutations in later generations. The mutations exert their influence on the esterase structure over large distances, in a manner that would be difficult to predict. The loops with the largest structural changes generally are not the sites of mutations. Similarly, none of the seven amino acid substitutions in the organophile are in the active site, even though the enzyme experiences significant changes in the organization of this site. In addition to reduction of surface loop flexibility, thermostability in the evolved esterase results from altered core packing, helix stabilization, and the acquisition of surface salt bridges, in agreement with other comparative studies of mesophilic and thermophilic enzymes. Crystallographic analysis of the wild type and its evolved counterparts reveals networks of mutations that collectively reorganize the active site. Interestingly, the changes that led to diversity within the alpha/beta hydrolase enzyme family and the reorganization seen in this study result from main-chain movements.

Biological Evolution↗

PubMed1999

Sequence homology and structural analysis of the clostridial neurotoxins.

The clostridial neurotoxins (CNTs), comprised of tetanus neurotoxin (TeNT) and the seven serotypes of botulinum neurotoxin (BoNT A-G), specifically bind to neuronal cells and disrupt neurotransmitter release by cleaving proteins involved in synaptic vesicle membrane fusion. In this study, multiple CNT sequences were analyzed within the context of the 1277 residue BoNT/A crystal structure to gain insight into the events of binding, pore formation, translocation, and catalysis that are required for toxicity. A comparison of the TeNT-binding domain structure to that of BoNT/A reveals striking differences in their surface properties. Further, the solvent accessibility of a key tryptophan in the C terminus of the BoNT/A-binding domain refines the location of the ganglioside-binding site. Data collected from a single frozen crystal of BoNT/A are included in this study, revealing slight differences in the binding domain orientation as well as density for a previously unobserved translocation domain loop. This loop and the conservation of charged residues with structural proximity to putative pore-forming sequences lend insight into the CNT mechanism of pore formation and translocation. The sequence analysis of the catalytic domain revealed an area near the active-site likely to account for specificity differences between the CNTs. It revealed also a tertiary structure, highly conserved in primary sequence, which seems critical to catalysis but is 30 A from the active-site zinc ion. This observation, along with an analysis of the 54 residue "belt" from the translocation domain are discussed with respect to the mechanism of catalysis.

Amino Acid Sequence↗

PubMed1999

NMR solution structure of alpha-conotoxin ImI and comparison to other conotoxins specific for neuronal nicotinic acetylcholine receptors.

Alpha-Conotoxins, peptides produced by predatory species of Conus marine snails, are potent antagonists of nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels involved in synaptic transmission. We determined the NMR solution structure of the smallest known alpha-conotoxin, ImI, a 12 amino acid peptide that binds specifically to neuronal alpha7-containing nAChRs in mammals. Calculation of the structure was based on a total of 80 upper distance constraints and 31 dihedral angle constraints resulting in 20 representative conformers with an average pairwise rmsd of 0.44 A from the mean structure for the backbone atoms N, Calpha, and C' of residues 2-11. The structure of ImI is characterized by two compact loops, defined by two disulfide bridges, which form distinct subdomains separated by a deep cleft. Two short 310-helical regions in the first loop are followed by a C-terminal beta-turn in the second. The two disulfide bridges and Ala 9 form a rigid hydrophobic core, orienting the other amino acid side chains toward the surface. Comparison of the three-dimensional structure of ImI to those of the larger, 16 amino acid alpha-conotoxins PnIA, PnIB, MII, and EpI-also specific for neuronal nAChRs-reveals remarkable similarity in local backbone conformations and relative solvent-accessible surface areas. The core scaffold is conserved in all five conotoxins, whereas the residues in solvent-exposed positions are highly variable. The second helical region, and the specific amino acids that the helix exposes to solvent, may be particularly important for binding and selectivity. This comparative analysis provides a three-dimensional structural basis for interpretation of mutagenesis data and structure-activity relationships for ImI as well other neuronal alpha-conotoxins.

