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Biomedical subjects

Glenn F King

Publications and source records attributed to Glenn F King.

At least 19 recordsLinked to original sources
PubMed2006

Modulation of insect Ca(v) channels by peptidic spider toxins.

Insects have a much smaller repertoire of voltage-gated calcium (Ca(V)) channels than vertebrates. Drosophila melanogaster harbors only a single ortholog of each of the vertebrate Ca(V)1, Ca(V)2, and Ca(V)3 subtypes, although its basal inventory is expanded by alternative splicing and editing of Ca(V) channel transcripts. Nevertheless, there appears to be little functional plasticity within this limited panel of insect Ca(V) channels, since severe loss-of-function mutations in genes encoding the pore-forming alpha1 subunits in Drosophila are embryonic lethal. Since the primary role of spider venom is to paralyze or kill insect prey, it is not surprising that most, if not all, spider venoms contain peptides that potently modify the activity of these functionally critical insect Ca(V) channels. Unfortunately, it has proven difficult to determine the precise ion channel subtypes recognized by these peptide toxins since insect Ca(V) channels have significantly different pharmacology to their vertebrate counterparts, and cloned insect Ca(V) channels are not available for electrophysiological studies. However, biochemical and genetic studies indicate that some of these spider toxins might ultimately become the defining pharmacology for certain subtypes of insect Ca(V) channels. This review focuses on peptidic spider toxins that specifically target insect Ca(V) channels. In addition to providing novel molecular tools for ion channel characterization, some of these toxins are being used as leads to develop new methods for controlling insect pests.

Amino Acid Sequence↗
PubMed2006

Mapping the phosphoinositide-binding site on chick cofilin explains how PIP2 regulates the cofilin-actin interaction.

Cofilin plays a key role in the choreography of actin dynamics via its ability to sever actin filaments and increase the rate of monomer dissociation from pointed ends. The exact manner by which phosphoinositides bind to cofilin and inhibit its interaction with actin has proven difficult to ascertain. We determined the structure of chick cofilin and used NMR chemical shift mapping and structure-directed mutagenesis to unambiguously locate its recognition site for phosphoinositides (PIs). This structurally unique recognition site requires both the acyl chain and head group of the PI for a productive interaction, and it is not inhibited by phosphorylation of cofilin. We propose that the interaction of cofilin with membrane-bound PIs abrogates its binding to both actin and actin-interacting protein 1, and facilitates spatiotemporal regulation of cofilin activity.

Actin Depolymerizing Factors↗
PubMed2006

Regulation of RhoGEF activity by intramolecular and intermolecular SH3 domain interactions.

RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22 human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions. The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3 domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used by other RhoGEF family members.

Amino Acid Motifs↗
PubMed2006

Domain architecture and structure of the bacterial cell division protein DivIB.

Bacterial cytokinesis requires the coordinated assembly of a complex of proteins, collectively known as the divisome, at the incipient division site. DivIB/FtsQ is a conserved component of the divisome in bacteria with cell walls, suggesting that it plays a role in synthesis and/or remodeling of septal peptidoglycan. We demonstrate that the extracytoplasmic region of DivIB comprises three discrete domains that we designate alpha, beta, and gamma from the N to C terminus. The alpha-domain is proximal to the cytoplasmic membrane and coincident with the polypeptide transport-associated domain that was proposed previously to function as a molecular chaperone. The beta-domain has a unique 3D fold, with no eukaryotic counterpart, and we show that it interconverts between two discrete conformations via cis-trans isomerization of a Tyr-Pro peptide bond. We propose that this isomerization might modulate protein-protein interactions of the flanking alpha- and gamma-domains. The C-terminal gamma-domain is unstructured in the absence of other divisomal proteins, but we show that it is critical for DivIB function.

Amino Acid Sequence↗
PubMed2006

Venom landscapes: mining the complexity of spider venoms via a combined cDNA and mass spectrometric approach.

