310 results on '"Iain D. Campbell"'
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2. A Calcium Dependent De-Adhesion Mechanism Regulates the Direction and Rate of Cell Migration: A Mathematical Model.
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Najl V. Valeyev, A. Kristina Downing, Andrei Skorinkin, Iain D. Campbell, and Nikolai V. Kotov
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- 2006
Catalog
3. A Christian's Pocket guide to Sin
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Iain D. Campbell
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- 2005
4. Conformational Changes in Talin on Binding to Anionic Phospholipid Membranes Facilitate Signaling by Integrin Transmembrane Helices.
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Antreas C. Kalli, Iain D. Campbell, and Mark S. P. Sansom
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- 2013
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5. A beleza e a Glória de Cristo
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Joel R. Beeke, David Murray, Iain D. Campbell, Richard D. Phillips, Gerald M. Bilkes, David Carmichael, Albert N. Martin, William Van Doodewaard, Joel R. Beeke, David Murray, Iain D. Campbell, Richard D. Phillips, Gerald M. Bilkes, David Carmichael, Albert N. Martin, and William Van Doodewaard more...
- Abstract
Para ser verdadeiramente cristão, temos de abraçar como doutrina e como experiência os principais dogmas da Reforma: sola gratia, sola fide, solus Christus, sola Scriptura e soli Deo Gloria. Não é a nossa salvação baseada somente na Escritura, somente por meio de Cristo, recebida somente pela fé, obra somente da graça e somente para a glória de Deus? No fulcro dessas veementes verdades está solus Christus (somente Cristo).'Somente Cristo'é nossa vida e a nossa salvação, a nossa beleza e a nossa glória. Cristo Jesus é o Senhor e Salvador glorioso, belo, que tem'pernas, colunas de mármore'(Ct 5.15), pois ele é forte e inabalável e é totalmente desejável (v.16). Nas palavras de Thomas Brooks,'Cristo é desejável, Cristo é muito desejável, Cristo é o mais desejável, Cristo é sempre desejável, Cristo é totalmente desejável'. Brooks também disse:'Cristo é o diamante mais brilhante no anel da glória'. Aproveite a festa espiritual servida em A Beleza e a Glória de Cristo, uma compilação das palestras da conferência anual do Seminário Teológico Reformado Puritano em agosto de 2010 em Grand Rapids, Michigan. Cada ensaio estabelece diante dos leitores as inesquecíveis riquezas do Senhor Jesus Cristo, a esperança da nossa glória e a glória da nossa esperança. Os tópicos incluem a beleza de Cristo profetizada e tipificada em Isaías e Cântico dos Cânticos; A glória de Cristo em sua encarnação, ministério terreno e morte na cruz; Cristo na teologia histórica e na vida cotidiana; A gloriosa exaltação de Cristo em sua ressurreição e em seu triunfo no livro do Apocalipse. Os colaboradores são David Murray, Iain Campbell, Richard Phillips, Gerald Bilkes, David Carmichael, Albert Martin, Joel Beeke, William VanDoodewaard, Ray Pennings e James Grier. more...
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- 2022
6. The Important Role of Calcium in Regulation of Adhesion Disassembly and Cell Migration: Mathematical Modelling.
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Najl V. Valeyev, Andrei Skorinkin, Kristy Downing, Iain D. Campbell, and Nikolai V. Kotov
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- 2005
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7. The evolution of protein NMR
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Iain D. Campbell
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Adhesion receptors ,Chemistry ,A protein ,Radiology, Nuclear Medicine and imaging ,Nanotechnology ,Nuclear magnetic resonance spectroscopy ,Data science - Abstract
Since the first NMR spectrum of a protein was published over 50 years ago, remarkable technical improvements have led to NMR being recognized as a uniquely powerful tool that can give a wide variety of useful information about macro- molecules and their interactions. This article gives an overview of NMR studies of proteins. It presents a personal historical perspective, briefly reviews the current status of the field and reflects on possible future directions. Two specific examples, related to multi-domain complexes and membrane-spanning adhesion receptors, are described to illustrate recent applications. Emphasis is placed on how general advances in molecular biology and technology are changing the nature and focus of NMR studies. more...
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- 2013
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8. Structural basis for the interaction between the cytoplasmic domain of the hyaluronate receptor layilin and the talin F3 subdomain
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Jaswir Basran, Igor L. Barsukov, David R. Critchley, Gordon C. K. Roberts, Clive R. Bagshaw, Kate L. Wegener, and Iain D. Campbell
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Talin ,animal structures ,Magnetic Resonance Spectroscopy ,Integrin ,Molecular Sequence Data ,macromolecular substances ,Biology ,Calorimetry ,Ligands ,environment and public health ,Fluorescence ,Protein Structure, Secondary ,Focal adhesion ,Mice ,Structure-Activity Relationship ,Structural Biology ,Animals ,Computer Simulation ,Amino Acid Sequence ,Cytoskeleton ,Molecular Biology ,Membrane Glycoproteins ,Cell adhesion molecule ,Kinase ,Adhesion ,Actin cytoskeleton ,Cell biology ,Protein Structure, Tertiary ,Kinetics ,Cytoplasm ,embryonic structures ,Mutation ,biology.protein ,Mutant Proteins ,Carrier Proteins ,Peptides ,Protein Binding - Abstract
Talin is a large cytoskeletal protein that is involved in coupling the integrin family of cell adhesion molecules to the actin cytoskeleton, colocalising with the integrins in focal adhesions (FAs). However, at the leading edge of motile cells, talin colocalises with the hyaluronan receptor layilin in what are thought to be transient adhesions, some of which subsequently mature into more stable FAs. During this maturation process, layilin is replaced with integrins, which are highly clustered in FAs, where localised production of PI(4,5)P(2) by type 1 phosphatidyl inositol phosphate kinase type 1gamma (PIPK1gamma) is thought to play a role in FA assembly. The talin FERM F3 subdomain binds both the integrin beta-subunit cytoplasmic domain and PIPK1gamma, and these interactions are understood in detail at the atomic level. The talin F3 domain also binds to short sequences in the layilin cytoplasmic domain, and here we report the structure of the talin/layilin complex, which shows that talin binds integrins, PIPK1gamma and layilin in similar although subtly different ways. Based on structure comparisons, we designed a set of talin F3 mutations that selectively affected the affinity of talin for its targets, as determined by stopped-flow fluorescence measurements. Such mutations will help to assess the importance of the interactions between talin and its various ligands in cell adhesion and migration. more...
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- 2016
9. The solution structure of EMILIN1 globular C1q domain reveals a disordered insertion necessary for interaction with the alpha4beta1 integrin
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Paolo Viglino, Simonetta Bot, Giuliana Verdone, Paola Spessotto, Alessandra Capuano, Gennaro Esposito, Francesco Bucciotti, Simon A. Colebrooke, Alessandra Corazza, Roberto Doliana, Alfonso Colombatti, Alessandra Silvestri, and Iain D. Campbell more...
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Models, Molecular ,Integrin ,Blood Pressure ,Integrin alpha4beta1 ,Biology ,Biochemistry ,Jurkat cells ,Protein Structure, Secondary ,Jurkat Cells ,Structure-Activity Relationship ,Cell Movement ,Cell Adhesion ,Homeostasis ,Humans ,Homology modeling ,Protein Structure, Quaternary ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Glycoproteins ,Membrane Glycoproteins ,Uterus ,EMILIN1 ,Cell Biology ,Ligand (biochemistry) ,Protein Structure, Tertiary ,Trophoblasts ,Mutagenesis, Site-Directed ,biology.protein ,C1q domain ,Biophysics ,Female ,Microfibril ,Elastin - Abstract
The extracellular matrix protein EMILIN1 (elastin microfibril interface located protein 1) is implicated in maintaining blood pressure homeostasis via the N-terminal elastin microfibril interface domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology model structure of the human 52-kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all of the gC1q (crystal) structures solved to date, the 10-stranded beta-sandwich fold of the gC1q domain is reduced to nine beta strands with a consequent increase in the size of the central cavity lumen. An unstructured loop, resulting from an insertion unique to EMILIN1 and EMILIN2 family members and located at the trimer apex upstream of the missing strand, specifically engages the alpha4beta1 integrin. Using both Jurkat T and EA.hy926 endothelial cells as well as site-directed mutagenesis, we demonstrate that the ability of alpha4beta1 integrins to recognize the trimeric EMILIN1 gC1q domain mainly depends on a single glutamic acid residue (Glu(933)). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILIN1 gC1q-alpha4beta1 represents a unique ligand/receptor system, with a requirement for a 3-fold arrangement of the interaction site. more...
