33 results on '"Perkins, Stephen J."'
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2. Chapter 6 X-Ray and Neutron Solution Scattering
- Author
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Perkins, Stephen J., primary
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- 1985
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3. THE STRUCTURE OF UBIQUINOL:CYTOCHROME REDUCTASE FROM NEUROSPORA MITOCHONDRIA
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WEISS, HANNS, primary, PERKINS, STEPHEN J., additional, and LEONARD, KEVIN, additional
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- 1983
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4. An updated interactive database for 1692 genetic variants in coagulation factor IX provides detailed insights into hemophilia B.
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Xu Z, Spencer HJ, Harris VA, and Perkins SJ
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- Humans, Mutation, Polymorphism, Genetic, Phenotype, Factor IX genetics, Factor IX chemistry, Hemophilia B diagnosis, Hemophilia B genetics
- Abstract
Background: Genetic variants in coagulation factor IX (FIX) are associated with hemophilia B, a rare bleeding disease. F9 variants are widespread across the gene and were summarized in our FIX variant database introduced in 2013., Objectives: We aimed to rationalize the molecular basis for 598 new F9 variants and 1645 new clinical cases, totaling 1692 F9 variants and 5358 related patient cases., Methods: New F9 variants were identified from publications and online resources, and compiled into a MySQL database for comparison with the human FIXa protein structure., Results: The new total of 1692 F9 variants correspond to 406 (88%) of the 461 FIX residues and now include 70 additional residues. They comprise 945 unique point variants, 281 deletions, 352 polymorphisms, 63 insertions, and 51 others. Most FIX variants were point variants, although their proportion (56%) has reduced compared to 2013 (73%); at the same time, the proportion of polymorphisms has increased from 5% to 21%. The 764 unique mild severity variants in the mature protein with known phenotypes include 74 (9.7%) quantitative type I variants and 116 (15.2%) predominantly qualitative type II variants. The remaining 574 variants types are unspecified. Inhibitors are associated with 152 hemophilia B cases out of 5358 patients (2.8%), an increase of 93 from the previous database., Conclusion: The even distribution of the F9 variants revealed few mutational hotspots, and most variants were associated with small perturbations in the FIX protein structure. The updated database will assist clinicians and researchers in assessing treatments for patients with hemophilia B., Competing Interests: Declaration of competing interest The authors state that they have no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2023
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5. An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics, and smFRET Identifies an Aggregation Mechanism.
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Codina N, Hilton D, Zhang C, Chakroun N, Ahmad SS, Perkins SJ, and Dalby PA
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- Cloning, Molecular, Hydrogen-Ion Concentration, Immunoglobulin Fab Fragments genetics, Immunoglobulin Fab Fragments metabolism, Kinetics, Mutagenesis, Site-Directed, Protein Unfolding, Sequence Alignment, Fluorescence Resonance Energy Transfer methods, Immunoglobulin Fab Fragments chemistry, Molecular Dynamics Simulation, Protein Aggregation, Pathological, Protein Conformation, Scattering, Small Angle, X-Ray Diffraction methods
- Abstract
Protein aggregation is the underlying cause of many diseases, and also limits the usefulness of many natural and engineered proteins in biotechnology. Better mechanistic understanding and characterization of aggregation-prone states is needed to guide protein engineering, formulation, and drug-targeting strategies that prevent aggregation. While several final aggregated states-notably amyloids-have been characterized structurally, very little is known about the native structural conformers that initiate aggregation. We used a novel combination of small-angle x-ray scattering (SAXS), atomistic molecular dynamic simulations, single-molecule Förster resonance energy transfer, and aggregation-prone region predictions, to characterize structural changes in a native humanized Fab A33 antibody fragment, that correlated with the experimental aggregation kinetics. SAXS revealed increases in the native state radius of gyration, R
g , of 2.2% to 4.1%, at pH 5.5 and below, concomitant with accelerated aggregation. In a cutting-edge approach, we fitted the SAXS data to full MD simulations from the same conditions and located the conformational changes in the native state to the constant domain of the light chain (CL ). This CL displacement was independently confirmed using single-molecule Förster resonance energy transfer measurements with two dual-labeled Fabs. These conformational changes were also found to increase the solvent exposure of a predicted APR, suggesting a likely mechanism through which they promote aggregation. Our findings provide a means by which aggregation-prone conformational states can be readily determined experimentally, and thus potentially used to guide protein engineering, or ligand binding strategies, with the aim of stabilizing the protein against aggregation., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)- Published
- 2019
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6. The solution structure of rabbit IgG accounts for its interactions with the Fc receptor and complement C1q and its conformational stability.