The complexity of Australian funnel-web spider venoms has been explored via the combined use of MALDI-TOF mass spectrometry coupled with chromatographic separation and the analysis of venom-gland cDNA libraries. The results show that these venoms are far more complex than previously realized. We show that the venoms of Australian funnel-web spiders contain many hundreds of peptides that follow a bimodal distribution, with about 75% of the peptides having a mass of 3000-5000 Da. The mass spectral data were validated by matching the experimentally observed masses with those predicted from peptide sequences derived from analysis of venom-gland cDNA libraries. We show that multiple isoforms of these peptides are found in small chromatographic windows, which suggests that the wide distribution of close molecular weights among the chromatographic fractions probably reflects a diversity of structures and physicochemical properties. The combination of all predicted and measured parameters permits the interpretation of three-dimensional 'venom landscapes' derived from LC-MALDI analysis. We propose that these venom landscapes might have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms.

Amino Acid Sequence↗
PubMed2005

Orally active acaricidal peptide toxins from spider venom.

Numerous species of ticks and mites (collectively known as acarines) are serious pests of animals, humans, and crops. There are few commercially available acaricides and major classes of these chemicals continue to be lost from the marketplace due to resistance development or deregistration by regulatory agencies. There is consequently a pressing need to isolate new and safe acaricidal compounds. In this study, we show that two families of peptide neurotoxins isolated from the venom of the Australian funnel-web spider Hadronyche versuta are lethal to the lone star tick Amblyomma americanum. These toxins, which are specific blockers of arthropod voltage-gated calcium channels, induce a pronounced phenotype characterized by an unusual gait that is rapidly followed by paralysis and death. Remarkably, one of these toxins, the calcium channel blocker omega-atracotoxin-Hv1a, is virtually equipotent whether the toxin is injected or fed to A. americanum.

Administration, Oral↗
PubMed2005

Were arachnids the first to use combinatorial peptide libraries?

Spiders, scorpions, and cone snails are remarkable for the extent and diversity of gene-encoded peptide neurotoxins that are expressed in their venom glands. These toxins are produced in the form of structurally constrained combinatorial peptide libraries in which there is hypermutation of essentially all residues in the mature-toxin sequence with the exception of a handful of strictly conserved cysteines that direct the three-dimensional fold of the toxin. This gene-based combinatorial peptide library strategy appears to have been first implemented by arachnids almost 400 million years ago, long before cone snails evolved a similar mechanism for generating peptide diversity.

Animals↗
PubMed2004

Scanning mutagenesis of omega-atracotoxin-Hv1a reveals a spatially restricted epitope that confers selective activity against insect calcium channels.

We constructed a complete panel of alanine mutants of the insect-specific calcium channel blocker omega-atracotoxin-Hv1a. Lethality assays using these mutant toxins identified three spatially contiguous residues, Pro10, Asn27, and Arg35, that are critical for insecticidal activity against flies (Musca domestica) and crickets (Acheta domestica). Competitive binding assays using radiolabeled omega-atracotoxin-Hv1a and neuronal membranes prepared from the heads of American cockroaches (Periplaneta americana) confirmed the importance of these three residues for binding of the toxin to target calcium channels presumably expressed in the insect membranes. At concentrations up to 10 microm, omega-atracotoxin-Hv1a had no effect on heterologously expressed rat Cav2.1, Cav2.2, and Cav1.2 calcium channels, consistent with the previously reported insect selectivity of the toxin. 30 microm omega-atracotoxin-Hv1a inhibited rat Cav currents by 10-34%, depending on the channel subtype, and this low level of inhibition was essentially unchanged when Asn27 and Arg35, which appears to be critical for interaction of the toxin with insect Cav channels, were both mutated to alanine. We propose that the spatially contiguous epitope formed by Pro10, Asn27, and Arg35 confers specific binding to insect Cav channels and is largely responsible for the remarkable phyletic selectivity of omega-atracotoxin-Hv1a. This epitope provides a structural template for rational design of chemical insecticides that selectively target insect Cav channels.

Amino Acid Substitution↗
PubMed2004

Differential hydrolysis of erythrocyte and mitochondrial membrane phospholipids by two phospholipase A2 isoenzymes (NK-PLA2-I and NK-PLA2-II) from the venom of the Indian monocled cobra Naja kaouthia.

