Biomedical subjects

S C Feil

Publications and source records attributed to S C Feil.

14 recordsLinked to original sources

PubMed2000

Cleaved antitrypsin polymers at atomic resolution.

Alpha1-antitrypsin deficiency, which can lead to both emphysema and liver disease, is a result of the accumulation of alpha1-antitrypsin polymers within the hepatocyte. A wealth of biochemical and biophysical data suggests that alpha1-antitrypsin polymers form via insertion of residues from the reactive center loop of one molecule into the beta-sheet of another. However, this long-standing hypothesis has not been confirmed by direct structural evidence. Here, we describe the first crystallographic evidence of a beta-strand linked polymer form of alpha1-antitrypsin: the crystal structure of a cleaved alpha1-antitrypsin polymer.

Biopolymers↗

PubMed1999

Crystal structure of the N-terminal, growth factor-like domain of Alzheimer amyloid precursor protein.

Amyloid precursor protein (APP) plays a central role in Alzheimer disease. A proteolytic-breakdown product of APP, called beta-amyloid, is a major component of the diffuse and fibrillar deposits found in Alzheimer diseased brains. The normal physiological role of APP remains largely unknown despite much work. A knowledge of its function will not only provide insights into the genesis of the disease but may also prove vital in the development of an effective therapy. Here we describe the 1.8 A resolution crystal structure of the N-terminal, heparin-binding domain of APP (residues 28-123), which is responsible, among other things, for stimulation of neurite outgrowth. The structure reveals a highly charged basic surface that may interact with glycosaminoglycans in the brain and an abutting hydrophobic surface that is proposed to play an important functional role such as dimerization or ligand binding. Structural similarities with cysteine-rich growth factors, taken together with its known growth-promoting properties, suggests the APP N-terminal domain could function as a growth factor in vivo.

Alzheimer Disease↗

PubMed1998

A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications.

BACKGROUND: Glutathione S-transferases (GSTs) are a multifunctional group of enzymes, widely distributed in aerobic organisms, that have a critical role in the cellular detoxification process. Unlike their mammalian counterparts, bacterial GSTs often catalyze quite specific reactions, suggesting that their roles in bacteria might be different. The GST from Proteus mirabilis (PmGST B1-1) is known to bind certain antibiotics tightly and reduce the antimicrobial activity of beta-lactam drugs. Hence, bacterial GSTs may play a part in bacterial resistance towards antibiotics and are the subject of intense interest. RESULTS: Here we present the structure of a bacterial GST, PmGST B1-1, which has been determined from two different crystal forms. The enzyme adopts the canonical GST fold although it shares less than 20% sequence identity with GSTs from higher organisms. The most surprising aspect of the structure is the observation that the substrate, glutathione, is covalently bound to Cys 10 of the enzyme. In addition, the highly structurally conserved N-terminal domain is found to have an additional beta strand. CONCLUSIONS: The crystal structure of PmGST B1-1 has highlighted the importance of a cysteine residue in the catalytic cycle. Sequence analyses suggest that a number of other GSTs share this property, leading us to propose a new class of GSTs - the beta class. The data suggest that the in vivo role of the beta class GSTs could be as metabolic or redox enzymes rather than conjugating enzymes. Compelling evidence is presented that the theta class of GSTs evolved from an ancestral member of the thioredoxin superfamily.

Amino Acid Sequence↗

PubMed1998

Movement of a loop in domain 3 of aerolysin is required for channel formation.