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Rayner LE, Kadkhodayi-Kholghi N, Heenan RK, Gor J, Dalby PA, and Perkins SJ
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- Animals, Neutron Diffraction, Protein Binding, Protein Conformation, Protein Multimerization, Rabbits, Scattering, Small Angle, Ultracentrifugation, X-Ray Diffraction, Complement C1q metabolism, Immunoglobulin G chemistry, Immunoglobulin G metabolism, Receptors, Fc metabolism
- Abstract
Solution structures for antibodies are critical to understand function and therapeutic applications. The stability of the solution structure of rabbit IgG in different buffers and temperatures was determined by analytical ultracentrifugation and X-ray and neutron scattering. Rabbit IgG showed a principally monomeric species, which is well resolved from small amounts of a dimeric species. The proportion of dimer increased with increased concentration, decreased temperature and heavy water from 8% to 25% in all buffers except for high salt (250 mM NaCl). The Guinier X-ray radius of gyration R(G) likewise increased with concentration in 137 mM NaCl buffer but was unchanged in 250 mM NaCl buffer. The Guinier neutron R(G) values increased as the temperature decreased. The X-ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2, whose positions did not change with concentration to indicate unchanged structures under all these conditions. The maximum dimension increased with concentration because of dimer formation. Constrained scattering modeling reproducibly revealed very similar asymmetric solution structures for monomeric rabbit IgG in different buffers, in which the Fab-Fc and Fab-Fab pairs were separated by maximally extended hinge structures. The dimer was best modeled by two pairs of Fab regions forming tip-to-tip contacts. The intact rabbit IgG structures explained the ability of its two ligands, the Fc receptor and complement C1q, to bind to the top of its Fc region that is fully accessible and unhindered by the Fab regions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
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- 2013
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7. Solution structure of TT30, a novel complement therapeutic agent, provides insight into its joint binding to complement C3b and C3d.
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Li K, Gor J, Holers VM, Storek MJ, and Perkins SJ
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- Amino Acid Sequence, Animals, Binding Sites, CHO Cells, Complement C3b metabolism, Complement C3d metabolism, Cricetinae, Crystallography, X-Ray, Ligands, Models, Molecular, Molecular Sequence Data, Protein Conformation, Receptors, Complement 3d chemistry, Receptors, Complement 3d genetics, Receptors, Complement 3d metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Solutions, Ultracentrifugation, Complement C3b chemistry, Complement C3d chemistry, Recombinant Proteins chemistry
- Abstract
A novel therapeutic reagent TT30 was designed to be effective in diseases of the alternative pathway of complement such as paroxysmal nocturnal hemoglobinuria and other diseases. TT30 is constructed from the first four short complement regulator (SCR) domains of complement receptor type 2 (CR2) that bind to complement C3d, followed by the first five SCR domains of complement factor H that bind to complement C3b. In order to assess how TT30 binds to C3d and C3b, we determined the TT30 solution structure by a combination of analytical ultracentrifugation, X-ray scattering and constrained modeling. The sedimentation coefficients and radius of gyration of TT30 were unaffected by citrate or phosphate-buffered saline buffers and indicate an elongated monomeric structure with a sedimentation coefficient of 3.1 S and a radius of gyration R(G) of 6.9 nm. Molecular modeling starting from 3000 randomized TT30 conformations showed that high-quality X-ray curve fits were obtained with extended SCR arrangements, showing that TT30 has a limited degree of inter-SCR flexibility in its solution structure. The best-fit TT30 structural models are readily merged with the crystal structure of C3b to show that the four CR2 domains extend freely into solution when the five complement factor H domains are bound within C3b. We reevaluated the solution structure of the CR2-C3d complex that confirmed its recent crystal structure. This recent CR2-C3d crystal structure showed that TT30 is able to interact readily with C3d ligands in many orientations when TT30 is bound to C3b., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
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- 2012
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8. Complement factor H-ligand interactions: self-association, multivalency and dissociation constants.
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Perkins SJ, Nan R, Li K, Khan S, and Miller A
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- C-Reactive Protein metabolism, Complement Activation, Heparin metabolism, Humans, Ligands, Protein Binding, Zinc metabolism, Complement C3b metabolism, Complement Factor H metabolism, Complement Pathway, Alternative immunology
- Abstract
Factor H (FH) is the major plasma regulator of the central complement protein C3b in the alternative pathway of complement activation. The elucidation of the FH interactions with five major ligands (below) is complicated by their weak μM dissociation constants K(D) and FH multivalency. We present the first survey of all the K(D) values for the major FH-ligand interactions and critically review their physiological significance. (i) FH self-association is presently well-established. We review multiple data sets that show that 5-14% of FH is self-associated in physiological conditions. FH self-association is significant for both laboratory investigations and physiological function.(ii) The FH-C3b complex shows low M affinity, meaning that the complex is not fully formed in plasma. In addition, C3, its hydrolysed form C3u, and its cleaved forms C3b and C3d show multimerisation. Current data favour a model when two C3b molecules bind independently to one FH molecule, as opposed to a1:1 stoichiometry where FH wraps itself around C3b.(iii) Heparin is often used as an analogue of the polyanionic host cell surface. The FH-heparin complex also shows a low M affinity, again meaning that complexes are not fully formed in vivo. The oligomeric FH-heparin complexes clarify a two-site interaction model of FH with host-cell surfaces.(iv) Reinvestigation of the FH and C-reactive protein (CRP) interaction revealed that this can only occur in plasma when CRP levels are elevated during acute-phase conditions. Given that CRP binds more weakly to the His402 allotype of FH than the Tyr402 allotype, this suggested a link with age-related macular degeneration (AMD).(v) FH activity is inhibited by zinc, which causes FH to aggregate strongly. High levels of bioavailable zinc occur in sub-retinal pigment epithelial deposits which lead to AMD. Excess zinc binds weakly to a central region of FH, explaining how zinc inhibits FH regulation of C3b., (Copyright © 2011 Elsevier GmbH. All rights reserved.)