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- 2016
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10. Effects of the N2144S mutation on backbone dynamics of a TB-cbEGF domain pair from human fibrillin-1
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Xuemei Yuan, Penny A. Handford, Jeremy Lack, Jörn M. Werner, A. Kristina Downing, V Knott, and Iain D. Campbell
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Models, Molecular ,Magnetic Resonance Spectroscopy ,Fibrillin-1 ,Mutant ,Molecular Sequence Data ,medicine.disease_cause ,Fibrillins ,Protein Structure, Secondary ,Marfan Syndrome ,Diffusion ,Motion ,Structural Biology ,medicine ,Humans ,Amino Acid Sequence ,Structural motif ,Pliability ,Molecular Biology ,Mutation ,Binding Sites ,Epidermal Growth Factor ,Chemistry ,Microfilament Proteins ,Intracellular Signaling Peptides and Proteins ,Ligand (biochemistry) ,Protein Structure, Tertiary ,Folding (chemistry) ,Crystallography ,Amino Acid Substitution ,Latent TGF-beta Binding Proteins ,Biophysics ,Calcium ,Microfibril ,Carrier Proteins ,Fibrillin ,Heteronuclear single quantum coherence spectroscopy - Abstract
The calcium-binding epidermal growth factor-like (cbEGF) module and the transforming growth factor β-binding protein-like (TB) module are the two major structural motifs found in fibrillin-1, the extracellular matrix (ECM) protein defective in the Marfan syndrome (MFS). An MFS-causing mutation, N2144S, which removes a calcium ligand in cbEGF32, does not detectably affect fibrillin-1 biosynthesis, rate of secretion, processing, or deposition of reducible fibrillin-1 into the ECM. Since the residue at position 2144 is normally engaged in calcium ligation, it is unable to mediate intermolecular interactions. We have shown previously that this mutation does not affect the folding properties of the TB or cbEGF domains in vitro, but does decrease calcium-binding in cbEGF and TB-cbEGF domain constructs. Here, we use NMR spectroscopy to probe the effects of the N2144S mutation on backbone dynamic properties of TB6-cbEGF32. Analysis of the backbone 15N relaxation data of wild-type TB6-cbEGF32 has revealed a flexible inter-domain linkage. Parallel dynamics analysis of the N2144S mutant has shown increased flexibility in the region joining the two domains as well as in the calcium-binding site at the N terminus of cbEGF32. This research demonstrates that a small change in peptide backbone flexibility, which does not enhance proteolytic susceptibility of the domain pair, is associated with an MFS phenotype. Flexibility of the TB-cbEGF linkage is likely to contribute to the biomechanical properties of fibrillin-rich connective tissue microfibrils, and may play a role in the microfibril assembly process. more...
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- 2016
11. Integrin structure, activation, and interactions
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Martin J. Humphries and Iain D. Campbell
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Models, Molecular ,Integrins ,Binding Sites ,Protein Conformation ,Protein subunit ,Integrin ,Biology ,Ligands ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Protein Structure, Tertiary ,Extracellular matrix ,Cytoplasm ,Cations ,Extracellular ,biology.protein ,Protein quaternary structure ,Cytoskeleton ,Intracellular ,Perspectives ,Signal Transduction - Abstract
Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large "head" on two "legs," with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains. These domains initiate the assembly of large signaling complexes and thereby bridge the extracellular matrix to the intracellular cytoskeleton. To allowcells to sample and respond to a dynamic pericellular environment, integrins have evolved a highly responsive receptor activation mechanism that is regulated primarily by changes in tertiary and quaternary structure. This review summarizes recent progress in the structural and molecular functional studies of this important class of adhesion receptor. © 2011 Cold Spring Harbor Laboratory Press. more...
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- 2016
12. Interface characterization of the type II module pair from fibronectin
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Jörn M. Werner, Iain D. Campbell, Steven P. Smith, Yasuhiro Hashimoto, and Andrew R. Pickford
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Models, Molecular ,Magnetic Resonance Spectroscopy ,Interface (Java) ,Protein Conformation ,In Vitro Techniques ,Bioinformatics ,Biochemistry ,Humans ,Binding Sites ,biology ,Chemistry ,business.industry ,Chemical shift ,Modular design ,Peptide Fragments ,Recombinant Proteins ,Characterization (materials science) ,Fibronectins ,Fibronectin ,Orientation (vector space) ,Crystallography ,Covalent bond ,biology.protein ,Thermodynamics ,Collagen ,business ,Linker - Abstract
The lone (1)F2(2)F2 modular pair of fibronectin is found in the collagen-binding region. This exclusive localization suggests the (1)F2(2)F2 pair plays an important role in the recognition of collagen. However, no information is currently available about the interaction between the two F2 modules and, thus, the orientation of their putative collagen-binding sites with respect to one another. Comparison of a variety of high-resolution NMR parameters from the F2 modules in isolation and the (1)F2(2)F2 pair was used to establish the extent of interaction between the F2 modules in the pair. Chemical shifts of the F2 modules and the (1)F2(2)F2 pair indicate that the structures of the modules are preserved in the pair and that, with the exception of the covalent linkage, they do not interact. (15)N NMR relaxation data identify significant motion occurring in the linker region of the (1)F2(2)F2 pair, and analyses of the anisotropic diffusion properties of the (1)F2(2)F2 pair are consistent with the modules in the F2 pair tumbling independent of one another. more...
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- 2016
13. The tail of integrin activation
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Iain D. Campbell and Nicholas J. Anthis
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Models, Molecular ,Talin ,Integrins ,Extracellular matrix assembly ,Integrin ,Biology ,Biochemistry ,CD49c ,Article ,Collagen receptor ,Cell biology ,Focal adhesion ,Integrin alpha M ,biology.protein ,Animals ,Humans ,Integrin, beta 6 ,Phosphorylation ,Molecular Biology ,Intracellular ,Protein Binding - Abstract
Integrins are essential adhesion receptors found on the surfaces of all metazoan cells. As regulators of cell migration and extracellular matrix assembly, these membrane-spanning heterodimers are critical for embryonic development, tissue repair and immune responses. Signals transmitted by integrins from outside to inside the cell promote cell survival and proliferation, but integrin affinity for extracellular ligands can also be controlled by intracellular cues. This bidirectional signaling is mediated by the short cytoplasmic tails of the two integrin subunits. Recent structural and functional studies of various integrin fragments and complexes between the cytoplasmic tails and intracellular proteins, such as talin, have provided new insight into the signaling processes centered around the tails, particularly inside-out integrin activation. more...
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- 2016
14. Preparation of recombinant fibronectin fragments for functional and structural studies
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Christopher J. Millard, Iain D. Campbell, A. Radu Aricescu, and David Staunton
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chemistry.chemical_classification ,biology ,Chemistry ,Cellular differentiation ,Cell ,law.invention ,Cell biology ,Extracellular matrix ,Fibronectin ,medicine.anatomical_structure ,law ,Recombinant DNA ,biology.protein ,medicine ,Protein folding ,Cell adhesion ,Glycoprotein - Abstract
Fibronectin, an ubiquitous extracellular matrix (ECM) glycoprotein, plays a major role in fundamental biological processes such as cell adhesion and migration, maintenance of normal cell morphology, cytoskeletal organization, and cell differentiation. Fibronectin is constructed from three types of independently folding protein module (Fn1, Fn2, and Fn3) and is found as a Fibrillar network in the ECM where it interacts with other ECM components and provides anchorage sites for cell surface integrin receptors. The mosaic nature of fibronectin permits it to be analyzed by a "dissection" strategy, where protein fragments generated by recombinant expression in E. coli, P. pastoris, and human cell lines are employed in structural and functional investigations. We describe methods suitable for the production of various fibronectin fragments for study by a variety of techniques including crystallography and electron microscopy but special mention is made of methods suitable for the production of samples for NMR studies. more...
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- 2016
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15. The integrin receptor in biologically relevant bilayers: insights from molecular dynamics simulations
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Ilpo Vattulainen, Iain D. Campbell, Tomasz Róg, Mark S.P. Sansom, Antreas C. Kalli, Tampere University, and Department of Physics
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Protein Conformation, alpha-Helical ,Talin ,0301 basic medicine ,Physiology ,Amino Acid Motifs ,Lipid Bilayers ,Integrin ,Biophysics ,Phosphatidylserines ,Platelet Glycoprotein GPIIb-IIIa Complex ,Molecular Dynamics Simulation ,Biology ,Lipid diffusion ,114 Physical sciences ,CD49c ,Article ,Collagen receptor ,03 medical and health sciences ,chemistry.chemical_compound ,Cell surface receptor ,Humans ,Protein Interaction Domains and Motifs ,Binding Sites ,Sequence Homology, Amino Acid ,030102 biochemistry & molecular biology ,Molecular dynamics simulations ,Phosphatidylethanolamines ,Cell Biology ,Phosphatidylserine ,Integrin alphaVbeta3 ,Sphingolipid ,Protein Structure, Tertiary ,Cell biology ,Protein Subunits ,Cholesterol ,030104 developmental biology ,chemistry ,Phosphatidylcholines ,biology.protein ,Thermodynamics ,Protein Conformation, beta-Strand ,lipids (amino acids, peptides, and proteins) ,Integrin, beta 6 ,Protein Multimerization ,Signal transduction ,Protein Binding - Abstract
Integrins are heterodimeric (αβ) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2–F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2–F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes. Electronic supplementary material The online version of this article (doi:10.1007/s00232-016-9908-z) contains supplementary material, which is available to authorized users. more...
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- 2016
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16. Efficient regulation of sigmaF, the first sporulation-specific sigma factor in B.subtilis
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Joanna Clarkson, Michael D. Yudkin, and Iain D. Campbell
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Spores, Bacterial ,chemistry.chemical_classification ,fungi ,Phosphatase ,Sigma Factor ,Bacillus subtilis ,Biology ,biology.organism_classification ,Adenosine Diphosphate ,Dephosphorylation ,Adenosine Triphosphate ,Spectrometry, Fluorescence ,Enzyme ,Bacterial Proteins ,chemistry ,Biochemistry ,Structural Biology ,Sigma factor ,Gene expression ,Biophysics ,Phosphorylation ,Molecular Biology - Abstract
Differential gene expression is established in the prespore and mother-cell compartments of Bacillus subtilis through the successive activation of a series of cell-type-specific sigma factors. Crucial to the success of this process is the control of the first prespore-specific sigma factor, sigmaF. sigmaF is regulated by the proteins SpoIIAB, SpoIIAA and SpoIIE. SpoIIAB forms an inhibitory complex with sigmaF, which can be dissociated by interaction with SpoIIAA. During this interaction SpoIIAA is phosphorylated. SpoIIE is a membrane-bound phosphatase that dephosphorylates SpoIIAA, thereby re-activating it. It is not understood how sigmaF is activated specifically in the prespore but not in the mother cell. Here, we use a recently developed fluorescence spectroscopy technique to follow in real time the formation of sigmaF.SpoIIAB complexes and their dissociation by SpoIIAA. We show that complete activation of sigmaF is induced by a tenfold increase in SpoIIE activity. This result demonstrates that relatively small changes in SpoIIE activity, which could arise from asymmetric septation, can achieve the all-or-nothing response in sigmaF activity required by the cell. For long-term sigmaF activation, we find that sustained SpoIIE activity is required to counteract the activity of SpoIIAB. Even though the continual phosphorylation and dephosphorylation of SpoIIAA by these two enzymes will expend some ATP, the formation of SpoIIAA.SpoIIAB.ADP complexes greatly diminishes the rate of the phosphorylation reaction, and thus minimizes the wastage of energy. These features provide a very efficient system for regulating sigmaF. more...
