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Paul N Barlow

Publications and source records attributed to Paul N Barlow.

At least 19 recordsLinked to original sources
PubMed2006

Critical role of the C-terminal domains of factor H in regulating complement activation at cell surfaces.

The plasma protein factor H primarily controls the activation of the alternative pathway of complement. The C-terminal of factor H is known to be involved in protection of host cells from complement attack. In the present study, we show that domains 19-20 alone are capable of discriminating between host-like and complement-activating cells. Furthermore, although factor H possesses three binding sites for C3b, binding to cell-bound C3b can be almost completely inhibited by the single site located in domains 19-20. All of the regulatory activities of factor H are expressed by the N-terminal four domains, but these activities toward cell-bound C3b are inhibited by isolated recombinant domains 19-20 (rH 19-20). Direct competition with the N-terminal site is unlikely to explain this because regulation of fluid phase C3b is unaffected by domains 19-20. Finally, we show that addition of isolated rH 19-20 to normal human serum leads to aggressive complement-mediated lysis of normally nonactivating sheep erythrocytes and moderate lysis of human erythrocytes, which possess membrane-bound regulators of complement. Taken together, the results highlight the importance of the cell surface protective functions exhibited by factor H compared with other complement regulatory proteins. The results may also explain why atypical hemolytic uremic syndrome patients with mutations affecting domains 19-20 can maintain complement homeostasis in plasma while their complement system attacks erythrocytes, platelets, endothelial cells, and kidney tissue.

Animals↗
PubMed2006

Disease-associated sequence variations congregate in a polyanion recognition patch on human factor H revealed in three-dimensional structure.

Mutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site.

Amino Acid Sequence↗
PubMed2005

Human C4b-binding protein, structural basis for interaction with streptococcal M protein, a major bacterial virulence factor.

Human C4b-binding protein (C4BP) protects host tissue, and those pathogens able to hijack this plasma glycoprotein, from complement-mediated destruction. We now show that the first two complement control protein (CCP) modules of the C4BP alpha-chain, plus the four residues connecting them, are necessary and sufficient for binding a bacterial virulence factor, the Streptococcus pyogenes M4 (Arp4) protein. Structure determination by NMR reveals two tightly coupled CCP modules in an elongated arrangement within this region of C4BP. Chemical shift perturbation studies demonstrate that the N-terminal, hypervariable region of M4 binds to a site including strand 1 of CCP module 2. This interaction is accompanied by an intermodular reorientation within C4BP. We thus provide a detailed picture of an interaction whereby a pathogen evades complement.

Amino Acid Sequence↗
PubMed2005

Structure of free MDM2 N-terminal domain reveals conformational adjustments that accompany p53-binding.

Critical to the inhibitory action of the oncogene product, MDM2, on the tumour suppressor, p53, is association of the N-terminal domain of MDM2 (MDM2N) with the transactivation domain of p53. The structure of MDM2N was previously solved with a p53-derived peptide, or small-molecule ligands, occupying its binding cleft, but no structure of the non-liganded MDM2N (i.e. the apo-form) has been reported. Here, we describe the solution structure and dynamics of apo-MDM2N and thus reveal the nature of the conformational changes in MDM2N that accompany binding of p53. The new structure suggests that p53 effects displacement of an N-terminal segment of apo-MDM2N that occludes access to the shallow end of the p53-binding cleft. MDM2N must also undergo an expansion upon binding, achieved through a rearrangement of its two pseudosymetrically related sub-domains resulting in outward displacements of the secondary structural elements that comprise the walls and floor of the p53-binding cleft. MDM2N becomes more rigid and stable upon binding p53. Conformational plasticity of the binding cleft of apo-MDM2N could allow the parent protein to bind specifically to several different partners, although, to date, all the known liganded structures of MDM2N are highly similar to one another. The results indicate that the more open conformation of the binding cleft of MDM2N observed in structures of complexes with small molecules and peptides is a more suitable one for ligand discovery and optimisation.

Animals↗
PubMed2005

Large-scale modelling as a route to multiple surface comparisons of the CCP module family.

Numerous mammalian proteins are constructed from a limited repertoire of module-types. Proteins belonging to the regulators of complement activation family--crucial for ensuring a complement-mediated immune response is targeted against infectious agents--are composed solely of complement control protein (CCP) modules. In the current study, CCP module sequences were grouped to allow selection of the most appropriate experimentally determined structures to serve as templates in an automated large-scale structure modelling procedure. The resulting 135 individual CCP module models, valuable in their own right, are available at the online database http://www.bru.ed.ac.uk/~dinesh/ccp-db.html. Comparisons of surface properties within a particular family of modules should be more informative than sequence alignments alone. A comparison of surface electrostatic features was undertaken for the first 28 CCP modules of complement receptor type 1 (CR1). Assignments to clusters based on surface properties differ from assignments to clusters based on sequences. This observation might reflect adaptive evolution of surface-exposed residues involved in protein-protein interactions. This illustrative example of a multiple surface-comparison was indeed able to pinpoint functional sites in CR1.