Amino Acid Sequence↗

PubMed1999

The structural basis of phenylketonuria.

The human phenylalanine hydroxylase gene (PAH) (locus on human chromosome 12q24.1) contains the expressed nucleotide sequence which encodes the hepatic enzyme phenylalanine hydroxylase (PheOH). The PheOH enzyme hydroxylates the essential amino acid l-phenylalanine resulting in another amino acid, tyrosine. This is the major pathway for catabolizing dietary l-phenylalanine and accounts for approximately 75% of the disposal of this amino acid. The autosomal recessive disease phenylketonuria (PKU) is the result of a deficiency of PheOH enzymatic activity due to mutations in the PAH gene. Of the mutant alleles that cause hyperphenylalaninemia or PKU 99% map to the PAH gene. The remaining 1% maps to several genes that encode enzymes involved in the biosynthesis or regeneration of the cofactor ((6R)-l-erythro-5,6,7,8-tetrahydrobiopterin) regenerating the cofactor (tetrahydrobiopterin) necessary for the hydroxylation reaction. The recently solved crystal structures of human phenylalanine hydroxylase provide a structural scaffold for explaining the effects of some of the mutations in the PAH gene and suggest future biochemical studies that may increase our understanding of the PKU mutations.

Amino Acid Sequence↗

PubMed1999

Structural basis of autoregulation of phenylalanine hydroxylase.

Phenylalanine hydroxylase converts phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an alpha-beta sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of phenylalanine, suggesting that phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.

Allosteric Regulation↗

PubMed1998

Crystallographic analysis of the human phenylalanine hydroxylase catalytic domain with bound catechol inhibitors at 2.0 A resolution.

The aromatic amino acid hydroxylases represent a superfamily of structurally and functionally closely related enzymes, one of those functions being reversible inhibition by catechol derivatives. Here we present the crystal structure of the dimeric catalytic domain (residues 117-424) of human phenylalanine hydroxylase (hPheOH), cocrystallized with various potent and well-known catechol inhibitors and refined at a resolution of 2.0 A. The catechols bind by bidentate coordination to each iron in both subunits of the dimer through the catechol hydroxyl groups, forming a blue-green colored ligand-to-metal charge-transfer complex. In addition, Glu330 and Tyr325 are identified as determinant residues in the recognition of the inhibitors. In particular, the interaction with Glu330 conforms to the structural explanation for the pH dependence of catecholamine binding to PheOH, with a pKa value of 5.1 (20 degreesC). The overall structure of the catechol-bound enzyme is very similar to that of the uncomplexed enzyme (rms difference of 0.2 A for the Calpha atoms). Most striking is the replacement of two iron-bound water molecules with catechol hydroxyl groups. This change is consistent with a change in the ligand field symmetry of the high-spin (S = 5/2) Fe(III) from a rhombic to a nearly axial ligand field symmetry as seen upon noradrenaline binding using EPR spectroscopy [Martinez, A., Andersson, K. K., Haavik, J., and Flatmark, T. (1991) Eur. J. Biochem. 198, 675-682]. Crystallographic comparison with the structurally related rat tyrosine hydroxylase binary complex with the oxidized cofactor 7,8-dihydrobiopterin revealed overlapping binding sites for the catechols and the cofactor, compatible with a competitive type of inhibition of the catechols versus BH4. The comparison demonstrates some structural differences at the active site as the potential basis for the different substrate specificity of the two enzymes.

Binding, Competitive↗

PubMed1998

Structural and kinetic evidence for strain in biological catalysis.

A classic hypothesis for enzyme catalysis is the induction of strain in the substrate. This notion was first expressed by Haldane with the lock and key analogy-"the key does not fit the lock perfectly but exercises a certain strain on it" (1). This mechanism has often been invoked to explain the catalytic efficiency of enzymes but has been difficult to establish conclusively (2-7). Here we describe X-ray crystallographic and mutational studies of an antibody metal chelatase which strongly support the notion that this antibody catalyzes metal ion insertion into the porphyrin ring by inducing strain. Analysis of the germline precursor suggests that this strain mechanism arose during the process of affinity maturation in response to a conformationally distorted N-alkylmesoporphyrin.