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- 2012
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9. Zinc binding to the Tyr402 and His402 allotypes of complement factor H: possible implications for age-related macular degeneration.
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Nan R, Farabella I, Schumacher FF, Miller A, Gor J, Martin AC, Jones DT, Lengyel I, and Perkins SJ
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- Amino Acid Sequence, Base Sequence, Binding Sites, Complement Factor H genetics, Humans, Models, Molecular, Molecular Sequence Data, Protein Binding, Retinal Drusen physiopathology, Scattering, Radiation, X-Rays, Complement Factor H chemistry, Histidine chemistry, Macular Degeneration metabolism, Tyrosine chemistry, Zinc chemistry
- Abstract
The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD. Heterozygous FH is inhibited by zinc, which causes FH to aggregate. Here, zinc binding to homozygous FH was studied. By analytical ultracentrifugation, large amounts of oligomers were observed with both the native Tyr402 and the AMD-risk His402 homozygous allotypes of FH and both the recombinant SCR-6/8 allotypes with Tyr/His402. X-ray scattering also showed that both FH and SCR-6/8 allotypes strongly aggregated at >10 μM zinc. The SCR-1/5 and SCR-16/20 fragments were less likely to bind zinc. These observations were supported by bioinformatics predictions. Starting from known zinc binding sites in crystal structures, we predicted 202 putative partial surface zinc binding sites in FH, most of which were in SCR-6. Metal site prediction web servers also suggested that SCR-6 and other domains bind zinc. Predicted SCR-6/8 dimer structures showed that zinc binding sites could be formed at the protein-protein interface that would lead to daisy-chained oligomers. It was concluded that zinc binds weakly to FH at multiple surface locations, most probably within the functionally important SCR-6/8 domains, and this explains why zinc inhibits FH activity. Given the high pathophysiological levels of bioavailable zinc present in subretinal deposits, we discuss how zinc binding to FH may contribute to deposit formation and inflammation associated with AMD., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
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- 2011
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10. Semi-rigid solution structures of heparin by constrained X-ray scattering modelling: new insight into heparin-protein complexes.
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Khan S, Gor J, Mulloy B, and Perkins SJ
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- Animals, Carbohydrate Conformation, Carbohydrate Sequence, Cattle, Crystallography, X-Ray, Disaccharides chemistry, In Vitro Techniques, Macromolecular Substances, Models, Molecular, Molecular Structure, Proteins chemistry, Scattering, Radiation, Solutions, Synchrotrons, Ultracentrifugation, X-Rays, Heparin chemistry
- Abstract
The anionic polysaccharides heparin and heparan sulphate play essential roles in the regulation of many physiological processes. Heparin is often used as an analogue for heparan sulphate. Despite knowledge of an NMR solution structure and 19 crystal structures of heparin-protein complexes for short heparin fragments, no structures for larger heparin fragments have been reported up to now. Here, we show that solution structures for six purified heparin fragments dp6-dp36 (where dp stands for degree of polymerisation) can be determined by a combination of analytical ultracentrifugation, synchrotron X-ray scattering, and constrained modelling. Analytical ultracentrifugation velocity data for dp6-dp36 showed sedimentation coefficients that increased linearly from 1.09 S to 1.84 S with size. X-ray scattering of dp6-dp36 gave radii of gyration R(G) that ranged from 1.33 nm to 3.12 nm and maximum lengths that ranged from 3.0 nm to 12.3 nm. The higher resolution of X-ray scattering revealed an increased bending of heparin with increased size. Constrained molecular modelling of 5000 randomised heparin conformers resulted in 9-15 best-fit structures for each of dp18, dp24, dp30, and dp36 that indicated flexibility and the presence of short linear segments in mildly bent structures. Comparisons of these solution structures with crystal structures of heparin-protein complexes revealed similar ranges of phi (phi) and psi (psi) angles between iduronate and glucosamine rings. We conclude that heparin in solution has a semi-rigid and extended conformation that is preformed for its optimal binding to protein targets without major conformational changes., (Copyright 2009 Elsevier Ltd. All rights reserved.)
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- 2010
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11. Multimeric interactions between complement factor H and its C3d ligand provide new insight on complement regulation.