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- 2016
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17. Exploiting the carboxylate chemical shift to resolve degenerate resonances in spectra of 13C-labelled glycosaminoglycans
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Paul L. DeAngelis, Andrew Almond, Iain D. Campbell, Charles D. Blundell, and Simon A. Colebrooke
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Carbon Isotopes ,Anomer ,Magnetic Resonance Spectroscopy ,Proton ,Stereochemistry ,Chemistry ,Chemical shift ,Molecular Sequence Data ,Carboxylic Acids ,General Chemistry ,Carbon-13 NMR ,Reference Standards ,Ring (chemistry) ,Sensitivity and Specificity ,Crystallography ,chemistry.chemical_compound ,Carbohydrate Sequence ,Proton NMR ,Carbohydrate Conformation ,Moiety ,General Materials Science ,Carboxylate ,Hyaluronic Acid ,Glycosaminoglycans - Abstract
Glycosaminoglycans (GAG) are important vertebrate extracellular matrix polysaccharides that comprise repeated units of an acidic and an N-acetylated sugar. The constituent acidic sugars are central to their biological functions, but have been largely inaccessible to NMR because the 1H resonances overlap with those from other residues. Here, pulse sequences that address this failure are developed using 13C-enriched oligosaccharides of the glycosaminoglycan, hyaluronan, as model systems. Two pulse sequences are presented that exploit the unique chemical shifts and scalar couplings present at the carboxylate moiety to filter out coherences from the N-acetylated sugars and produce simple spectra containing only resonances from the acidic sugars. The first sequence uses one-bond couplings to correlate the carboxylate carbon with the adjacent carbon and its directly attached proton, while the second sequence exploits a long-range coupling to correlate the carboxylate carbon with the anomeric proton and carbon of the same residue. In addition, inclusion of an isotropic mixing block into these sequences allows resonances from the otherwise degenerate ring protons to be resolved. Spectra from the hyaluronan tetra- and hexasaccharides show that all glucuronic acid (GlcA) residues can be resolved from one another, allowing nuclei to be assigned in a sequence-specific manner. However, in some spectra, resonances are observed at positions not predicted by spin-operator analysis, and simulations reveal that these additional magnetisation transfers result from strong-coupling. These experiments represent a foundation from which new structural and biochemical information can be obtained in a sequence-specific manner for the acidic sugar residues in hyaluronan and other glycosaminoglycans. Copyright © 2005 John Wiley & Sons, Ltd. more...
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- 2016
18. Cooling overall spin temperature: protein NMR experiments optimized for longitudinal relaxation effects
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Iain D. Campbell and Michaël Deschamps
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Nuclear and High Energy Physics ,Magnetic Resonance Spectroscopy ,Spins ,Chemistry ,Temperature ,Biophysics ,Analytical chemistry ,Proteins ,Nuclear magnetic resonance spectroscopy ,Condensed Matter Physics ,Biochemistry ,Free induction decay ,Excited state ,Spin diffusion ,Molecule ,Transverse relaxation-optimized spectroscopy ,Protons ,Heteronuclear single quantum coherence spectroscopy - Abstract
In experiments performed on protonated proteins at high fields, 80% of the NMR spectrometer time is spent waiting for the (1)H atoms to recover their polarization after recording the free induction decay. Selective excitation of a fraction of the protons in a large molecule has previously been shown to lead to faster longitudinal relaxation for the selected protons [K. Pervushin, B. Vögeli, A. Eletsky, Longitudinal (1)H relaxation optimization in TROSY NMR spectroscopy, J. Am. Chem. Soc. 124 (2002) 12898-12902; P. Schanda, B. Brutscher, Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds, J. Am. Chem. Soc. 127 (2005) 8014-8015; H.S. Attreya, T. Szyperski, G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment, Proc. Natl. Acad. Sci. USA 101 (2004) 9642-9647]. The pool of non-selected protons acts as a "thermal bath" and spin-diffusion processes ("flip-flop" transitions) channel the excess energy from the excited pool to the non-selected protons in regions of the molecule where other relaxation processes can dissipate the excess energy. We present here a sensitivity enhanced HSQC sequence (COST-HSQC), based on one selective E-BURP pulse, which can be used on protonated (15)N enriched proteins (with or without (13)C isotopic enrichment). This experiment is compared to a gradient sensitivity enhanced HSQC with a water flip-back pulse (the water flip-back pulse quenches the spin diffusion between (1)H(N) and (1)H(alpha) spins). This experiment is shown to have significant advantages in some circumstances. Some observed limitations, namely sample overheating with short recovery delays and complex longitudinal relaxation behaviour are discussed and analysed. more...
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- 2016
19. The eighth FIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth FIII domain
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Iain D. Campbell, Christopher F. van der Walle, Helen J. Mardon, Richard Fairless, Harri Altroff, and Judith Asselin
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Models, Molecular ,Protein Conformation ,Recombinant Fusion Proteins ,Integrin ,Enzyme-Linked Immunosorbent Assay ,Plasma protein binding ,Ligands ,Biochemistry ,Receptors, Fibronectin ,Cell Adhesion ,Humans ,Cloning, Molecular ,Binding site ,Cell adhesion ,Molecular Biology ,Guanidine ,Glutathione Transferase ,Integrin binding ,Binding Sites ,Dose-Response Relationship, Drug ,biology ,Chemistry ,Cell Biology ,Immunohistochemistry ,Fibronectins ,Protein Structure, Tertiary ,Fibronectin ,Kinetics ,Mutation ,Biophysics ,biology.protein ,Thermodynamics ,Integrin, beta 6 ,Signal transduction ,Protein Binding ,Signal Transduction - Abstract
Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha(5)beta(1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha(5)beta(1) interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin alpha(5)beta(1) to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin alpha(5)beta(1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs. more...
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- 2016
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20. FOURIER-TRANSFORM NMR PULSE METHODS FOR MEASUREMENT OF SLOW-EXCHANGE RATES
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Christopher M. Dobson, R. G. Ratcliffe, Iain D. Campbell, and Robert J. P. Williams
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Hydrogen ,Chemistry ,General Engineering ,Analytical chemistry ,chemistry.chemical_element ,Lower temperature ,symbols.namesake ,Fourier transform ,Transverse relaxation ,Bloch equations ,Slow exchange ,Curve fitting ,symbols ,Atomic physics - Abstract
The seven-membered ring of Valium exists in two equivalent conformations. Exchange between the two forms exchanges two coupled nonequivalent hydrogen. atoms in the ring and this system has been used to examine NMR Fourier transform pulse methods for measuring rates of slow exchange. Experiments are reported both for the exchanging system (at 320 K) and for the effectively nonexchanging system at a lower temperature (270 K). The selective inversion of one exchanging resonance allows the determination of all the rate and relaxation constants by an iterative fitting of the data to the general solution of the modified Bloch equations. However, the use of a combination of non-selective inversion, selective inversion, and presaturation sequences is shown to provide definitive and consistent values for the rate and relaxation constants without the need for difficult curve fitting to several variables. By this means the rate of exchange in a 100 m M Valium solution in deuterochloroform at 320 K is found to be 5.4 ± 0.2 sec − . An appendix discusses the application of transverse relaxation measurements and concludes that for the slow exchange in Valium this method of determining the rate of exchange is inferior to the measurement of longitudinal relaxation. more...
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- 2016
21. The effects of dissolved oxygen upon amide proton relaxation and chemical shift in a perdeuterated protein
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Jonathan Boyd, Tobias S. Ulmer, and Iain D. Campbell
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Nuclear and High Energy Physics ,Magnetic Resonance Spectroscopy ,Hydrogen bond ,Chemistry ,Chemical shift ,Biophysics ,Analytical chemistry ,Amide proton ,chemistry.chemical_element ,Hydrogen Bonding ,Nuclear magnetic resonance spectroscopy ,Condensed Matter Physics ,Biochemistry ,Oxygen ,Amides ,Fibronectins ,Relaxation rate ,Humans ,Protons ,Hyperfine structure ,Bar (unit) - Abstract
The effects of dissolved molecular oxygen upon amide proton ((1)H(N)) longitudinal and transverse relaxation rates and chemical shifts were studied for a small protein domain, the second type 2 module of fibronectin ((2)F2)-isotopically enriched to 99% (2)H, 98% (15)N. Longitudinal relaxation rate enhancements, R(O(2))((1)H(N)), of individual backbone (1)H(N) nuclei varied up to 14 fold between a degassed and oxygenated (1 bar) solution, indicating that the oxygen distribution within the protein is inhomogeneous. On average, smaller relaxation rate enhancements were observed for (1)H(N) nuclei associated with the core of the protein compared to (1)H(N) nuclei closer to the surface, suggesting restricted oxygen accessibility to some regions. In agreement with an O(2)-(1)H(N) hyperfine interaction in the extreme narrowing limit, the (1)H(N) transverse relaxation rates showed no significant change, up to an oxygen pressure of 9.5 bar (the maximum pressure used in this study). For most (1)H(N) resonances, small deltadelta(O(2))((1)H(N)) hyperfine chemical shifts could be detected between oxygen pressures of 1 bar and 9.5 bar. more...