Amino Acid Sequence↗

PubMed1998

Partial characterization and three-dimensional-structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria.

The molecular basis for the metabolic defect in patients with phenylketonuria has been characterized for seven missense point mutations (R252G/Q, L255V/S, A259V/T and R270S) and a termination mutation (G272X) in an evolutionarily conserved motif of exon 7 in the catalytic domain of the human phenylalanine hydroxylase (hPAH) gene. The mutations were expressed in three heterologous in vitro systems. When expressed as fusion proteins with maltose-binding protein in Escherichia coli five of the mutant proteins demonstrated a defect in the normal ability of hPAH to fold and assemble as homotetramer/dimer, and they were mostly recovered as inactive aggregated forms. Only for the R252Q and L255V mutants were catalytically active tetramer and dimer recovered and for R252G some dimer, i.e. 20% (R252Q, tetramer), 44% (L255V, tetramer) and 4.4% (R252G, dimer) of the activity for the respective wild-type (wt) forms. When expressed by a coupled in vitro transcription-translation system, all the mutant enzymes were recovered as a mixture of non-phosphorylated and phosphorylated forms with a low homospecific activity (i.e. maximum 11% of wt-hPAH for the L255V mutant). When transiently expressed in human embryonic kidney (A293) cells a very low level of immunoreactive PAH protein was recovered in spite of normal PAH mRNA levels. All these mutations resulted in variant hPAH proteins which revealed a defect in oligomerization, an increased sensitivity to limited proteolysis in vitro, reduced cellular stability and a variable reduction in their catalytic activity. All these effects seem to result from structural perturbations of the monomer, and based on the crystal structure of the catalytic domain of hPAH, an explanation is provided for the impact of the mutations on the folding and oligomerization of the monomers.

Amino Acid Sequence↗

PubMed1998

Crystal structure of tyrosine hydroxylase with bound cofactor analogue and iron at 2.3 A resolution: self-hydroxylation of Phe300 and the pterin-binding site.

TyrOH is a non-heme iron enzyme which uses molecular oxygen to hydroxylate tyrosine to form L-dihydroxyphenylalanine (L-DOPA), and tetrahydrobiopterin to form 4a-hydroxybiopterin, in the rate-limiting step of the catecholamine biosynthetic pathway. The 2.3 A crystal structure of the catalytic and tetramerization domains of rat tyrosine hydroxylase (TyrOH) in the presence of the cofactor analogue 7,8-dihydrobiopterin and iron shows the mode of pterin binding and the proximity of its hydroxylated 4a carbon to the required iron. The pterin binds on one face of the large active-site cleft, forming an aromatic pi-stacking interaction with Phe300. This phenylalanine residue of TyrOH is found to be hydroxylated in the meta position, most likely through an autocatalytic process, and to consequently form a hydrogen bond to the main-chain carbonyl of Gln310 which anchors Phe300 in the active site. The bound pterin forms hydrogen bonds from N-8 to the main-chain carbonyl of Leu295, from O-4 to Tyr371 and Glu376, from the C-1' OH to the main-chain amides of Leu294 and Leu295, and from the C-2' hydroxyl to an iron-coordinating water. The part of the pterin closest to the iron is the O-4 carbonyl oxygen at a distance of 3.6 A. The iron is 5.6 A from the pterin 4a carbon which is hydroxylated in the enzymatic reaction. No structural changes are observed between the pterin bound and the nonliganded enzyme. On the basis of these structures, molecular oxygen could bind in a bridging position optimally between the pterin C-4a and iron atom prior to substrate hydroxylation. This structure represents the first report of close interactions between pterin and iron in an enzyme active site.

