Biomedical subjects

James M McDonnell

Publications and source records attributed to James M McDonnell.

10 recordsLinked to original sources

PubMed2006

The intrinsic flexibility of IgE and its role in binding FcepsilonRI.

The interaction between IgE and its high affinity cellular receptor (FcepsilonRI) is an essential step in the development of allergic responses. Studies have identified the third constant domain of IgE (Cepsilon3) as the receptor binding region. The Cepsilon3 domain has unusual structural features; it was found to be a 'molten globule' structure in an isolated form, only assuming a well structured form upon binding to FcepsilonRI. The conformational flexibility intrinsic to the receptor binding portion of the molecule may be useful to IgE in allowing the large allosteric changes postulated to be required for FcepsilonRI engagement. If allosteric inhibitors can be developed then the dynamic properties of the Cepsilon3 domain may provide opportunities for therapeutic intervention in allergic disorders.

Binding Sites↗

PubMed2006

Cancer-associated glycoforms of gelatinase B exhibit a decreased level of binding to galectin-3.

Gelatinase B (MMP-9) and galectin-3 are widely known to participate in tumor cell invasion and metastasis. Glycans derived from MMP-9 expressed in MCF-7 breast cancer and THP-1 myeloid leukemia cells were compared with those from MMP-9 expressed in natural neutrophils. The many O-linked glycans of neutrophil gelatinase B presented a cluster of mainly galactosylated core II structures, 46% of which were ligands for galectin-3; 11% contained two to three N-acetyllactosamine repeating units that are high-affinity ligands for the lectin. The glycan epitopes thus provide MMP-9 with both high-affinity and (presumably) high-avidity interactions with galectin-3. In contrast, the O-glycans released from MMP-9 expressed in MCF-7 and THP-1 cells were predominantly sialylated core I structures. Only 10% of MCF-7 and THP-1 gelatinase B O-glycans were ligands for galectin-3 and contained only a maximum single N-acetyllactosamine repeat. Consistent with the glycan analysis, surface plasmon resonance binding assays indicated that the cancer-associated glycoforms of MMP-9 bound galectin-3 with an affinity and avidity significantly reduced compared with those of the natural neutrophil MMP-9. Galectin-3 exists as a multimer that also binds laminin, providing a means of localizing neutrophil MMP-9 in the extracellular matrix (ECM). The analytical data presented here suggest that MMP-9 glycoforms secreted by tumor cells are unlikely to be tethered at the site of secretion, thus promoting more extensive cleavage of the ECM and providing a rationale for the contribution that gelatinase B makes to cancer cell metastasis.

Animals↗

PubMed2006

Direct analysis of a GPCR-agonist interaction by surface plasmon resonance.

Despite their clinical importance, detailed analysis of ligand binding at G-protein coupled receptors (GPCRs) has proved difficult. Here we successfully measure the binding of a GPCR, neurotensin receptor-1 (NTS-1), to its ligand, neurotensin (NT), using surface plasmon resonance (SPR). Specific responses were observed between NT and purified, detergent-solublised, recombinant NTS-1, using a novel configuration where the biotinylated NT ligand was immobilised on the biosensor surface. This SPR approach shows promise as a generic approach for the study of ligand interactions with other suitable GPCRs.

Animals↗

PubMed2006

Catalytic folding of the Cepsilon3 domain by its high affinity receptor.

The interaction of immunoglobulin E (IgE) with its cellular receptor FcepsilonRIalpha is a central regulator of allergy. Structural studies have identified the third domain (Cepsilon3) of the constant region of epsilon heavy chain as the receptor binding region. The isolated Cepsilon3 domain is a "molten globule" that becomes structured upon binding of the FcepsilonRIalpha ligand. In this study, fluorescence and nuclear magnetic resonance spectroscopies are used to characterise the role of soluble FcepsilonRIalpha in the folding of the monomeric Cepsilon3 domain of IgE. Soluble FcepsilonRIalpha is shown to display characteristic properties of a catalyst for the folding of Cepsilon3, with the rate of Cepsilon3 folding being dependent on the concentration of the receptor.

Anilino Naphthalenesulfonates↗

PubMed2006

NMR determination that an extended BH3 motif of pro-apoptotic BID is specifically bound to BCL-XL.

The BH3 motif of the pro-survival family of proteins, BCL, is also present in pro-apoptotic proteins like BID and BAX. Homo- and hetero-oligomerization interactions of the BH3 motif are generally recognized as the critical component of their apoptotic activities. In full-length BID, the putative hydrophobic binding surface of its BH3 motif is substantially occluded by intramolecular contacts, many of which are removed on BID's transformation to tBID by cleavage with caspase 8, required for tBID's pro-apoptotic action on mitochondria, thereby releasing cytochrome c.

Amino Acid Motifs↗

PubMed2005

The structure of human CD23 and its interactions with IgE and CD21.