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- 2016
22. Phosphorylation induces subtle structural changes in SpoIIAA, a key regulator of sporulation
- Author
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Michael D. Yudkin, Iain D. Campbell, and Joanna Clarkson
- Subjects
inorganic chemicals ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Mutant ,Regulator ,macromolecular substances ,Biology ,Crystallography, X-Ray ,Biochemistry ,environment and public health ,Bacterial Proteins ,Histidine ,Phosphorylation ,Molecular Biology ,Binding properties ,Cell Biology ,Protein Structure, Tertiary ,enzymes and coenzymes (carbohydrates) ,Kinetics ,Mutation ,Biophysics ,bacteria ,Sporulation in Bacillus subtilis ,Bacillus subtilis ,Research Article - Abstract
The phosphorylation state of SpoIIAA is a key factor in the regulation of sporulation in Bacillus subtilis. Previous crystallographic studies had led to the conclusion that phosphorylation alters the binding affinity of SpoIIAA for its partner proteins solely through the additional charge and bulk of the phosphoryl group: small structural changes observed elsewhere in the protein were considered to be random fluctuations rather than the result of phosphorylation. The results presented in the present paper show that NMR studies detect the same subtle structural changes in solution as those seen in the crystal, strongly implying that they are the direct result of phosphorylation. These subtle structural changes are similar to those that occur in a non-phosphorylated mutant that is defective in binding to one of its partner proteins. We propose that the structural changes which occur in SpoIIAA on phosphorylation act in concert with the phosphoryl group to alter its binding properties. more...
- Published
- 2016
23. Preparation of isotopically labeled recombinant fragments of fibronectin for functional and structural study by heteronuclear nuclear magnetic resonance spectroscopy
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Iain D. Campbell, Jeremy R. Bright, Jennifer R. Potts, and Andrew R. Pickford
- Subjects
Fibronectin ,Nuclear magnetic resonance ,Biochemistry ,Heteronuclear molecule ,biology ,law ,Chemistry ,Recombinant DNA ,biology.protein ,Nuclear magnetic resonance spectroscopy ,law.invention - Published
- 2016
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24. Studies of focal adhesion assembly
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Iain D. Campbell
- Subjects
Talin ,Integrins ,Protein Conformation ,Filamins ,Integrin ,Focal adhesion assembly ,macromolecular substances ,Plasma protein binding ,Filamin ,Biochemistry ,Models, Biological ,Focal adhesion ,Protein structure ,Contractile Proteins ,Cell Adhesion ,Humans ,biology ,Chemistry ,Microfilament Proteins ,RNA-Binding Proteins ,Phosphoproteins ,Cell biology ,Fibronectins ,Fibronectin ,DNA-Binding Proteins ,biology.protein ,Phosphorylation ,Protein Binding - Abstract
Recent studies of some proteins involved in the formation of focal adhesions are described. These include fibronectin, integrins, talin, Dok1 and filamin. Emphasis is placed on features that facilitate regulated assembly of complexes; these include a modular construction and flexible regions that provide interaction sites whose affinity can be adjusted by conformational masking and phosphorylation. © 2008 Biochemical Society. more...
- Published
- 2016
25. NMR analysis of interacting soluble forms of the cell-cell recognition molecules CD2 and CD48
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Paul C. Driscoll, Helen R. Mott, M. S. B. Mcalister, Simon J. Davis, P A van der Merwe, and Iain D. Campbell
- Subjects
Magnetic Resonance Spectroscopy ,Chemistry ,Stereochemistry ,Protein Conformation ,Chinese hamster ovary cell ,Chemical shift ,Cell-cell recognition ,CD2 Antigens ,Nuclear magnetic resonance spectroscopy ,CHO Cells ,Cell Communication ,CD48 Antigen ,Hydrogen-Ion Concentration ,Biochemistry ,Rats ,Mice ,Protein structure ,Heteronuclear molecule ,Solubility ,Antigens, CD ,Cricetinae ,Animals ,Humans ,Binding site ,Heteronuclear single quantum coherence spectroscopy - Abstract
The T cell glycoprotein, CD2, is one of the best characterized molecules mediating recognition at the cell surface. The ligands of murine and human CD2 are CD48 and CD58, respectively, and interactions between these molecules have been shown to influence antigen recognition and T cell activation. The CD58 binding site of human CD2 has been characterized in mutational studies, and here we use heteronuclear NMR spectroscopy to identify the rat CD48 binding site of the N-terminal domain of rat CD2 (CD2d1). The NMR spectrum of bacterially expressed CD2d1, assigned initially at pH 4.3 in the course of determining the three-dimensional solution structure of this domain [Driscoll, P.C., et al. (1991) Nature 353, 762-765], has been reassigned as a two-dimensional 15N-1H heteronuclear single-quantum coherence (HSQC) spectrum at neutral pH. The CD48 binding surface was identified by monitoring perturbations in the line widths and chemical shifts of cross peaks in the HSQC spectrum of CD2d1 during titrations with a soluble form of CD48 expressed in Chinese hamster ovary cells. This first solution NMR analysis of interacting cell surface molecules shows that the ligand binding site extends across an area of ca. 700-800 A2 of the GFCC'C" face corresponding almost exactly to lattice contacts in crystals of soluble CD2 first proposed as a model of the interaction of CD2 with its ligands. The analysis finds no evidence for any large-scale structural changes in domain 1 of CD2 to accompany CD48 binding. Comparisons of the human and rat CD2 ligand binding sites suggest that species- and ligand-specific binding may be determined by as few as three amino acid residues, corresponding to Thr37, Leu38, and Glu41 in rat CD2 (Lys42, Lys43, and Gln46 in human CD2). more...
- Published
- 2016
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26. Shape and dynamics of a calcium-binding protein investigated by nitrogen-15 NMR relaxation
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Jörn M. Werner, Iain D. Campbell, and A. Kristina Downing
- Subjects
Protein structure ,chemistry ,Epidermal growth factor ,Calcium-binding protein ,Diffusion ,Biophysics ,chemistry.chemical_element ,Nuclear magnetic resonance spectroscopy ,Microfilament Protein ,Nitrogen ,Isotopes of nitrogen - Published
- 2016
27. Integrin binding immunoglobulin type filamin domains have variable stability
- Author
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Pengju Jiang and Iain D. Campbell
- Subjects
Models, Molecular ,Integrins ,Protein Folding ,Adhesion receptors ,Protein Conformation ,Filamins ,Integrin ,Immunoglobulins ,macromolecular substances ,Filamin ,Biochemistry ,Contractile Proteins ,Drug Stability ,Humans ,Binding site ,Nuclear Magnetic Resonance, Biomolecular ,Actin ,Integrin binding ,Binding Sites ,biology ,Chemistry ,Microfilament Proteins ,Hydrogen-Ion Concentration ,Peptide Fragments ,Protein Structure, Tertiary ,Cytoplasm ,Biophysics ,biology.protein ,Thermodynamics ,Antibody - Abstract
Filamin, a large modular protein composed mainly of many immunoglobulin-like domains, is a potent cross-linker of actin filaments. The region containing immunoglobulin type modules 19-21 makes up the binding site for the cytoplasmic tails of the integrin adhesion receptors. Here we investigate the stability of the Ig-like filamin domains using NMR studies over a range of pH and temperature. We show that the 21st Ig-like module (FLNa21) is partly unfolded even under physiological conditions and when attached to FLNa20. It is, however, appreciably stabilized upon binding to integrins. FLNa21 is noticeably less stable than neighboring homologous modules, such as FLNa19 and FLNa17. This variability in stability could be related to the known sensitivity of filamin to cell-mediated mechanical forces. more...
- Published
- 2016
28. Physical evidence for the induced release of the Bacillus subtilis transcription factor, sigma(F), from its inhibitory complex
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Iain D. Campbell, Joanna Clarkson, and Michael D. Yudkin
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Base Sequence ,biology ,fungi ,Mutant ,Reproducibility of Results ,Sigma Factor ,Bacillus subtilis ,biology.organism_classification ,Inhibitory postsynaptic potential ,Kinetics ,Spectrometry, Fluorescence ,Bacterial Proteins ,Biochemistry ,Structural Biology ,Gene expression ,Biophysics ,bacteria ,Phosphorylation ,Molecular Biology ,Fluorescence response ,Sporulation in Bacillus subtilis ,Transcription factor ,DNA Primers - Abstract
The release of the transcription factor sigma(F) from its inhibitory complex with SpoIIAB is a key regulatory step in the control of sporulation in Bacillus subtilis as it initiates a pattern of differential gene expression in the mother cell and prespore compartments. The sigma(F).SpoIIAB complex is dissociated by the unphosphorylated form of the protein SpoIIAA, the alternative binding partner of SpoIIAB. Here, we employ fluorescence spectroscopy to examine the mechanism by which SpoIIAA acts on the sigma(F).SpoIIAB complex. We constructed a mutant of sigma(F), sigma(F)-W46L, which displayed a reproducible fluorescence response on binding to SpoIIAB. Using this mutant we were able to quantify the amount of sigma(F) bound to SpoIIAB in real time. The results provide physical evidence for the "induced release" mechanism of sigma(F) activation. We demonstrate that SpoIIAA interacts directly with the sigma(F).SpoIIAB complex, greatly decreasing the affinity of SpoIIAB for sigma(F) and thus causing the release of the latter. We also demonstrate that sigma(F) is released before SpoIIAA is phosphorylated and that release occurs on a similar time scale to the binding of SpoIIAA to SpoIIAB. more...