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Okemefuna AI, Li K, Nan R, Ormsby RJ, Sadlon T, Gordon DL, and Perkins SJ
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- Complement C3d isolation & purification, Complement Factor H isolation & purification, Kinetics, Models, Biological, Models, Molecular, Protein Binding, Surface Plasmon Resonance, Ultracentrifugation, X-Ray Diffraction, Complement C3d immunology, Complement C3d metabolism, Complement Factor H immunology, Complement Factor H metabolism, Protein Multimerization
- Abstract
Activation of C3 to C3b signals the start of the alternative complement pathway. The C-terminal short complement regulator (SCR)-20 domain of factor H (FH), the major serum regulator of C3b, possesses a binding site for C3d, a 35-kDa physiological fragment of C3b. Size distribution analyses of mixtures of SCR-16/20 or FH with C3d by analytical ultracentrifugation in 50 and 137 mM NaCl buffer revealed a range of discrete peaks, showing that multimeric complexes had formed at physiologically relevant concentrations. Surface plasmon resonance studies showed that native FH binds C3d in two stages. An equilibrium dissociation constant K(D)(1) of 2.6 microM in physiological buffer was determined for the first stage. Overlay experiments indicated that C3d formed multimeric complexes with FH. X-ray scattering showed that the maximum dimension of the C3d complexes with SCR-16/20 at 29 nm was not much longer than that of the unbound SCR-16/20 dimer. Molecular modelling suggested that the ultracentrifugation and scattering data are most simply explained in terms of associating dimers of each of SCR-16/20 and C3d. We conclude that the physiological interaction between FH and C3d is not a simple 1:1 binding stoichiometry between the two proteins that is often assumed. Because the multimers involve the C-terminus of FH, which is bound to host cell surfaces, our results provide new insight on FH regulation during excessive complement activation, both in the fluid phase and at host cell surfaces decorated by C3d.
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- 2009
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12. Electrostatic interactions contribute to the folded-back conformation of wild type human factor H.
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Okemefuna AI, Nan R, Gor J, and Perkins SJ
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- Complement Factor H chemistry, Humans, Models, Molecular, Protein Conformation, Protein Structure, Quaternary, Ultracentrifugation methods, X-Ray Diffraction methods, Protein Folding, Static Electricity
- Abstract
Factor H (FH), a major serum regulator of C3b in the complement alternative pathway, is composed of 20 short complement regulator (SCR) domains. Earlier solution structures for FH showed that this has a folded-back domain arrangement and exists as oligomers. To clarify the molecular basis for this, analytical ultracentrifugation and X-ray scattering studies of native FH were performed as a function of NaCl concentration and pH. The sedimentation coefficient for the FH monomer decreased from 5.7 S to 5.3 S with increase in NaCl concentration, showing that weak electrostatic inter-domain interactions affect its folded-back structure. FH became more elongated at pH 9.4, showing the involvement of histidine residue(s) in its folded-back structure. Similar studies of partially deglycosylated FH suggested that oligosaccharides were not significant in determining the FH domain structure. The formation of FH oligomers decreased with increased NaCl concentration, indicating that electrostatic interactions also affect this. X-ray scattering showed that the maximum length of FH increased from 32 nm in low salt to 38 nm in high salt. Constrained X-ray scattering modelling was used to generate significantly improved FH molecular structures at medium resolution. In 50 mM NaCl, the modelled structures showed that inter-SCR domain contacts are likely, while these contacts are fewer in 250 mM NaCl. The results of this study show that the conformation of FH is affected by its local environment, and this may be important for its interactions with C3b and when bound to polyanionic cell surfaces.
- Published
- 2009
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13. Uncontrolled zinc- and copper-induced oligomerisation of the human complement regulator factor H and its possible implications for function and disease.
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Nan R, Gor J, Lengyel I, and Perkins SJ
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- Complement Factor H chemistry, Humans, Models, Molecular, Molecular Weight, Scattering, Radiation, Titrimetry, X-Rays, Copper pharmacology, Macular Degeneration metabolism, Protein Multimerization drug effects, Zinc pharmacology
- Abstract
Polymorphisms in factor H (FH), a major regulator of complement activation, and the accumulation of high zinc concentrations in the outer retina are both associated with age-related macular degeneration. FH is inhibited by zinc, which causes FH to aggregate. To investigate this, we quantitatively studied zinc-induced FH self-association by X-ray scattering and analytical ultracentrifugation to demonstrate uncontrolled FH oligomerisation in conditions corresponding to physiological levels of FH and pathological levels of zinc in the outer retina. By scattering, FH at 2.8-7.0 microM was unaffected until [Zn] increased to 20 microM, whereupon the radius of gyration, RG, values increased from 9 to 15 nm at [Zn]=200 microM. The maximum dimension of FH increased from 32 to 50 nm, indicating that compact oligomers had formed. By ultracentrifugation, size-distribution analyses showed that monomeric FH at 5.57 S was the major species at [Zn] up to 60 microM. At [Zn] above 60 microM, a series of large oligomers were formed, ranging up to 100 S in size. Oligomerisation was reversed by ethylenediaminetetraacetic acid. Structurally distinct large oligomers were observed for Cu, while Ni, Cd and Fe showed low amounts of oligomers and Mg and Ca showed no change. Fluid-phase assays showed reduced FH activities that correlated with increased oligomer formation. The results were attributed to different degrees of stabilisation of weak self-dimerisation sites in FH by transition metals. The relevance of metal-induced FH oligomer formation to complement regulation and age-related macular degeneration is discussed.