We previously demonstrated that venom from the Indian monocled cobra Naja kaouthia is a rich source of phospholipase A2 enzymes, and we purified and characterized a major PLA2 isoenzyme (NK-PLA2-I) from N. kaouthia venom. In the present study, we report the purification and biochemical characterization of a second PLA2 isoenzyme (NK-PLA2-II) from the same venom. A comparison of the membrane phospholipid hydrolysis patterns by these two PLA2s has revealed that they cause significantly more damage to mitochondrial membranes (NK-PLA2-I > NK-PLA2-II) as compared to erythrocyte membranes due to more efficient binding of the enzymes to mitochondrial membranes. Fatty acid release patterns by these PLA2s from the membrane phospholipid PC-pools indicate that NK-PLA2-I does not discriminate between saturated and unsaturated fatty acids whereas NK-PLA2-II shows a preference for unsaturated fatty acids during the initial phase of attack. The current investigation provides new insight into the molecular arrangement of NK-PLA2-sensitive domains in erythrocyte and mitochondrial membranes and highlights the contribution of polar, but uncharged, amino acids such as serine and cysteine in NK-PLA2 induced membrane damage.

Animals↗
PubMed2004

Structure and mechanism of action of Sda, an inhibitor of the histidine kinases that regulate initiation of sporulation in Bacillus subtilis.

Histidine kinases are used extensively in prokaryotes to monitor and respond to changes in cellular and environmental conditions. In Bacillus subtilis, sporulation-specific gene expression is controlled by a histidine kinase phosphorelay that culminates in phosphorylation of the Spo0A transcription factor. Sda provides a developmental checkpoint by inhibiting this phosphorelay in response to DNA damage and replication defects. We show that Sda acts at the first step in the relay by inhibiting autophosphorylation of the histidine kinase KinA. The structure of Sda, which we determined using NMR, comprises a helical hairpin. A cluster of conserved residues on one face of the hairpin mediates an interaction between Sda and the KinA dimerization/phosphotransfer domain. This interaction stabilizes the KinA dimer, and the two proteins form a stable heterotetramer. The data indicate that Sda forms a molecular barricade that inhibits productive interaction between the catalytic and phosphotransfer domains of KinA.

Amino Acid Sequence↗
PubMed2004

The wonderful world of spiders: preface to the special Toxicon issue on spider venoms.

Spiders are remarkable for their complete reliance on predation as a trophic strategy. Their evolutionary success is due in large part to production of a complex venom that is designed to rapidly subdue or kill their prey. This issue provides an overview of the fascinating complexity of these venoms, a realistic account of the danger they pose to humans, and an examination of their immense but largely untapped potential for drug and insecticide discovery.

Animals↗
PubMed2004

Australian funnel-web spiders: master insecticide chemists.

Arthropods are the most diverse animal group on the planet. Their ability to inhabit a vast array of ecological niches has inevitably brought them into conflict with humans. Although only a small minority are classified as pest species, they nevertheless destroy about a quarter of the world's annual crop production and transmit an impressive array of pathogens of human and veterinary public health importance. Arthropod pests have been controlled almost exclusively with chemical insecticides since the introduction of DDT in the 1940s. However, the evolution of resistance to many insecticides, coupled with increased awareness of the potential environmental and human and animal health impacts of these chemicals, has stimulated the search for new insecticidal compounds, novel molecular targets, and alternative control methods. Spider venoms are complex chemical cocktails that have evolved to kill or paralyze arthropod prey, and they represent a largely untapped reservoir of insecticidal compounds. This review focuses on several families of invertebrate-specific peptide neurotoxins that were isolated from the venom of Australian funnel-web spiders. These peptides are promising insecticide leads because of their selectivity for invertebrates and activity on previously unvalidated targets. These toxins should facilitate the development of novel target-based screens for new insecticide leads, while their mapped pharmacophores will provide templates for rational design of mimetics that act at these target sites. Furthermore, genes encoding these toxins can be used to improve the efficacy of insect-specific viruses.