- Published
- 2016
29. Biophysical Analysis of Kindlin-3 Reveals an Elongated Conformation and Maps Integrin Binding to the Membrane-distal β-Subunit NPXY Motif
- Author
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Anna K. Füzéry, Robert J.C. Gilbert, Luke A. Yates, Iain D. Campbell, and Roman Bonet
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Talin ,Integrins ,Magnetic Resonance Spectroscopy ,metabolism [Recombinant Proteins] ,Protein Conformation ,chemistry [Recombinant Proteins] ,Amino Acid Motifs ,Plasma protein binding ,metabolism [Cytoskeletal Proteins] ,Biochemistry ,Mice ,0302 clinical medicine ,Protein structure ,X-Ray Diffraction ,Structural Biology ,Sf9 Cells ,Protein Isoforms ,0303 health sciences ,FERM domain ,Integrin beta1 ,kindlin-3 protein, mouse ,metabolism [Antigens, CD29] ,Recombinant Proteins ,3. Good health ,Pleckstrin homology domain ,Kindlins ,Protein Binding ,Protein subunit ,Integrin ,Biophysics ,Biology ,Biophysical Phenomena ,03 medical and health sciences ,ddc:570 ,chemistry [Protein Isoforms] ,Scattering, Small Angle ,chemistry [Talin] ,Cell Adhesion ,metabolism [Protein Subunits] ,Animals ,chemistry [Membrane Proteins] ,Amino Acid Sequence ,Molecular Biology ,metabolism [Protein Isoforms] ,genetics [Cytoskeletal Proteins] ,030304 developmental biology ,Integrin binding ,chemistry [Protein Subunits] ,Small-angle X-ray Scattering ,Antigens, CD29 ,Membrane Proteins ,Cell Biology ,metabolism [Talin] ,NMR ,Protein Structure, Tertiary ,Cytoskeletal Proteins ,Protein Subunits ,Structural biology ,Mutation ,biology.protein ,chemistry [Antigens, CD29] ,chemistry [Cytoskeletal Proteins] ,metabolism [Membrane Proteins] ,030217 neurology & neurosurgery - Abstract
Background: Kindlins are essential co-activators of integrins. Results: Kindlin-3 has an elongated structure and forms a ternary complex with the Talin head and integrin β-tails. The Kindlin-3-tail interface involves a membrane-distal NPXY motif on the tail. Conclusion: New information about the conformation and interactions of Kindlin-3 has been obtained. Significance: The solution structure and protein/protein interactions of Kindlin-3 give insight into its role., Kindlin-3, a 75-kDa protein, has been shown to be critical for hemostasis, immunity, and bone metabolism via its role in integrin activation. The Kindlin family is hallmarked by a FERM domain comprised of F1, F2, and F3 subdomains together with an N-terminal F0 domain and a pleckstrin homology domain inserted in the F2 domain. Recombinant Kindlin-3 was cloned, expressed, and purified, and its domain organization was studied by x-ray scattering and other techniques to reveal an extended conformation. This unusual elongated structure is similar to that found in the paralogue Talin head domain. Analytical ultracentrifugation experiments indicated that Kindlin-3 forms a ternary complex with the Talin and β-integrin cytoplasmic tails. NMR showed that Kindlin-3 specifically recognizes the membrane-distal tail NPXY motif in both the β1A and β1D isoforms, although the interaction is stronger with β1A. An upstream Ser/Thr cluster in the tails also plays a critical role. Overall these data support current biological, clinical, and mutational data on Kindlin-3/β-tail binding and provide novel insights into the overall conformation and interactions of Kindlin-3. more...
- Published
- 2012
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30. The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended
- Author
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Iain D. Campbell, Salla Ruskamo, Gregor Hofmann, Robert J.C. Gilbert, Ulla Pentikäinen, Jari Ylänne, and Pengju Jiang
- Subjects
Models, Molecular ,genetics [Receptors, Dopamine D3] ,metabolism [Recombinant Proteins] ,Protein Conformation ,genetics [Antigens, CD18] ,chemistry [Recombinant Proteins] ,Plasma protein binding ,Crystallography, X-Ray ,Ligands ,Filamin ,metabolism [Antigens, CD18] ,metabolism [Cytoskeletal Proteins] ,Biochemistry ,filamins ,Contractile Proteins ,Protein structure ,metabolism [Peptide Fragments] ,FLNA ,chemistry [Antigens, CD18] ,genetics [Cell Adhesion Molecules] ,Small-angle X-ray scattering ,Microfilament Proteins ,genetics [Contractile Proteins] ,Recombinant Proteins ,chemistry [Receptors, Dopamine D3] ,FBLIM1 protein, human ,ddc:540 ,Domain (ring theory) ,Dimerization ,Protein Binding ,chemistry [Contractile Proteins] ,Filamins ,Antigens, CD18 ,metabolism [Cell Adhesion Molecules] ,Biology ,Scattering, Small Angle ,metabolism [Receptors, Dopamine D3] ,Humans ,chemistry [Microfilament Proteins] ,Protein Interaction Domains and Motifs ,metabolism [Mutant Proteins] ,DRD3 protein, human ,Molecular Biology ,metabolism [Contractile Proteins] ,Actin ,genetics [Cytoskeletal Proteins] ,Cryoelectron Microscopy ,Mutagenesis ,ta1182 ,Receptors, Dopamine D3 ,metabolism [Microfilament Proteins] ,Cell Biology ,chemistry [Cell Adhesion Molecules] ,genetics [Peptide Fragments] ,Peptide Fragments ,Cytoskeletal Proteins ,Crystallography ,chemistry [Mutant Proteins] ,chemistry [Peptide Fragments] ,CD18 Antigens ,Biophysics ,chemistry [Cytoskeletal Proteins] ,Mutant Proteins ,genetics [Microfilament Proteins] ,Cell Adhesion Molecules - Abstract
Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16–21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16–24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties. more...
- Published
- 2012
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31. Assembly of a Filamin Four-domain Fragment and the Influence of Splicing Variant-1 on the Structure
- Author
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Ulla Pentikäinen, Salla Ruskamo, Heikki Takala, Pengju Jiang, Jari Ylänne, and Iain D. Campbell
- Subjects
Models, Molecular ,Filamins ,Protein domain ,Biology ,Filamin ,Biochemistry ,Protein Structure, Secondary ,Structure-Activity Relationship ,Contractile Proteins ,Protein structure ,Humans ,FLNA ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Microfilament Proteins ,Alternative splicing ,ta1182 ,Signal transducing adaptor protein ,Cell Biology ,Actin cytoskeleton ,Molecular biology ,Protein Structure, Tertiary ,Cell biology ,Alternative Splicing ,Protein Structure and Folding ,RNA splicing - Abstract
Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed. more...
- Published
- 2011
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32. The Structure of the Talin/Integrin Complex at a Lipid Bilayer: An NMR and MD Simulation Study
- Author
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Mark S.P. Sansom, Iain D. Campbell, Benjamin T. Goult, Nicholas J. Anthis, Kate L. Wegener, and Antreas C. Kalli
- Subjects
Models, Molecular ,Platelet Membrane Glycoprotein IIb ,Talin ,Magnetic Resonance Spectroscopy ,Membrane lipids ,Lipid Bilayers ,Molecular Sequence Data ,Integrin ,macromolecular substances ,Molecular Dynamics Simulation ,Biology ,Article ,Membrane Lipids ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,Structural Biology ,Integrin complex ,Animals ,Humans ,Amino Acid Sequence ,Lipid bilayer ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Sequence Homology, Amino Acid ,Integrin beta1 ,Bilayer ,Transmembrane protein ,Protein Structure, Tertiary ,3. Good health ,Cell biology ,Multiprotein Complexes ,Mutation ,biology.protein ,Protein Multimerization ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Summary Integrins are cell surface receptors crucial for cell migration and adhesion. They are activated by interactions of the talin head domain with the membrane surface and the integrin β cytoplasmic tail. Here, we use coarse-grained molecular dynamic simulations and nuclear magnetic resonance spectroscopy to elucidate the membrane-binding surfaces of the talin head (F2-F3) domain. In particular, we show that mutations in the four basic residues (K258E, K274E, R276E, and K280E) in the F2 binding surface reduce the affinity of the F2-F3 for the membrane and modify its orientation relative to the bilayer. Our results highlight the key role of anionic lipids in talin/membrane interactions. Simulation of the F2-F3 in complex with the α/β transmembrane dimer reveals information for its orientation relative to the membrane. Our studies suggest that the perturbed orientation of talin relative to the membrane in the F2 mutant would be expected to in turn perturb talin/integrin interactions., Graphical Abstract Highlights ► Simulations and NMR define the interactions of the talin head domain with membranes ► Talin binds to anionic lipids in a bilayer via the F2 subdomain ► Mutations in the F2 lipid-binding site may perturb talin/integrin interactions more...