- Published
- 2008
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14. Solution structure of the complex formed between human complement C3d and full-length complement receptor type 2.
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Li K, Okemefuna AI, Gor J, Hannan JP, Asokan R, Holers VM, and Perkins SJ
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- Buffers, Humans, Kinetics, Models, Molecular, Protein Binding drug effects, Scattering, Radiation, Sodium Chloride pharmacology, Solutions, Ultracentrifugation, X-Rays, Complement C3d chemistry, Receptors, Complement 3d chemistry
- Abstract
Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B-cells through its binding to C3d, a cleavage fragment of the major complement component C3. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains in a partially folded-back but flexible structure. Here, the effect of C3d binding to CR2 was determined by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient of unbound CR2 is 4.03 S in 50 mM NaCl. Because this agrees well with a value of 3.93 S in 137 mM NaCl, the overall CR2 structure is unaffected by change in ionic strength. Unbound C3d exists in monomer-dimer and monomer-trimer equilibria in 50 mM NaCl, but as a monomer only in 137 mM NaCl. In c(s) size-distribution analyses, an equimolar mixture of the CR2-C3d complex in 50 mM NaCl revealed a single peak shifted to 4.52 S when compared to unbound CR2 at 4.03 S to show that the complex had formed. The CR2-C3d complex in 137 mM NaCl showed two peaks at 2.52 S and 4.07 S to show that this had dissociated. Solution structural models for the CR2 SCR-1/2 complex with C3d and CR2 SCR-1/15 were superimposed. These gave an average sedimentation coefficient of 4.57 S for the complex, in good agreement with the observed value of 4.52 S. It is concluded that CR2 does not detectably change conformation when C3d is bound to it. Consistent with previous analyses, its C3d complex is not formed in physiological salt conditions. The implications of these solution results for its immune role are discussed. To our knowledge, this is the first solution structural study of a large multidomain SCR protein CR2 bound to its physiological ligand C3d.
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- 2008
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15. Implications of the progressive self-association of wild-type human factor H for complement regulation and disease.
- Author
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Nan R, Gor J, and Perkins SJ
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- Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Complement Factor H isolation & purification, Dimerization, Hemolytic-Uremic Syndrome etiology, Hemolytic-Uremic Syndrome pathology, Histidine metabolism, Humans, Models, Molecular, Molecular Sequence Data, Molecular Weight, Optic Disk Drusen etiology, Optic Disk Drusen pathology, Polymorphism, Genetic, Protein Binding, Protein Structure, Tertiary, Scattering, Radiation, Synchrotrons, Ultracentrifugation, X-Rays, Complement Factor H chemistry, Complement Factor H genetics, Complement Factor H metabolism, Gene Expression Regulation
- Abstract
Factor H (FH) is a major regulator of complement alternative pathway activation. It is composed of 20 short complement regulator (SCR) domains and is genetically associated as a risk factor for age-related macular degeneration. Previous studies on FH suggested that it existed in monomeric and dimeric forms. Improved X-ray scattering and analytical ultracentrifugation methodology for wild-type FH permitted a clarification of these oligomeric properties. Data at lower concentrations revealed a dependence of the X-ray radius of gyration values on concentration that corresponded to the weak self-association of FH. Global sedimentation equilibrium fits indicated that a monomer-dimer equilibrium best described the data up to 1.3 mg/ml with a fitted dissociation constant K(D) of 28 microM and that higher oligomers formed at increased concentrations. The K(D) showed that about 85-95% of serum FH will be monomeric in the absence of other factors. Size-distribution analyses in sedimentation velocity experiments showed that monomeric FH was the major species but that as many as six oligomeric forms co-existed with it. The data were explained in terms of two weak dimerisation sites recently identified in the SCR-6/8 and SCR-16/20 fragments of FH with similar K(D) values. These observations indicate a mechanism for the progressive self-association of FH and may be relevant for complement regulation and the formation of drusen deposits that are associated with age-related macular degeneration.
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- 2008
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16. The partly folded back solution structure arrangement of the 30 SCR domains in human complement receptor type 1 (CR1) permits access to its C3b and C4b ligands.
