155 research outputs found
Mechanisms governing precise protein biotinylation
No full textAbstract not availableLouise M. Sternicki, Kate L. Wegener, John B. Bruning, Grant W. Booker and Steven W. Polya
Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase
No full textData source: Supplementary data, https://doi.org/10.1016/j.tube.2015.02.046
Link to a related website: https://digital.library.adelaide.edu.au/dspace/bitstream/2440/92432/2/hdl_92432.pdf, Open Access via UnpaywallAbstract not availableWanisa Salaemae, Min Y. Yap, Kate L. Wegener, Grant W. Booker,
Matthew C.J. Wilce, Steven W. Polya
Structural diversity in integrin/talin interactions
No full textThe adhesion of integrins to the extracellular matrix is regulated by binding of the cytoskeletal protein talin to the cytoplasmic tail of the β-integrin subunit. Structural studies of this interaction have hitherto largely focused on the β3-integrin, one member of the large and diverse integrin family. Here, we employ NMR to probe interactions and dynamics, revealing marked structural diversity in the contacts between β1A, β1D, and β3 tails and the Talin1 and Talin2 isoforms. Coupled with analysis of recent structures of talin/β tail complexes, these studies elucidate the thermodynamic determinants of this heterogeneity and explain why the Talin2/β1D isoforms, which are co-localized in striated muscle, form an unusually tight interaction. We also show that talin/integrin affinity can be enhanced 1000-fold by deleting two residues in the β tail. Together, these studies illustrate how the integrin/talin interaction has been fine-tuned to meet varying biological requirements.Nicholas J. Anthis, Kate L. Wegener, David R. Critchley, and Iain D. Campbellhttp://www.cell.com/structure/hom
Bioactive dahlein peptides from the skin secretions of the Australian aquatic frog Litoria dahlii: sequence determination by electrospray mass spectrometry
No full textThe definitive version may be found at www.wiley.comEleven dahlein peptides are present in the skin secretion of the Australian aquatic frog Litoria dahlii. All peptides have been sequenced using a combination of electrospray mass spectrometry (ES-MS) and Lys-C digestion/MS, with each sequence confirmed by automated Edman sequencing. The 13-residue dahlein 1 peptides (e.g. dahlein 1.1 GLFDIIKNIVSTL-NH(2)) exhibit weak wide-spectrum antimicrobial activity but no significant activity in the anticancer testing program of the National Cancer Institute (Washington). There are no potent antimicrobial peptides present in the glandular secretion, but the dahleins 5 strongly inhibit the formation of NO by neuronal nitric oxide synthase (e.g. dahlein 5.1 GLLGSIGNAIGAFIANKLKP-OH).Kate L. Wegener, Craig S. Brinkworth, John H. Bowie, John C. Wallace, Michael J. Tyle
The solution structure of frenatin 3, a neuronal nitric oxide synthase inhibitor from the giant tree frog, Litoria infrafrenata
No full textThe definitive version may be found at www.wiley.comThe peptide frenatin 3 is a major component of the skin secretion of the Australian giant tree frog, Litoria infrafrenata. Frenatin 3 is 22 amino acids in length, and shows neither antimicrobial nor anticancer activity. It inhibits the production of nitric oxide by the enzyme neuronal nitric oxide synthase at a micromolar concentration by binding to its regulatory protein, Ca2+ calmodulin, a protein known to recognize and bind amphipathic alpha-helices. The solution structure of frenatin 3 has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. In trifluoroethanol/water mixtures, the peptide forms an amphipathic alpha-helix over residues 1-14 while the C-terminal eight residues are more flexible and less structured. The flexible region may be responsible for the lack of antimicrobial activity. In water, frenatin 3 exhibits some alpha-helical character in its N-terminal region.Craig S. Brinkworth, John A. Carver, Kate L. Wegener, Jason Doyle, Lyndon E. Llewellyn and John H. Bowi
The molecular basis of filamin binding to integrins and competition with talin
No full textThe ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin β cytoplasmic tail forms an extended β strand that interacts with β strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.Tiila Kiema, Yatish Lad, Pengju Jiang, Camilla L. Oxley, Massimiliano Baldassarre, Kate L. Wegener, Iain D. Campbell, Jari Ylänne and David A. Calderwoodhttp://www.sciencedirect.com/science/journal/1097276
The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis: The solution structure of aurein 1.2