Animals↗

PubMed1998

Mlc1p is a light chain for the unconventional myosin Myo2p in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth. We explore the role of the neck region and its associated light chains in regulating Myo2p function. Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Delta6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain. We exploit this mutant in a characterization of the light chains of Myo2p. First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p. Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p. MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency. Reduced levels of MYO2 overcome the haploinsufficiency of MLC1. The mutant MYO2-Delta6IQ is able to suppress haploinsufficiency but not deletion of MLC1. We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p. Overexpression of MYO2 is toxic, causing a severe decrease in growth rate. When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain. High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p. Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region.

Amino Acid Sequence↗

PubMed1998

Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria.

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.

Amino Acid Sequence↗

PubMed1998

Structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors.

A series of influenza neuraminidase inhibitors with the cyclohexene scaffold containing lipophilic side chains have been synthesized and evaluated for influenza A and B neuraminidase inhibitory activity. The size and geometry of side chains have been modified systematically in order to investigate structure-activity relationships of this class of compounds. The X-ray crystal structures of several analogues complexed with neuraminidase revealed that the lipophilic side chains bound to the hydrophobic pocket consisted of Glu276, Ala246, Arg224, and Ile222 of the enzyme active site. The structure-activity relationship studies of this series have also demonstrated remarkably different inhibitory potency between influenza A and B neuraminidase. This indicated that the lipophilic side chains had quite different hydrophobic interactions with influenza A and B neuraminidase despite their complete homology in the active site. Influenza B neuraminidase appeared to be much more sensitive toward the increased steric bulkiness of inhibitors compared to influenza A neuraminidase. From the extensive structure-activity relationship investigation reported in this article, GS 4071 emerged as one of the most potent influenza neuraminidase inhibitors against both influenza A and B strains.

Acetamides↗

PubMed1998

Immunological origins of binding and catalysis in a Diels-Alderase antibody.

The three-dimensional structure of an antibody (39-A11) that catalyzes a Diels-Alder reaction has been determined. The structure suggests that the antibody catalyzes this pericyclic reaction through a combination of packing and hydrogen-bonding interactions that control the relative geometries of the bound substrates and electronic distribution in the dienophile. A single somatic mutation, serine-91 of the light chain to valine, is largely responsible for the increase in affinity and catalytic activity of the affinity-matured antibody. Structural and functional studies of the germ-line precursor suggest that 39-A11 and related antibodies derive from a family of germ-line genes that have been selected throughout evolution for the ability of the encoded proteins to form a polyspecific combining site. Germ line-encoded antibodies of this type, which can rapidly evolve into high-affinity receptors for a broad range of structures, may help to expand the binding potential associated with the structural diversity of the primary antibody repertoire.

Amino Acid Sequence↗

PubMed1998

The human D1A dopamine receptor: heterologous expression in Saccharomyces cerevisiae and purification of the functional receptor.

Functional human D1A dopamine receptor has been expressed in Saccharomyces cerevisiae. The primary sequence of the receptor was modified to include two affinity tags at the C-terminus of the protein, a FLAG tag (DYKDDDDK), and a His6 tag (HHHHHH). These modifications allowed for purification to near homogeneity using immobilized metal affinity chromatography and immunoaffinity chromatography. Radioligand binding demonstrated that the purified and reconstituted receptor binds the antagonist [3H]SCH23390 with an affinity (KD = 8.0 +/- 3.2 nM) comparable to that of the native receptor.

Amino Acid Sequence↗

PubMed1998

Unraveling the structures and modes of action of bacterial toxins.

The mechanism by which a soluble protein converts into a protein that spans a membrane remains a central question in understanding the molecular mechanism of toxicity of bacterial protein toxins. Using crystallographic structures of soluble toxins as templates, the past year has seen a number of experiments that are designed to probe the membrane state using other structural methods. In addition, crystallographic information concerning the clostridial neurotoxins has emerged, suggesting a novel mechanism of pore formation and new relationships between toxin binding domains.

Bacterial Toxins↗