Amino Acid Sequence↗
PubMed2005

Polychromatic selective population inversion for TROSY experiments with large proteins.

This paper presents polychromatic selective polarization inversion (PC-SPI) as an alternative to the polarization transfer methods recently developed for the application of NMR to large biological molecules. Theoretical and numerical considerations indicate that PC-SPI has the potential for more efficient polarization transfer under conditions of rapid transverse relaxation compared to J coupling- and cross-correlated relaxation-based transfers. The main advantage offered by the method presented here is the maintenance of near-optimal trajectories of inversion of the individual components of the spin magnetization while using broadband optimized pulses. A 2D experiment was implemented combining PC-SPI with TROSY-based chemical shift correlation. The experiment was applied to detect (15)N-(1)H chemical shift correlation spectra of a 200 kDa complex consisting of an 80% (2)H- and uniformly (15)N,(13)C-labeled 22 kDa portion of complement receptor type 1 and unlabeled C3b of complement (180 kDa).

Binding Sites↗
PubMed2005

Opportunities for new therapies based on the natural regulators of complement activation.

While the complement system is an essential component of immunity, shutting down all or part of it could be beneficial in a wide range of clinical situations. Designer, small-molecule, protease inhibitors and antagonists of protein-protein interactions are under development, while an approach based on a humanized monoclonal antibody to the C5 component works effectively against the later stages of complement activation and is close to completing clinical trials. The cobra venom factor depletes plasma of essential complement components, and a humanized (nonimmunogenic) version is being sought. Perhaps the most promising approach to comprehensive complement downregulation, however, is the exploitation of innate regulators of complement activation, with two products in clinical trials. The potential for more efficacious complement blockers of this kind is growing because of better targeting, but a deeper knowledge at the atomic level of mechanisms of action of these regulators is needed to underpin a rational approach to design of still more potent complement inhibitors.

Complement Activation↗
PubMed2004

Functional insights from the structure of the multifunctional C345C domain of C5 of complement.

The complement protein C5 initiates assembly of the membrane attack complex. This remarkable process results in lysis of target cells and is fundamental to mammalian defense against infection. The 150-amino acid residue domain at the C terminus of C5 (C5-C345C) is pivotal to C5 function. It interacts with enzymes that convert C5 to C5b, the first step in the assembly of the membrane attack complex; it also binds to the membrane attack complex components C6 and C7 with high affinity. Here a recombinant version of this C5-C345C domain is shown to adopt the oligosaccharide/oligonucleotide binding fold, with two helices packed against a five-stranded beta-barrel. The structure is compared with those from the netrin-like module family that have a similar fold. Residues critical to the interaction with C5-convertase cluster on a mobile, hydrophobic inter-strand loop that protrudes from the open face of the beta-barrel. The opposite, helix-dominated face of C5-C345C carries a pair of exposed hydrophobic side chains adjacent to a striking negatively charged patch, consistent with affinity for positively charged factor I modules in C6 and C7. Modeling of homologous domains from complement proteins C3 and C4, which do not participate in membrane attack complex assembly, suggests that this provisionally identified C6/C7-interacting face is indeed specific to C5.

Amino Acid Sequence↗
PubMed2004

An atomic resolution model for assembly, architecture, and function of the Dr adhesins.

Pathogenic bacteria possess adhesion protein complexes that play essential roles in targeting host cells and in propagating infection. Although each family of adhesion proteins is generally associated with a specific human disease, the Dr family from Escherichia coli is a notable exception, as its members are associated with both diarrheal and urinary tract infections. These proteins are reported to form both fimbrial and afimbrial structures at the bacterial cell surface and target a common host cell receptor, the decay-accelerating factor (DAF or CD55). Using the newly solved three-dimensional structure of AfaE, we have constructed a robust atomic resolution model that reveals the structural basis for assembly by donor strand complementation and for the architecture of capped surface fibers.

Adhesins, Escherichia coli↗
PubMed2004

Structural analysis of the complement control protein (CCP) modules of GABA(B) receptor 1a: only one of the two CCP modules is compactly folded.

The gamma-aminobutyric acid type B (GABA(B)) receptor is a heterodimeric G-protein-coupled receptor. In humans, three splice variants of the GABA(B) receptor 1 (R1) subunit differ in having one, both, or neither of two putative complement control protein (CCP) modules at the extracellular N terminus, prior to the GABA-binding domain. The in vivo function of these predicted modules remains to be discovered, but a likely association with extracellular matrix proteins is intriguing. The portion of the GABA(B) R1a variant encompassing both of its CCP module-like sequences has been expressed, as have the sequences corresponding to each individual module. Each putative CCP module exhibits the expected pattern of disulfide formation. However, the second module (CCP2) is more compactly folded than the first, and the three-dimensional structure of this more C-terminal module (expressed alone) was solved on the basis of NMR-derived nuclear Overhauser effects. This revealed a strong similarity to previously determined CCP module structures in the regulators of complement activation. The N-terminal module (CCP1) displayed conformational heterogeneity under a wide range of conditions whether expressed alone or together with CCP2. Several lines of evidence indicated the presence of native disorder in CCP1, despite the fact that recombinant CCP1 contributes to binding to the extracellular matrix protein fibulin-2. Thus, we have shown that the two CCP modules of GABA(B) R1a have strikingly different structural properties, reflecting their different functions.