- Author
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Furtado PB, Huang CY, Ihyembe D, Hammond RA, Marsh HC, and Perkins SJ
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- Amino Acid Sequence, Binding Sites, Consensus Sequence, Conserved Sequence, Cysteine chemistry, Humans, Ligands, Models, Molecular, Molecular Sequence Data, Peptide Fragments genetics, Peptide Fragments isolation & purification, Peptide Fragments metabolism, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Repetitive Sequences, Amino Acid, Rotation, Scattering, Radiation, Sequence Homology, Amino Acid, Tryptophan chemistry, Ultracentrifugation, X-Rays, Complement C3b chemistry, Complement C4 chemistry, Peptide Fragments chemistry, Protein Folding, Receptors, Complement chemistry
- Abstract
Human complement receptor type 1 (CR1, CD35) is a type I membrane-bound glycoprotein that belongs to the regulators of complement activity (RCA) family. The extra-cellular component of CR1 is comprised of 30 short complement regulator (SCR) domains, whereas complement receptor type 2 (CR2) has 15 SCR domains and factor H (FH) has 20 SCR domains. The domain arrangement of a soluble form of CR1 (sCR1) was studied by X-ray scattering and analytical ultracentrifugation. The radius of gyration R(G) of sCR1 of 13.4(+/-1.1) nm is not much greater than those for CR2 and FH, and its R(G)/R(0) anisotropy ratio is 3.76, compared to ratios of 3.67 for FH and 4.1 for CR2. Unlike CR2, but similar to FH, two cross-sectional R(G) ranges were identified that gave R(XS) values of 4.7(+/-0.2) nm and 1.2(+/-0.7) nm, respectively, showing that the SCR domains adopt a range of conformations including folded-back ones. The distance distribution function P(r) showed that the most commonly occurring distance in sCR1 is at 11.5 nm. Its maximum length of 55 nm is less than double those for CR2 or FH, even though sCR1 has twice the number of SCR domains compared to CR2 Sedimentation equilibrium experiments gave a mean molecular weight of 235 kDa for sCR1. This is consistent with the value of 245 kDa calculated from its composition including 14 N-linked oligosaccharide sites, and confirmed that sCR1 is a monomer in solution. Sedimentation velocity experiments gave a sedimentation coefficient of 5.8 S. From this, the frictional ratio (f/f(0)) of sCR1 was calculated to be 2.29, which is greater than those of 1.96 for CR2 and 1.77 for FH. The constrained scattering modelling of the sCR1 solution structure starting from homologous SCR domain structures generated 5000 trial conformationally randomised models, 43 of which gave good scattering fits to show that sCR1 has a partly folded-back structure. We conclude that the inter-SCR linkers show structural features in common with those in FH, but differ from those in CR2, and the SCR arrangement in CR1 will permit C3b or C4b to access all three ligand sites.
- Published
- 2008
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17. The regulatory SCR-1/5 and cell surface-binding SCR-16/20 fragments of factor H reveal partially folded-back solution structures and different self-associative properties.
- Author
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Okemefuna AI, Gilbert HE, Griggs KM, Ormsby RJ, Gordon DL, and Perkins SJ
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- Amino Acid Sequence, Binding Sites, Complement C3 metabolism, Consensus Sequence, Conserved Sequence, Dimerization, Heparin metabolism, Humans, Models, Molecular, Molecular Sequence Data, Peptide Fragments genetics, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Scattering, Radiation, Static Electricity, Ultracentrifugation, X-Rays, Complement Factor H chemistry, Complement Factor H metabolism, Peptide Fragments chemistry, Peptide Fragments metabolism, Protein Folding
- Abstract
Factor H (FH) is a plasma glycoprotein that plays a central role in regulation of the alternative pathway of complement. It is composed of 20 short complement regulator (SCR) domains. The SCR-1/5 fragment is required for decay acceleration and cofactor activity, while the SCR-16/20 fragment possesses binding sites for complement C3d and heparin. X-ray scattering and analytical ultracentrifugation showed that SCR-1/5 was monomeric, while SCR-16/20 formed dimers. The Guinier radius of gyration R(G) of 4.3 nm for SCR-1/5 and those of 4.7 nm and about 7.8 nm for monomeric and dimeric SCR-16/20, respectively, showed that their structures are partially folded back and bent. The distance distribution function P(r) showed that SCR-1/5 has a maximum dimension of 15 nm while monomeric and dimeric SCR-16/20 are 17 nm and about 27 nm long, respectively. The sedimentation coefficient of 2.4 S for SCR-1/5 showed no concentration-dependence, while that for SCR-16/20 was 2.8 S for the monomer and 3.9 S for the dimer. Sedimentation equilibrium data showed that SCR-1/5 is monomeric while SCR-16/20 exhibited a weak monomer-dimer equilibrium with a dissociation constant of 16 microM. The constrained scattering and sedimentation modelling of SCR-1/5 and SCR-16/20 showed that partially folded-back and bent flexible SCR arrangements fitted both data sets better than extended linear arrangements, and that the dimer was best modelled in the SCR-16/20 model by an end-to-end association of two SCR-20 domains. The SCR-1/5 and SCR-16/20 models were conformationally similar to the previously determined partially folded-back structure for intact wild-type FH, hence suggesting a partial explanation of the intact FH structure. Comparison of the SCR-16/20 model with the crystal structure of C3b clarified reasons for the distribution of mutations leading to atypical haemolytic uraemic syndrome.