- Published
- 2010
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33. Implications for Collagen Binding from the Crystallographic Structure of Fibronectin 6FnI1–2FnII7FnI
- Author
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Richard W. Farndale, Ioannis Vakonakis, Ulrich Schwarz-Linek, Michèle C. Erat, Andrew R. Pickford, and Iain D. Campbell
- Subjects
Protein Structure ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Context (language use) ,Crystallography, X-Ray ,Biochemistry ,Pichia ,Collagen receptor ,Extracellular matrix ,03 medical and health sciences ,X-ray Crystallography ,0302 clinical medicine ,Protein structure ,Cell Movement ,Animals ,Binding site ,Protein Interactions ,10. No inequality ,Fibronectin ,Molecular Biology ,030304 developmental biology ,Extracellular Matrix Proteins ,0303 health sciences ,Binding Sites ,biology ,Circular Dichroism ,Cell Biology ,NMR ,Recombinant Proteins ,Fibronectins ,Gelatin Binding Domain ,Protein Structure and Folding ,Solvents ,Biophysics ,biology.protein ,Collagen ,Peptides ,Molecular Biophysics ,030217 neurology & neurosurgery ,Type I collagen ,Protein Binding ,Binding domain - Abstract
Collagen and fibronectin (FN) are two abundant and essential components of the vertebrate extracellular matrix; they interact directly with cellular receptors and affect cell adhesion and migration. Past studies identified a FN fragment comprising six modules, (6)FnI(1-2)FnII(7-9)FnI, and termed the gelatin binding domain (GBD) as responsible for collagen interaction. Recently, we showed that the GBD binds tightly to a specific site within type I collagen and determined the structure of domains (8-9)FnI in complex with a peptide from that site. Here, we present the crystallographic structure of domains (6)FnI(1-2)FnII(7)FnI, which form a compact, globular unit through interdomain interactions. Analysis of NMR titrations with single-stranded collagen peptides reveals a dominant collagen interaction surface on domains (2)FnII and (7)FnI; a similar surface appears involved in interactions with triple-helical peptides. Models of the complete GBD, based on the new structure and the (8-9)FnI·collagen complex show a continuous putative collagen binding surface. We explore the implications of this model using long collagen peptides and discuss our findings in the context of FN interactions with collagen fibrils. more...
- Published
- 2010
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34. Asparaginyl β-Hydroxylation of Proteins Containing Ankyrin Repeat Domains Influences Their Stability and Function
- Author
-
Iain D. Campbell, Ivan Prokes, Adam P. Hardy, Christopher J. Schofield, and Leanne Kelly
- Subjects
Models, Molecular ,Molecular Sequence Data ,Hydroxylation ,Mixed Function Oxygenases ,Substrate Specificity ,Structure-Activity Relationship ,chemistry.chemical_compound ,Protein structure ,Structural Biology ,Catalytic Domain ,Structure–activity relationship ,Amino Acid Sequence ,Asparagine ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,Protein Stability ,Temperature ,Proteins ,Ankyrin Repeat ,Protein Structure, Tertiary ,Amino acid ,Repressor Proteins ,chemistry ,Biochemistry ,Ankyrin repeat ,Heteronuclear single quantum coherence spectroscopy - Abstract
Recent reports have provided evidence that the beta-hydroxylation of conserved asparaginyl residues in ankyrin repeat domain (ARD) proteins is a common posttranslational modification in animal cells. Here, nuclear magnetic resonance (NMR) and other biophysical techniques are used to study the effect of asparaginyl beta-hydroxylation on the structure and stability of 'consensus' ARD proteins. The NMR analyses support previous work suggesting that a single beta-hydroxylation of asparagine can stabilize the stereotypical ARD fold. A second asparaginyl beta-hydroxylation causes further stabilization. In combination with mutation studies, the biophysical analyses reveal that the stabilizing effect of beta-hydroxylation is, in part, mediated by a hydrogen bond between the asparaginyl beta-hydroxyl group and the side chain of a conserved aspartyl residue, two residues to the N-terminal side of the target asparagine. Removal of this hydrogen bond resulted in reduced stabilization by hydroxylation. Formation of the same hydrogen bond is also shown to be a factor in inhibiting binding of hydroxylated ARDs to factor-inhibiting hypoxia-inducible factor (FIH). The effects of hydroxylation appear to be predominantly localized to the target asparagine and proximal residues, at least in the consensus ARD protein. The results reveal that thermodynamic stability is a factor in determining whether a particular ARD protein is an FIH substrate; a consensus ARD protein with three ankyrin repeats is an FIH substrate, while more stable consensus ARD proteins, with four or five ankyrin repeats, are not. However, NMR studies reveal that the consensus protein with four ankyrin repeats is still able to bind to FIH, suggesting that FIH may interact in cells with natural ankyrin repeats without resulting hydroxylation. Overall, the work provides novel biophysical insights into the mechanism by which asparaginyl beta-hydroxylation stabilizes the ARD proteins and reduces their binding to FIH. more...
- Published
- 2009
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35. The structure of an integrin/talin complex reveals the basis of inside-out signal transduction
- Author
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Feng Ye, Nicholas J. Anthis, Kate L. Wegener, Benjamin T. Goult, David R. Critchley, Edward D. Lowe, Ioannis Vakonakis, Iain D. Campbell, Mark H. Ginsberg, Neil Bate, and Chungho Kim
- Subjects
Models, Molecular ,Talin ,Integrins ,animal structures ,Macromolecular Substances ,Molecular Sequence Data ,Integrin ,CHO Cells ,macromolecular substances ,Biology ,Models, Biological ,CD49c ,Article ,General Biochemistry, Genetics and Molecular Biology ,Focal adhesion ,Cell membrane ,Cricetulus ,Cricetinae ,medicine ,Animals ,Amino Acid Sequence ,Cell adhesion ,Molecular Biology ,Sequence Homology, Amino Acid ,General Immunology and Microbiology ,General Neuroscience ,Cell Membrane ,Cell Polarity ,Talin binding ,Protein Structure, Tertiary ,Cell biology ,medicine.anatomical_structure ,Integrin alpha M ,biology.protein ,Integrin, beta 6 ,Protein Binding ,Signal Transduction - Abstract
Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that uniquely mediate inside-out signal transduction, whereby adhesion to the extracellular matrix is activated from within the cell by direct binding of talin to the cytoplasmic tail of the beta integrin subunit. Here, we report the first structure of talin bound to an authentic full-length beta integrin tail. Using biophysical and whole cell measurements, we show that a specific ionic interaction between the talin F3 domain and the membrane-proximal helix of the beta tail disrupts an integrin alpha/beta salt bridge that helps maintain the integrin inactive state. Second, we identify a positively charged surface on the talin F2 domain that precisely orients talin to disrupt the heterodimeric integrin transmembrane (TM) complex. These results show key structural features that explain the ability of talin to mediate inside-out TM signalling. more...
- Published
- 2009
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36. The Structure of the N-Terminus of Kindlin-1: A Domain Important for αIIbβ3 Integrin Activation
- Author
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Igor L. Barsukov, Mohamed Bouaouina, Iain D. Campbell, David A. Calderwood, David S. Harburger, Bipin Patel, Benjamin T. Goult, Nicholas J. Anthis, David R. Critchley, Gordon C. K. Roberts, and Neil Bate more...
- Subjects
Models, Molecular ,Talin ,0302 clinical medicine ,Ezrin ,Protein structure ,Structural Biology ,Radixin ,FACS, fluorescence-activated cell sorting ,MFI, mean fluorescence intensity ,focal adhesion ,GFP, green fluorescent protein ,0303 health sciences ,biology ,FERM domain ,HSQC, heteronuclear single quantum coherence ,Platelet Glycoprotein GPIIb-IIIa Complex ,Cell biology ,Neoplasm Proteins ,Biochemistry ,030220 oncology & carcinogenesis ,FA, focal adhesions ,integrin ,Integrin ,PH, pleckstrin homology ,Molecular Sequence Data ,Sequence alignment ,macromolecular substances ,CHO, Chinese hamster ovary ,Article ,Focal adhesion ,03 medical and health sciences ,NOE, nuclear Overhauser enhancements ,PDB, Protein Data Bank ,NOESY, nuclear Overhauser enhancement spectroscopy ,Protein Interaction Domains and Motifs ,structure ,Amino Acid Sequence ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,kindlin ,Sequence Homology, Amino Acid ,fungi ,F0–F3, FERM subdomains 0–3 ,Membrane Proteins ,Protein Structure, Tertiary ,Mutagenesis, Insertional ,FERM, four-point-one, ezrin, radixin, moesin ,biology.protein - Abstract
The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the beta-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced alphaIIbbeta3 integrin activation and for the localization of kindlin-1 to focal adhesions. more...
- Published
- 2009
37. An Integrin Phosphorylation Switch
- Author
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Edward D. Lowe, Camilla L. Oxley, Nicholas J. Anthis, Ioannis Vakonakis, Iain D. Campbell, and Kate L. Wegener
- Subjects
Phosphotyrosine binding ,Integrin ,macromolecular substances ,Cell Biology ,Plasma protein binding ,Biology ,Biochemistry ,CD49c ,Talin binding ,Cell biology ,Collagen receptor ,Integrin alpha M ,biology.protein ,Integrin, beta 6 ,Molecular Biology - Abstract
Integrins play a fundamental role in cell migration and adhesion; knowledge of how they are regulated and controlled is vital for understanding these processes. Recent work showed that Dok1 negatively regulates integrin activation, presumably by competition with talin. To understand how this occurs, we used NMR spectroscopy and x-ray crystallography to investigate the molecular details of interactions with integrins. The binding affinities of β3 integrin tails for the Dok1 and talin phosphotyrosine binding domains were quantified using 15N-1H hetero-nuclear single quantum correlation titrations, revealing that the unphosphorylated integrin tail binds more strongly to talin than Dok1. Chemical shift mapping showed that unlike talin, Dok1 exclusively interacts with the canonical NPXY motif of the β3 integrin tail. Upon phosphorylation of Tyr747 in the β3 integrin tail, however, Dok1 then binds much more strongly than talin. Thus, we show that phosphorylation of Tyr747 provides a switch for integrin ligand binding. This switch may represent an in vivo mechanism for control of integrin receptor activation. These results have implications for the control of integrin signaling by proteins containing phosphotyrosine binding domains. more...