No full textPublished in European Journal of Biochemistry, 2000; 267:5330-5341 at www.interscience.wiley.comSeventeen aurein peptides are present in the secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aurea, and 16 from the corresponding secretion of the related Southern Bell Frog L. raniformis. Ten of these peptides are common to both species. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. These peptides are named in three groups (aureins 1±3) according to their sequences. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), aurein 2.2 (GLFDIVKKVVGALGSLNH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). Both L. aurea and L. raniformis have endoproteases that deactivate the major membrane-active aurein peptides by removing residues from both the N- and C-termini of the peptides. The most abundant degradation products have two residues missing from the N-terminal end of the peptide. The solution structure of the basic peptide, aurein 1.2, has been determined by NMR spectroscopy to be an amphipathic a-helix with well-defined hydrophilic and hydrophobic regions. Certain of the aurein peptides (e.g. aureins 1.2 and 3.1) show anticancer activity in the NCI test regime, with LC50 values in the 102521024 m range. The aurein 1 peptides have only 13 amino-acid residues: these are the smallest antibiotic and anticancer active peptides yet reported from an anuran. The longer aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5.1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH) show neither antibacterial nor anticancer activity.Tomas Rozek, Kate L. Wegener, John H. Bowie, Ian N. Olver, John A. Carver, John C. Wallace and Michael J. Tyle
The structure of an interdomain complex that regulates talin activity
No full textTalin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316–326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with β3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.Benjamin T. Goult, Neil Bate, Nicholas J. Anthis, Kate L. Wegener, Alexandre R. Gingras, Bipin Patel, Igor L. Barsukov, Iain D. Campbell, Gordon C. K. Roberts and David R. Critchle
Precipitant-ligand exchange technique reveals the ADP binding mode in Mycobacterium tuberculosis dethiobiotin synthetase
No full textDethiobiotin synthetase from Mycobacterium tuberculosis (MtDTBS) is a promising antituberculosis drug target. Small-molecule inhibitors that target MtDTBS provide a route towards new therapeutics for the treatment of antibiotic-resistant tuberculosis. Adenosine diphosphate (ADP) is an inhibitor of MtDTBS; however, structural studies into its mechanism of inhibition have been unsuccessful owing to competitive binding to the enzyme by crystallographic precipitants such as citrate and sulfate. Here, a crystallographic technique termed precipitant-ligand exchange has been developed to exchange protein-bound precipitants with ligands of interest. Proof of concept for the exchange method was demonstrated using cytidine triphosphate (CTP), which adopted the same binding mechanism as that obtained with traditional crystal-soaking techniques. Precipitant-ligand exchange also yielded the previously intractable structure of MtDTBS in complex with ADP solved to 2.4 Å resolution. This result demonstrates the utility of precipitant-ligand exchange, which may be widely applicable to protein crystallography.Andrew P. Thompson, Kate L. Wegener, Grant W. Booker, Steven W. Polyak and John B. Brunin
A bimane‐based peptide staple for combined helical induction and fluorescent imaging
No full textThe thiol-selective fluorescent imaging agent, dibromobimane, has been repurposed to crosslink cysteine- and homocysteine- containing peptides, with the resulting bimane linker acting as both a structural constraint and a fluorescent tag. Macro- cyclisation was conducted on nine short peptides containing two cysteines and/or homocysteines, both on-resin and in buffered aqueous solution, to give macrocycles ranging in size from 16 (i,i +2) to 31 (i,i + 7) atoms. The structures were defined by CD, NMR structure calculations by using Xplor-NIH, NMR secondary shift and J HαNH analyses to reveal helical structure in the i,i + 4 (1, 2), and i,i + 3 (5) constrained peptides. Cellular- uptake studies were conducted with three of the macrocycles. Subsequent confocal imaging revealed punctate fluorescence within the cytosol indicative of peptides trapped in endocytic vesicles. These studies demonstrate that dibromobimane is an effective tool for defining secondary structure within short peptides, whilst simultaneously introducing a fluorescent tag suitable for common cell-based experiments.Aimee J. Horsfall, Kylie R. Dunning, Kelly L. Keeling, Denis B. Scanlon, Kate L. Wegener, Andrew D. Abel
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