The low-affinity immunoglobulin E (IgE) receptor, CD23 (FcepsilonRII), binds both IgE and CD21 and, through these interactions, regulates the synthesis of IgE, the antibody isotype that mediates the allergic response. We have determined the three-dimensional structure of the C-type lectin domain of CD23 in solution by nuclear magnetic resonance spectroscopy. An analysis of concentration-dependent chemical shift perturbations have allowed us to identify the residues engaged in self-association to the trimeric state, whereas ligand-induced changes have defined the binding sites for IgE and CD21. The results further reveal that CD23 can bind both ligands simultaneously. Despite the C-type lectin domain structure, none of the interactions require calcium. We also find that IgE and CD23 can interact to form high molecular mass multimeric complexes. The interactions that we have described provide a solution to the paradox that CD23 is involved in both up- and down-regulation of IgE and provide a structural basis for the development of inhibitors of allergic disease.

Binding Sites↗

PubMed2005

Structural characterization of Ca2+/CaM in complex with the phosphorylase kinase PhK5 peptide.

Phosphorylase kinase (PhK) is a large hexadecameric enzyme consisting of four copies of four subunits: (alphabetagammadelta)4. An intrinsic calmodulin (CaM, the delta subunit) binds directly to the gamma protein kinase chain. The interaction site of CaM on gamma has been localized to a C-terminal extension of the kinase domain. Two 25-mer peptides derived from this region, PhK5 and PhK13, were identified previously as potential CaM-binding sites. Complex formation between Ca2+/CaM with these two peptides was characterized using analytical gel filtration and NMR methods. NMR chemical shift perturbation studies showed that while PhK5 forms a robust complex with Ca2+/CaM, no interactions with PhK13 were observed. 15N relaxation characteristics of Ca2+/CaM and Ca2+/CaM/PhK5 complexes were compared with the experimentally determined structures of several Ca2+/CaM/peptide complexes. Good fits were observed between Ca2+/CaM/PhK5 and three structures: Ca2+/CaM complexes with peptides from endothelial nitric oxide synthase, with smooth muscle myosin light chain kinase and CaM kinase I. We conclude that the PhK5 site is likely to have a direct role in Ca2+-regulated control of PhK activity through the formation of a classical 'compact' CaM complex.

Amino Acid Sequence↗

PubMed2004

The key role of protein flexibility in modulating IgE interactions.

The interaction between IgE and its high affinity receptor (FcepsilonRI) is a critical step in the development of allergic responses. Detailed characterization of the IgE-FcepsilonRI interaction may offer insights into possible modes of inhibiting the interaction, which could thereby act as a potential therapy for allergy. In this study, NMR, CD, and fluorescence spectroscopies have been used to characterize structurally the Cepsilon3 domain of IgE and its interaction with other protein ligands, namely, Cepsilon2, Cepsilon4, sFcepsilonRIalpha, and CD23. We have shown that the recombinant Cepsilon3 domain exists alone in solution as a "molten globule." On interaction with sFcepsilonRIalpha, Cepsilon3 adopts a folded tertiary structure, as shown by the release of the fluorescent probe 8-anilinonaphthalene-1-sulfonate and by characteristic changes in the (1)H, (15)N heteronuclear single quantum coherence NMR spectrum. However, the interactions between the Cepsilon3 domain and Cepsilon2, Cepsilon4, or CD23 do not induce such folding and would therefore be expected to involve only local interaction surfaces. The conformational flexibility of the Cepsilon3 domain of the whole IgE molecule may play a role in allowing fine tuning of the affinity and specificity of IgE for a variety of different physiological ligands and may be involved in the conformational change of IgE postulated to occur on interaction with FcepsilonRI.

Base Sequence↗

PubMed2004

Screening and optimizing protein production in E. coli.

Significant improvements in the technologies used for protein production have been driven by impending genome-scale proteomics projects. These initiatives have favored Escherichia coli-based expression systems, which allow rapid cloning and expression of proteins at low cost. The range of commercially available molecular biology kits, vectors, affinity tags, and host cell lines have increased dramatically in recent years. For the structural biology community, where protein production is often a rate-limiting step, these developments have made the process of producing and purifying large amounts of protein for structural studies simpler and faster. The large-scale automated screening approaches for optimizing protein production employed by structural genomics initiatives can be adapted to a more practical targeted approach appropriate for individual structural biology groups. This chapter describes simple, rapid screening methods for testing optimal vector/host combinations using a 96-well format.

Cloning, Molecular↗

PubMed2002

Mutagenesis within human FcepsilonRIalpha differentially affects human and murine IgE binding.

Soluble fragments of the alpha-chain of FcepsilonRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcepsilonRIalpha is used in this study, in conjunction with the crystal structure of the FcepsilonRIalpha/IgE complex, to define the relative importance of specific residues within human FcepsilonRIalpha for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcepsilonRIalpha and attenuate allergic disease. Three residues within the C-C' region of the FcepsilonRIalpha2 domain and two residues within the alpha2 proximal loops of the alpha1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three alpha2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in alpha1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the alpha1/alpha2 interface. Our results demonstrate that the effects of mutations in human FcepsilonRIalpha on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.

Animals↗