- Published
- 2008
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18. Associative and structural properties of the region of complement factor H encompassing the Tyr402His disease-related polymorphism and its interactions with heparin.
- Author
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Fernando AN, Furtado PB, Clark SJ, Gilbert HE, Day AJ, Sim RB, and Perkins SJ
- Subjects
- Amino Acid Sequence, Dimerization, Humans, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Structure, Quaternary, Protein Structure, Tertiary, Scattering, Radiation, Structure-Activity Relationship, Ultracentrifugation, X-Ray Diffraction, Complement Factor H chemistry, Heparin metabolism, Histidine chemistry, Macular Degeneration genetics, Polymorphism, Genetic, Tyrosine chemistry
- Abstract
Factor H (FH) is a major complement control protein in serum. The seventh short complement regulator (SCR-7) domain of the 20 in FH is associated with age-related macular degeneration through a Tyr402His polymorphism. The recombinant SCR-6/8 domains containing either His402 or Tyr402 and their complexes with a heparin decasaccharide were studied by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient is concentration dependent, giving a value of 2.0 S at zero concentration and a frictional ratio f/f(o) of 1.2 for both allotypes. The His402 allotype showed a slightly greater self-association than the Tyr402 allotype, and small amounts of dimeric SCR-6/8 were found for both allotypes in 50 mM, 137 mM and 250 mM NaCl buffers. Sedimentation equilibrium data were interpreted in terms of a monomer-dimer equilibrium with a dissociation constant of 40 microM for the His402 form. The Guinier radius of gyration R(G) of 3.1-3.3 nm and the R(G)/R(O) ratio of 2.0-2.1 showed that SCR-6/8 is relatively extended in solution. The distance distribution function P(r) showed a maximum dimension of 10 nm, which is less than the length expected for a linear domain arrangement. The constrained scattering and sedimentation modelling of FH SCR-6/8 showed that bent SCR arrangements fit the data better than linear arrangements. Previously identified heparin-binding residues were exposed on the outside curvature of this bent domain structure. Heparin caused the formation of a more linear structure, possibly by binding to residues in the linker. It was concluded that the His402 allotype may self-associate more readily than the Tyr402 allotype, SCR-6/8 is partly responsible for the folded-back structure of intact FH, and SCR-6/8 changes conformation upon heparin binding.
- Published
- 2007
- Full Text
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19. The 15 SCR flexible extracellular domains of human complement receptor type 2 can mediate multiple ligand and antigen interactions.
- Author
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Gilbert HE, Asokan R, Holers VM, and Perkins SJ
- Subjects
- Animals, Baculoviridae genetics, Computer Simulation, Crystallography, X-Ray, DNA, Complementary, Humans, Ligands, Models, Molecular, Molecular Weight, Neutrons, Nuclear Magnetic Resonance, Biomolecular, Pliability, Protein Conformation, Protein Structure, Tertiary, Receptors, Complement 3d genetics, Receptors, Complement 3d isolation & purification, Scattering, Radiation, Spodoptera cytology, Synchrotrons, Ultracentrifugation, X-Rays, Antigens metabolism, Receptors, Complement 3d chemistry, Receptors, Complement 3d metabolism
- Abstract
Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (+/- 13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (+/- 0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-alpha. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d-antigen complexes with the B cell receptor.
- Published
- 2006
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20. Purification, properties and extended solution structure of the complex formed between human immunoglobulin A1 and human serum albumin by scattering and ultracentrifugation.