Amino Acid Sequence↗
PubMed2004

Positioning of the MinE binding site on the MinD surface suggests a plausible mechanism for activation of the Escherichia coli MinD ATPase during division site selection.

Division site selection in Escherichia coli requires that the MinD protein interact with itself and with MinC and MinE. MinD is a member of the NifH-ArsA-Par-MinD subgroup of ATPases. The MinE-MinD interaction results in activation of MinD ATPase activity in the presence of membrane vesicles. The sites within MinD responsible for its interaction with MinC and MinE were studied by site-directed mutagenesis and yeast two-hybrid analysis, guided by the known three-dimensional structure of MinD proteins. This provided evidence that MinC and MinE bind to overlapping sites on the MinD surface. The results also suggested that MinE and the invariant Lys11 residue in the ATPase P-loop of MinD compete for binding to a common site within the MinD structure, thereby providing a plausible structural basis for the ability of MinE to activate the ATPase activity of MinD.

Adenosine Triphosphatases↗
PubMed2003

The MinD membrane targeting sequence is a transplantable lipid-binding helix.

MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably also Archaea. It was recently demonstrated that membrane localization of MinD is mediated by an 8-12-residue C-terminal motif termed the membrane targeting sequence or MTS. In this study we show that the MinD MTS is a transplantable lipid-binding motif that can effectively target heterologous proteins to the cell membrane. We demonstrate that eubacterial MTSs interact directly with lipid bilayers as an amphipathic helix, with a distinct preference for anionic phospholipids. Moreover, we provide evidence that the phospholipid preference of each MTS, as well as its affinity for biological membranes, has been evolutionarily "tuned" to its specific role in different bacteria. We propose a model to describe how the MTS is coupled to ATP binding to regulate the reversible membrane association of Escherichia coli MinD during its pole-to-pole oscillation cycle.

Adenosine Triphosphatases↗
PubMed2002

Membrane localization of MinD is mediated by a C-terminal motif that is conserved across eubacteria, archaea, and chloroplasts.

MinD is a widely conserved ATPase that has been demonstrated to play a pivotal role in selection of the division site in eubacteria and chloroplasts. It is a member of the large ParA superfamily of ATPases that are characterized by a deviant Walker-type ATP-binding motif. MinD localizes to the cytoplasmic face of the inner membrane in Escherichia coli, and its association with the inner membrane is a prerequisite for membrane recruitment of the septation inhibitor MinC. However, the mechanism by which MinD associates with the membrane has proved enigmatic; it seems to lack a transmembrane domain and the amino acid sequence is devoid of hydrophobic tracts that might predispose the protein to interaction with lipids. In this study, we show that the extreme C-terminal region of MinD contains a highly conserved 8- to 12-residue sequence motif that is essential for membrane localization of the protein. We provide evidence that this motif forms an amphipathic helix that most likely mediates a direct interaction between MinD and membrane phospholipids. A model is proposed whereby the membrane-targeting motif mediates the rapid cycles of membrane attachment-release-reattachment that are presumed to occur during pole-to-pole oscillation of MinD in E. coli.

Adenosine Triphosphatases↗
PubMed2002

Division site placement in E.coli: mutations that prevent formation of the MinE ring lead to loss of the normal midcell arrest of growth of polar MinD membrane domains.

The MinE protein functions as a topological specificity factor in determining the site of septal placement in Escherichia coli. MinE assembles into a membrane-associated ring structure near midcell and directs the localization of MinD and MinC into a membrane- associated polar zone that undergoes a characteristic pole-to-pole oscillation cycle. Single (green fluorescent protein) and double label (yellow fluorescent protein/cyan fluorescent protein) fluorescence labeling experiments showed that mutational alteration of a site on the alpha-face of MinE led to a failure to assemble the MinE ring, associated with loss of the ability to support a normal pattern of division site placement. The absence of the MinE ring did not prevent the assembly and disassembly of the MinD polar zone. Mutant cells lacking the MinE ring were characterized by the growth of MinD polar zones past their normal arrest point near midcell. The results suggested that the MinE ring acts as a stop-growth mechanism to prevent the MinCD polar zone from extending beyond the midcell division site.

Adenosine Triphosphatases↗