- Published
- 2008
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38. The Wondrous Cross : Four Sermons on the Theme 'When I Survey the Wondrous Cross'
- Author
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Iain D Campbell and Iain D Campbell
- Subjects
- Devotional literature
- Abstract
Every believer knows that the cross is of great importance, but in the busyness of life and pressing in of so many other issues it can somehow lose its significance. In four chapters Iain D. Campbell takes us back to the cross, using the words of'When I Survey the Wondrous Cross'as a guide. Salvation, assurance, the righteousness of God and justification are all presented; the great love of God demonstrated in the suffering of the Lord Jesus Christ and our necessary response are made clear. It will not take long to read this little book, but the refreshing view of the Saviour and his work it offers will be a rich blessing to all. more...
- Published
- 2014
39. Structural Analysis of the Interactions Between Paxillin LD Motifs and α-Parvin
- Author
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Maria K. Hoellerer, Sonja Lorenz, Iain D. Campbell, Martin E.M. Noble, Edward D. Lowe, and Ioannis Vakonakis
- Subjects
Models, Molecular ,PROTEINS ,Amino Acid Motifs ,Molecular Sequence Data ,Antiparallel (biochemistry) ,Calponin homology domain ,Leucine-Rich Repeat Proteins ,Article ,Focal adhesion ,03 medical and health sciences ,Structural Biology ,Protein Interaction Mapping ,Humans ,Degeneracy (biology) ,Actinin ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Paxillin ,030304 developmental biology ,0303 health sciences ,biology ,Sequence Homology, Amino Acid ,030302 biochemistry & molecular biology ,Microfilament Proteins ,Signal transducing adaptor protein ,3. Good health ,Biochemistry ,biology.protein ,Biophysics ,Linker ,Protein Binding ,Signal Transduction - Abstract
The adaptor protein paxillin contains five conserved leucine-rich (LD) motifs that interact with a variety of focal adhesion proteins, such as alpha-parvin. Here, we report the first crystal structure of the C-terminal calponin homology domain (CH(C)) of alpha-parvin at 1.05 A resolution and show that it is able to bind all the LD motifs, with some selectivity for LD1, LD2, and LD4. Cocrystal structures with these LD motifs reveal the molecular details of their interactions with a common binding site on alpha-parvin-CH(C), which is located at the rim of the canonical fold and includes part of the inter-CH domain linker. Surprisingly, this binding site can accommodate LD motifs in two antiparallel orientations. Taken together, these results reveal an unusual degree of binding degeneracy in the paxillin/alpha-parvin system that may facilitate the assembly of dynamic signaling complexes in the cell. more...
- Published
- 2008
40. Solution Structure and Sugar-Binding Mechanism of Mouse Latrophilin-1 RBL: a 7TM Receptor-Attached Lectin-Like Domain
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Tobias Langenhan, Andreas Russ, Iain D. Campbell, Simone Prömel, and Ioannis Vakonakis
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Models, Molecular ,Receptors, Peptide ,PROTEINS ,Molecular Sequence Data ,Carbohydrates ,Plasma protein binding ,Biology ,Rhamnose ,Article ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,Latrophilin 1 ,Mice ,0302 clinical medicine ,Protein structure ,Structural Biology ,Cell surface receptor ,Lectins ,Animals ,Amino Acid Sequence ,Receptor ,Molecular Biology ,Peptide sequence ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,0303 health sciences ,Sequence Homology, Amino Acid ,Rhamnose binding ,Transmembrane protein ,3. Good health ,Cell biology ,Protein Structure, Tertiary ,Solutions ,Biochemistry ,Amino Acid Substitution ,SIGNALING ,CELLBIO ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Latrophilin-1 (Lat-1), a target receptor for alpha-Latrotoxin, is a putative G protein-coupled receptor implicated in synaptic function. The extracellular portion of Lat-1 contains a rhamnose binding lectin (RBL)-like domain of unknown structure. RBL domains, first isolated from the eggs of marine species, are also found in the ectodomains of other metazoan transmembrane proteins, including a recently discovered coreceptor of the neuronal axon guidance molecule SLT-1/Slit. Here, we describe a structure of this domain from the mouse Lat-1. RBL adopts a unique alpha/beta fold with long structured loops important for monosaccharide recognition, as shown in the structure of a complex with L-rhamnose. Sequence alignments and mutagenesis show that residues important for carbohydrate binding are often absent in other receptor-attached examples of RBL, including the SLT-1/Slit coreceptor. We postulate that this domain class facilitates direct protein-protein interactions in many transmembrane receptors. more...
- Published
- 2008
41. Structure of three tandem filamin domains reveals auto-inhibition of ligand binding
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Pengju Jiang, C.H. Coles, David A. Calderwood, Tiila R. Kiema, Olli T. Pentikäinen, Yatish Lad, Iain D. Campbell, Jari Ylänne, and Organization, European Molecular Biology
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Models, Molecular ,Integrins ,animal structures ,integrin ,Filamins ,Integrin ,macromolecular substances ,Plasma protein binding ,Ligands ,Filamin ,Biochemistry ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Filamin binding ,Contractile Proteins ,Humans ,Binding site ,Cell adhesion ,Cytoskeleton ,Molecular Biology ,X-ray crystallography ,030304 developmental biology ,Integrin binding ,0303 health sciences ,General Immunology and Microbiology ,biology ,General Neuroscience ,Microfilament Proteins ,030302 biochemistry & molecular biology ,cell adhesion ,cytoskeleton ,filamin ,Protein Structure, Tertiary ,Cell biology ,biology.protein ,Protein Binding - Abstract
Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 A resolution structure of a three-domain fragment of human filamin A (IgFLNa19-21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a beta-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20-IgFLNa21 interaction enhances filamin binding to integrin beta-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin-ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure. more...
- Published
- 2007
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42. The Croonian lecture 2006 Structure of the living cell
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Iain D. Campbell
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Models, Molecular ,Structure (mathematical logic) ,0303 health sciences ,cell migration ,Protein Conformation ,Cells ,Review ,Living cell ,Biology ,Data science ,General Biochemistry, Genetics and Molecular Biology ,Toolbox ,03 medical and health sciences ,0302 clinical medicine ,microscopy ,macromolecules ,structure ,General Agricultural and Biological Sciences ,Molecular Biology ,Neuroscience ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
The smallest viable unit of life is a single cell. To understand life, we need to visualize the structure of the cell as well as all cellular components and their complexes. This is a formidable task that requires sophisticated tools. These have developed from the rudimentary early microscopes of 350 years ago to a toolbox that includes electron microscopes, synchrotrons, high magnetic fields and vast computing power. This lecture briefly reviews the development of biophysical tools and illustrates how they begin to unravel the ‘molecular logic of the living state’. more...
- Published
- 2007
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43. The mechanism of cell differentiation in Bacillus subtilis
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Joanna Clarkson, Iain D. Campbell, Michael D. Yudkin, and Dagmar Iber
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Cell type ,Cellular differentiation ,Cell ,Sigma Factor ,Bacillus subtilis ,Biology ,Cell fate determination ,Allosteric Regulation ,Bacterial Proteins ,medicine ,Cell Lineage ,Phosphorylation ,Protein kinase A ,Transcription factor ,Spores, Bacterial ,Multidisciplinary ,Cell Differentiation ,DNA-Directed RNA Polymerases ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,Phosphoric Monoester Hydrolases ,Cell biology ,medicine.anatomical_structure ,Biochemistry ,Holoenzymes ,Protein Kinases ,Sporulation in Bacillus subtilis - Abstract
Sporulation in Bacillus subtilis serves as a model for the development of two different cell types from a single cell. Although much information has been accumulated about the mechanisms that initiate the developmental programmes, important questions remain that can be answered only by quantitative analysis. Here we develop, with the help of existing and new experimental results, a mathematical model that reproduces published in vitro experiments and explains how the activation of the key transcription factor is regulated. The model identifies the difference in volume between the two cell types as the primary trigger for determining cell fate. It shows that this effect depends on the allosteric behaviour of a key protein kinase and on a low rate of dephosphorylation by the corresponding phosphatase; both predicted effects are confirmed experimentally. more...
- Published
- 2006
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44. Structural insight into binding ofStaphylococcus aureusto human fibronectin
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Jennifer R. Potts, Iain D. Campbell, Joern M. Werner, Nicola A. G. Meenan, Ulrich Schwarz-Linek, Ewa S. Pilka, and Andrew R. Pickford
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Staphylococcus aureus ,Biophysics ,Peptide ,Peptide binding ,Fibronectin binding protein A ,medicine.disease_cause ,Biochemistry ,Protein Structure, Secondary ,Bacterial protein ,Structure-Activity Relationship ,Structural Biology ,Genetics ,medicine ,Humans ,Adhesins, Bacterial ,Nuclear Magnetic Resonance, Biomolecular ,Fibronectin ,Molecular Biology ,chemistry.chemical_classification ,biology ,Cell Biology ,NMR ,Fibronectins ,chemistry ,Residual dipolar coupling ,biology.protein ,MSCRAMM ,Peptides ,Protein Binding - Abstract
Staphylococcus aureus possesses cell-wall attached proteins that bind the human protein fibronectin (Fn). An intermodule interface between the 4F1 and 5F1 modules in the N-terminal domain of Fn is maintained on bacterial peptide binding but there is a small change in the intermodule orientation and alignment of β-strands that are predicted to bind the peptide. The module pair is elongated, as in the unbound state. Combined with evidence that residues in both 4F1 and 5F1 are directly involved in peptide binding, this observation supports the hypothesis that, when bound to intact Fn, the bacterial protein adopts an unusual, highly extended conformation. more...