Animals↗
PubMed2004

Backbone dynamics of complement control protein (CCP) modules reveals mobility in binding surfaces.

The regulators of complement activation (RCA) are critical to health and disease because their role is to ensure that a complement-mediated immune response to infection is proportionate and targeted. Each protein contains an uninterrupted array of from four to 30 examples of the very widely occurring complement control protein (CCP, or sushi) module. The CCP modules mediate specific protein-protein and protein-carbohydrate interactions that are key to the biological function of the RCA and, paradoxically, provide binding sites for numerous pathogens. Although structural and mutagenesis studies of CCP modules have addressed some aspects of molecular recognition, there have been no studies of the role of molecular dynamics in the interaction of CCP modules with their binding partners. NMR has now been used in the first full characterization of the backbone dynamics of CCP modules. The dynamics of two individual modules-the 16th of the 30 modules of complement receptor type 1 (CD35), and the N-terminal module of membrane cofactor protein (CD46)-as well as their solution structures, are compared. Although both examples share broadly similar three-dimensional structures, many structurally equivalent residues exhibit different amplitudes and timescales of local backbone motion. In each case, however, regions of the module-surface implicated by mutagenesis as sites of interactions with other proteins include several mobile residues. This observation suggests further experiments to explore binding mechanisms and identify new binding sites.

Amino Acid Sequence↗
PubMed2003

Solution structure of a functionally active fragment of decay-accelerating factor.

The second and third modules of human decay accelerating factor (DAF) are necessary and sufficient to accelerate decay of the classical pathway (CP) convertase of complement. No structure of a mammalian protein with decay-accelerating activity has been available to date. We therefore determined the solution structure of DAF modules 2 and 3 (DAF approximately 2,3). Structure-guided analysis of 24 mutants identified likely contact points between DAF and the CP convertase. Three (R96, R69, and a residue in the vicinity of L171) lie on DAF approximately 2,3's concave face. A fourth, consisting of K127 and nearby R100, is on the opposite face. Regions of module 3 remote from the semiflexible 2-3 interface seem not to be involved in binding to the CP convertase. DAF thus seems to occupy a groove on the CP convertase such that both faces of DAF close to the 2-3 junction (including a positively charged region that encircles the protein at this point) interact simultaneously. Alternative pathway convertase interactions with DAF require additional regions of CCP 3 lying away from the 2-3 interface, consistent with the established additional requirement of module 4 for alternative pathway regulation.

Binding Sites↗
PubMed2002

Vaccinia virus complement control protein is monomeric, and retains structural and functional integrity after exposure to adverse conditions.

Vaccinia virus complement control protein (VCP) possesses the ability to inhibit both classical and alternative pathways of complement activation, as well as bind to heparin or heparan sulfate proteoglycans, making it a unique multifunctional protein with therapeutic potential. Recently, the structure of the complete molecule of VCP was determined by X-ray crystallography. Two or three VCP molecules were packed within the unit cells of both crystal forms. Using gel filtration, VCP has now been shown to exist as a monomer in solution. To test the stability of this molecule, VCP was studied by nuclear magnetic resonance (NMR) over a range of temperatures and by differential scanning calorimetry (DSC). It was also subjected to adverse physical conditions, including, freeze-thawing, changes in pH, changes in temperature, and storage at room temperature. VCP melts fully reversibly, and it maintained its 3-D structure and the ability to inhibit serum-induced hemolysis of sheep red blood cells after exposure to many extreme conditions. The robustness of VCP may be rationalized in terms of its architecture.

Animals↗
PubMed2002

Structure of the C3b binding site of CR1 (CD35), the immune adherence receptor.

Complement receptor type 1 (CR1 or CD35) is a multiple modular protein that mediates the immune adherence phenomenon, a fundamental event for destroying microbes and initiating an immunological response. It fulfills this role through binding C3b/C4b-opsonized foreign antigens. The structure of the principal C3b/C4b binding site (residues 901-1095) of CR1 is reported, revealing three complement control protein modules (modules 15-17) in an extended head-to-tail arrangement with flexibility at the 16-17 junction. Structure-guided mutagenesis identified a positively charged surface region on module 15 that is critical for C4b binding. This patch, together with basic side chains of module 16 exposed on the same face of CR1, is required for C3b binding. These studies reveal the initial structural details of one of the first receptor-ligand interactions to be identified in immunobiology.

Binding Sites↗