Aerolysin is a channel-forming toxin that must oligomerize in order to become insertion-competent. Modeling based on the crystal structure of the proaerolysin dimer and electron microscopic images of the oligomer indicated that a loop in domain 3 must move away from the beta-sheet that forms the main body of the protein before oligomerization can proceed. In order to determine if movement actually occurs, strategically located amino acids in the loop and in the sheet were replaced with cysteines by site-directed mutagenesis. A double mutant was produced in which the new cysteines, at position 253 on the loop and position 300 in the sheet, were close enough together to allow formation of a disulfide bridge. The double mutant was unable to oligomerize, and it was completely inactive, showing not only that the bridge had formed but also that movement of the loop was essential for formation of the oligomer. The existence of the bridge was confirmed by X-ray crystallography. The reduced form of the protein and the single mutants T253C and A300C were as active as wild type, indicating that the amino acid replacements themselves had no functional consequences. Labeling studies using an environment-sensitive fluorescent sulfhydryl-reactive probe confirmed that the structure of the protein changes in the loop region as a consequence of proteolytic activation of proaerolysin, a step which also must precede oligomerization.

Aeromonas↗

PubMed1998

Aerolysin--a paradigm for membrane insertion of beta-sheet protein toxins?

The determination of the crystal structure of the bacterial protein proaerolysin provided the first view of a pore-forming toxin constructed mainly from beta-sheet. The structure that was obtained and subsequent crystallographic and biochemical studies have together allowed us to explain how the toxin is transformed from a water-soluble dimer to a heptameric transmembrane pore. Recent discoveries of structural similarities between aerolysin and other toxins suggest that the structure/function studies we have made may prove useful in understanding the actions of a number of pore-forming proteins.

Amino Acid Sequence↗

PubMed1997

Crystallization, structural determination and analysis of a novel parasite vaccine candidate: Fasciola hepatica glutathione S-transferase.

Glutathione S-transferases (GSTs) represent the major class of detoxifying enzymes from parasitic helminths. As a result, they are candidates for chemotherapeutic and vaccine design. Indeed, GSTs from Fasciola hepatica have been found to be effective for vaccinating sheep and cattle against fasciolosis. This helminth contains at least seven GST isoforms, of which four have been cloned. The cloned isoforms (Fh51, Fh47, Fh7 and Fh1) all belong to the mu class of GSTs, share greater than 71% sequence identity, yet display distinct substrate specificities. Crystals of Fh47 were obtained using the hanging drop vapour diffusion technique. The crystals belong to space group I4122, with one monomer in the asymmetric unit, which corresponds to a very high solvent content of approximately 75%. The physiological dimer is generated via a crystallographic 2-fold rotation. The three-dimensional structure of Fh47 was solved by molecular replacement using the Schistosoma japonicum glutathione S-transferase (Sj26) crystal structure as a search model. The structure adopts the canonical GST fold comprising two domains: an N-terminal glutathione-binding domain, consisting of a four-stranded beta-sheet and three helices whilst the C-terminal domain is entirely alpha-helical. The presence of Phe19 in Fh47 results in a 6 degrees interdomain rotation in comparison to Sj26, where the equivalent residue is a leucine. Homology models of Fh51, Fh7 and Fh1, based on the Fh47 crystal structure, reveal critical differences in the residues lining the xenobiotic binding site, particularly at residue positions 9, 106 and 204. In addition, differences amongst the isoforms in the non-substrate binding site were noted, which may explain the observed differential binding of large ligands. The major immunogenic epitopes of Fh47 were surprisingly found not to reside on the most solvent-exposed regions of the molecule.

Amino Acid Sequence↗

PubMed1997

Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form.

The mechanisms by which proteins gain entry into membranes is a fundamental problem in biology. Here, we present the first crystal structure of a thiol-activated cytolysin, perfringolysin O, a member of a large family of toxins that kill eukaryotic cells by punching holes in their membranes. The molecule adopts an unusually elongated shape rich in beta sheet. We have used electron microscopy data to construct a detailed model of the membrane channel form of the toxin. The structures reveal a novel mechanism for membrane insertion. Surprisingly, the toxin receptor, cholesterol, appears to play multiple roles: targeting, promotion of oligomerization, triggering a membrane insertion competent form, and stabilizing the membrane pore.

Cell Membrane↗

PubMed1997

Regulation and crystallization of phosphorylated and dephosphorylated forms of truncated dimeric phenylalanine hydroxylase.

Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.

Animals↗

PubMed1996

Giant protein kinases: domain interactions and structural basis of autoregulation.

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.

Amino Acid Sequence↗

PubMed1996

Crystallization and preliminary X-ray analysis of a thiol-activated cytolysin.

We present the first reported crystallization of a member of the thiol-activated family of protein toxins. Perfringolysin O, a virulence factor of Clostridium perfringens, has been crystallized in two different forms by the hanging drop vapor diffusion method. In one form the toxin crystallizes with PEG 20000 in the orthorhombic space group C222(1) with cell dimensions of a = 47.8 A, b = 182.0 A and c = 175.5 A and the crystals diffract to beyond 2.5 A resolution. In the second form the toxin crystallizes in a large variety of organic solvents including malt whisky. This crystal form belongs to the orthorhombic space group P222(1) with unit cell dimensions a = 47.1 A, b = 166.1 A and c = 214.0 A and with diffraction observed to 2.4 A resolution.

Bacterial Toxins↗

PubMed1996

Ca2+/S100 regulation of giant protein kinases.

Protein phosphorylation by protein kinases plays a central regulatory role in cellular processes and these kinases are themselves tightly regulated. One common mechanism of regulation involves Ca2+-binding proteins (CaBP) such as calmodulin (CaM). Here we report a Ca2+-effector mechanism for protein kinase activation by demonstrating the specific and >1,000-fold activation of the myosin-associated giant protein kinase twitchin by Ca2+/S100A1(2). S100A1(2) is a member of a large CaBP family that is implicated in various cellular processes, including cell growth, differentiation and motility, but whose molecular actions are largely unknown. The S100A1(2)-binding site is a part of the autoregulatory sequence positioned in the active site that is responsible for intrasteric autoinhibition of twitchin kinase; the mechanism of autoinhibition based on the crystal structures of two twitchin kinase fragments is described elsewhere. Ca2+/S100 represents a likely physiological activator for the entire family of giant protein kinases involved in muscle contractions and cytoskeletal structure.

Amino Acid Sequence↗

PubMed1996

Crystallization and preliminary X-ray analysis of a bacterial glutathione transferase.

Crystals of a bacterial glutathione S-transferase from Proteus mirabilis have been grown from polyethylene glycol by the hanging-drop vapour-diffusion method. Successful crystallization required the presence of the substrate glutathione. The crystals belong to the tetragonal space group P4 with cell dimensions a = b = 90.9 and c = 117.3 A. They contain between three and six monomers in the asymmetric unit and diffract to beyond 2.3 A resolution.

Journal Article↗

PubMed1995

Crystal structure of a theta-class glutathione transferase.

Glutathione S-transferases (GSTs) are a family of enzymes involved in the cellular detoxification of xenotoxins. Cytosolic GSTs have been grouped into four evolutionary classes for which there are representative crystal structures of three of them. Here we report the first crystal structure of a theta-class GST. So far, all available GST crystal structures suggest that a strictly conserved tyrosine near the N-terminus plays a critical role in the reaction mechanism and such a role has been convincingly demonstrated by site-directed mutagenesis. Surprisingly, the equivalent residue in the theta-class structure is not in the active site, but its role appears to have been replaced by either a nearby serine or by another tyrosine residue located in the C-terminal domain of the enzyme.

Amino Acid Sequence↗

PubMed1994

Crystallization and preliminary X-ray diffraction studies of a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina.

Crystals of a glutathione S-transferase from the Australian sheep blowfly Lucilia cuprina have been grown from ammonium sulphate by the hanging drop vapour diffusion method. Successful crystallization required the presence of the inhibitor S-hexylglutathione. The crystals belong to the tetragonal space group P4(1)22 (or P4(3)22) with cell dimensions of a = b = 88.1 A and c = 66.9 A. They contain one monomer in the asymmetric unit and diffract beyond 2.8 A resolution.

Animals↗