- Published
- 2005
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45. Binding, Domain Orientation, and Dynamics of the Lck SH3−SH2 Domain Pair and Comparison with Other Src-Family Kinases
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Finn Bauer, Gregor Hofmann, Silke Hoffmann, H. Sticht, Kristian Schweimer, Jörn M. Werner, Edith Hofinger, Anke Kiessling, Paul Rösch, and Iain D. Campbell
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animal structures ,chemistry [Proto-Oncogene Proteins c-fyn] ,chemistry [src-Family Kinases] ,macromolecular substances ,Ligands ,Proto-Oncogene Proteins c-fyn ,SH2 domain ,environment and public health ,Biochemistry ,SH3 domain ,src Homology Domains ,HAMP domain ,Protein structure ,FYN ,ddc:570 ,metabolism [Lymphocyte Specific Protein Tyrosine Kinase p56(lck)] ,Humans ,metabolism [Peptides] ,Nuclear Magnetic Resonance, Biomolecular ,Chemistry ,hemic and immune systems ,Protein Structure, Tertiary ,src-Family Kinases ,Protein kinase domain ,Lymphocyte Specific Protein Tyrosine Kinase p56(lck) ,Biophysics ,chemistry [Lymphocyte Specific Protein Tyrosine Kinase p56(lck)] ,FYN protein, human ,Peptides ,Protein Binding ,Proto-oncogene tyrosine-protein kinase Src ,Binding domain - Abstract
The catalytic activity of Src-family kinases is regulated by association with its SH3 and SH2 domains. Activation requires displacement of intermolecular contacts by SH3/SH2 binding ligands resulting in dissociation of the SH3 and SH2 domains from the kinase domain. To understand the contribution of the SH3-SH2 domain pair to this regulatory process, the binding of peptides derived from physiologically relevant SH2 and SH3 interaction partners was studied for Lck and its relative Fyn by NMR spectroscopy. In contrast to Fyn, activating ligands do not induce communication between SH2 and SH3 domains in Lck. This can be attributed to the particular properties of the Lck SH3-SH2 linker which is shown to be extremely flexible thus effectively decoupling the behavior of the SH3 and SH2 domains. Measurements on the SH32 tandem from Lck further revealed a relative domain orientation that is distinctly different from that found in the Lck SH32 crystal structure and in other Src kinases. These data suggest that flexibility between SH2 and SH3 domains contributes to the adaptation of Src-family kinases to specific environments and distinct functions. more...
- Published
- 2005
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46. Towards a Structure for a TSG-6·Hyaluronan Complex by Modeling and NMR Spectroscopy
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David J. Mahoney, Andrew Almond, Charles D. Blundell, Paul L. DeAngelis, Iain D. Campbell, and Anthony J. Day
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chemistry.chemical_classification ,Stereochemistry ,Sequence alignment ,Cell Biology ,Plasma protein binding ,Nuclear magnetic resonance spectroscopy ,Biochemistry ,Amino acid ,Conserved sequence ,Crystallography ,Protein structure ,chemistry ,Molecular Biology ,Peptide sequence ,Ternary complex - Abstract
The Link module from human TSG-6, a hyaladherin with roles in ovulation and inflammation, has a hyaluronan (HA)-binding groove containing two adjacent tyrosine residues that are likely to form CH-π stacking interactions with sequential rings in the sugar. We have used this observation to construct a model of a protein·HA complex, which was then tested against existing experimental information and by acquisition of new NMR data sets of [13C, 15N]HA (8-mer) complexed with unlabeled protein. A major finding of this analysis was that acetamido side chains of two GlcNAc rings fit into hydrophobic pockets on either side of the adjacent tyrosines, providing a selectivity mechanism of HA over other polysaccharides. Furthermore, two basic residues have a separation that matches that of glucuronic acids in the sugar, consistent with the formation of salt bridges; NMR experiments at a range of pH values identified protein groups that titrate due to their proximity to a free carboxylate in HA. Sequence alignment and construction of homology models for all human Link modules in their HA-bound states revealed that many of these features are conserved across the superfamily, thus allowing the prediction of functionally important residues. In the case of cartilage link protein, its two Link modules were docked together (using bound HA as a guide), identifying hydrophobic residues likely to form an intra-Link module interface as well as amino acids that could be involved in supporting intermolecular interactions between link proteins and chondroitin sulfate proteoglycans. Here, we propose a mechanism for ternary complex formation that generates higher order helical structures, as may exist in cartilage aggregates. more...
- Published
- 2005
- Full Text
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47. Engaging with Keller : Thinking Through the Theology of an Influential Evangelical
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Iain D Campbell, William M Schweitzer, Iain D Campbell, and William M Schweitzer
- Abstract
Tim Keller's name is known across the evangelical world. His work as a pastor-teacher has found expression both in the urban ministries of Redeemer Presbyterian Church in New York, and in his many writings. Keller's books, in turn, have spawned Bible study courses and generated a great measure of discussion about key biblical concepts, as he has sought to make the gospel relevant for a modern generation. In this collection of essays, written from within the same evangelical constituency, several writers engage with different aspects of Keller's thought. While indebted to Keller in many ways, they also wish to examine his position in the light of Scripture and to work constructively as well as critically with his published works. That such an influential figure should be the subject of discussion is not surprising; what will be surprising to many is that not all evangelicals are prepared to accept without question all of Keller's conclusions or formulations. This is a book to stimulate discussion and to remind us that God's Word must always be our final judge in matters of theology, evangelism and apologetics. more...
- Published
- 2013
48. Molecular Recognition of Paxillin LD Motifs by the Focal Adhesion Targeting Domain
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Gilles Labesse, Maria K. Hoellerer, Martin E.M. Noble, Jörn M. Werner, Stefan T. Arold, and Iain D. Campbell
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Amino Acid Motifs ,Focal adhesion ,03 medical and health sciences ,Molecular recognition ,Structural Biology ,Homology modeling ,Binding site ,Cytoskeleton ,Molecular Biology ,Paxillin ,030304 developmental biology ,Focal Adhesions ,0303 health sciences ,biology ,Cadherin ,Chemistry ,030302 biochemistry & molecular biology ,Vinculin ,Cadherins ,Phosphoproteins ,Protein Structure, Tertiary ,Cell biology ,Cytoskeletal Proteins ,Biochemistry ,biology.protein ,Peptides - Abstract
Focal adhesions (FAs) are large submembrane signaling complexes formed at sites of cellular attachment to the extracellular matrix. The interaction of LD motifs with their targets plays an important role in the assembly of FAs. We have determined the molecular basis for the recognition of two paxillin LD motifs by the FA targeting (FAT) domain of FA kinase using a combination of X-ray crystallography, solution NMR, and homology modeling. The four-helix FAT domain displays two LD binding sites on opposite sites of the molecule that bind LD peptides in a helical conformation. Threading studies suggest that the LD-interacting domain of p95PKL shares a common four-helical core with the FAT domain and the tail of vinculin, defining a structural family of LD motif binding modules. more...
- Published
- 2003
- Full Text
- View/download PDF
49. Domain-Specific Interactions of Talin with the Membrane-Proximal Region of the Integrin β3 Subunit
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Iain D. Campbell, Tobias S. Ulmer, Mark H. Ginsberg, and David A. Calderwood
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Talin ,biology ,Extracellular matrix assembly ,Cell Membrane ,Integrin ,Integrin beta3 ,Cell migration ,macromolecular substances ,Biochemistry ,CD49c ,Molecular biology ,Collagen receptor ,Cell biology ,Integrin alpha M ,biology.protein ,Animals ,Integrin, beta 6 ,Chickens ,Nuclear Magnetic Resonance, Biomolecular ,Actin ,Protein Binding - Abstract
Activation (affinity regulation) of integrin adhesion receptors controls cell migration and extracellular matrix assembly. Talin connects integrins with actin filaments and influences integrin affinity by binding to the integrins' short cytoplasmic beta-tail. The principal beta-tail binding site in talin is a FERM domain, comprised of three subdomains (F1, F2, and F3). Previous studies of integrin alphaIIbbeta3 have shown that both F2 and F3 bind the beta3 tail, but only F3, or the F2-F3 domain pair, induces activation. Here, talin-induced perturbations of beta3 NMR resonances were examined to explore integrin activation mechanisms. F3 and F2-F3, but not F2, distinctly perturbed the membrane-proximal region of the beta3 tail. All domains also perturbed more distal regions of the beta3 tail that appear to form the major interaction surface, since the beta3(Y747A) mutation suppressed those effects. These results suggest that perturbation of the beta3 tail membrane-proximal region is associated with talin-mediated integrin activation. more...
- Published
- 2003
- Full Text
- View/download PDF
50. SH3-SH2 Domain Orientation in Src Kinases
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Jörn M. Werner, Iain D. Campbell, and Tobias S. Ulmer
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animal structures ,Kinase ,Chemistry ,macromolecular substances ,SH2 domain ,environment and public health ,Crystallography ,FYN ,Structural Biology ,Residual dipolar coupling ,Biophysics ,Active state ,Molecular Biology ,Src family ,Proto-oncogene tyrosine-protein kinase Src - Abstract
The regulatory domains of Src family kinases SH3 and SH2 suppress Src activity when bound to the catalytic domain. Here, the isolated SH3-SH2 fragment from the Src family member Fyn (FynSH32) is studied by NMR. The properties of this fragment are expected to be similar to the domains in the active state, where they are dissociated from the catalytic domain. Crosscommunication between SH3 and SH2 of FynSH32, measured by chemical shift perturbation, was found to be small. Diffusion and alignment anisotropy measurements showed that SH3 and SH2 of peptide-bound FynSH32 are significantly coupled but still exhibit some interdomain flexibility. The observed average domain orientation indicates that a large SH3-SH2 domain closure is required to reach the inactive state. The implications of these results for Src regulation are discussed. more...
- Published
- 2002
- Full Text
- View/download PDF
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