- Author
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Almogren A, Furtado PB, Sun Z, Perkins SJ, and Kerr MA
- Subjects
- Humans, Immunoglobulin A genetics, Immunoglobulin A metabolism, Models, Molecular, Molecular Sequence Data, Monocytes metabolism, Neutrophils metabolism, Peptide Hydrolases metabolism, Proteus mirabilis enzymology, Scattering, Radiation, Serum Albumin metabolism, Ultracentrifugation, Immunoglobulin A chemistry, Protein Conformation, Serum Albumin chemistry
- Abstract
Immunoglobulin A (IgA) is unique amongst antibodies in being able to form polymeric structures that may possess important functions in the pathology of specific diseases. IgA also forms complexes with other plasma proteins, the IgA1-human serum albumin (HSA) complex (IgA1-HSA) being typical. We have purified this complex using a novel two-step purification based on thiophilic chromatography and gel filtration, and characterised this. HSA is linked covalently to the tailpiece of IgA1 by a disulphide bond between Cys471 in IgA1 and Cys34 in HSA. IgA1-HSA binds to IgA receptors on neutrophils and monocytes, and elicits a respiratory burst that is comparable in magnitude to that of monomeric IgA1. The solution arrangement of IgA1-HSA was identified by X-ray scattering and ultracentrifugation. The radius of gyration R(G) of 7.5(+/-0.3) nm showed that IgA1-HSA is more extended in solution than IgA1 (R(G) of 6.1-6.2 nm). Its distance distribution function P(r) showed two peaks that indicated a well-separated solution structure similar to that for IgA1, and a maximum dimension of 25 nm, which is greater than that of 21 nm for IgA1. Sedimentation equilibrium showed that the IgA1:HSA stoichiometry is 1:1. Sedimentation velocity resulted in a sedimentation coefficient of 6.4S and a frictional ratio of 1.87, which is greater than that of 1.56 for IgA1. The constrained modelling of the IgA1-HSA structure using known structures for IgA1 and HSA generated 2432 conformationally randomised models of which 52 gave good scattering fits. The HSA structure was located at the base of the Fc fragment in IgA1 in an extended arrangement. Such a structure accounts for the functional activity of IgA1-HSA, and supports our previous modelling analysis of the IgA1 solution structure. The IgA1-HSA complex may suggest the potential for creating a new class of targeted therapeutic reagents based on the coupling of IgA1 to carrier proteins.
- Published
- 2006
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21. Extended flexible linker structures in the complement chimaeric conjugate CR2-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy.
- Author
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Gilbert HE, Aslam M, Guthridge JM, Holers VM, and Perkins SJ
- Subjects
- Amino Acid Sequence, Animals, Humans, Immunoglobulin G genetics, Mice, Models, Molecular, Molecular Sequence Data, Receptors, Complement 3d genetics, Recombinant Fusion Proteins genetics, Scattering, Radiation, Sequence Alignment, Ultracentrifugation, Immunoglobulin G chemistry, Protein Conformation, Receptors, Complement 3d chemistry, Recombinant Fusion Proteins chemistry
- Abstract
Complement receptor 2 (CR2; CD21) is a membrane-bound regulator of complement activation, being comprised of 15 or 16 short complement repeat (SCR) domains. A recombinant glycosylated human CR2 SCR 1-2 domain pair was engineered with the Fc fragment of a mouse IgG1 antibody to create a chimaera CR2-Ig containing the major ligand binding domains. Such a chimaera has therapeutic potential as a complement inhibitor or immune modulator. X-ray and neutron scattering and analytical ultracentrifugation identified its domain structure in solution, and provided a comparison with controversial folded-back crystal structures for deglycosylated CR2 SCR 1-2. The radius of gyration R(G) of CR2-Ig was determined to be 5.39(+/-0.14) nm and 5.29(+/-0.01) nm by X-ray and neutron scattering, respectively. The maximum dimension of CR2-Ig was determined to be 17 nm. The molecular mass of CR2-Ig ranged between 101,000 Da and 107,000 Da as determined by neutron scattering and sedimentation equilibrium, in good agreement with the sequence-derived value of 106,600 Da. Sedimentation velocity gave a sedimentation coefficient of 4.49(+/-0.11) S. Stereochemically complete models for CR2-Ig were constructed from crystal structures for the CR2 SCR 1-2 and mouse IgG1 Fc fragments. The two SCR domains and the Fc fragment were joined by randomised conformational peptides. The analysis of 35,000 possible CR2-Ig models showed that only those models in which the two SCR domains were arranged in an open V-shape in random orientations about the Fc fragment accounted for the scattering and sedimentation data. It was not possible to define one single conformational family of Fab-like fragment relative to the Fc fragment. This flexibility is attributed to the relatively long linker sequence and the absence of the antibody light chain from CR2-Ig. The modelling also confirmed that the structure of CR2 SCR 1-2 is more extended in solution than in its crystal structure.
- Published
- 2006
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22. Semi-extended solution structure of human myeloma immunoglobulin D determined by constrained X-ray scattering.
- Author
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Sun Z, Almogren A, Furtado PB, Chowdhury B, Kerr MA, and Perkins SJ
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Humans, Immunoglobulin D isolation & purification, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Quaternary, Sequence Alignment, Solutions chemistry, Ultracentrifugation, Immunoglobulin D chemistry, Multiple Myeloma chemistry, Multiple Myeloma immunology
- Abstract
Human immunoglobulin D (IgD) occurs most abundantly as a membrane-bound antibody on the surface of mature B cells (mIgD). IgD possesses the longest hinge sequence of all the human antibody isotypes, with 64 residues connecting the Fab and Fc fragments. A novel rapid purification scheme of secreted IgD from the serum of an IgD myeloma patient using thiophilic (T-gel) and lectin affinity chromatography gave a stable, homogeneous IgD preparation. Synchrotron X-ray scattering and analytical ultracentrifugation of IgD identified the solution arrangement of its Fab and Fc fragments, and thereby its hinge structure. The Guinier X-ray radius of gyration R(G) of 6.9(+/-0.1)nm showed that IgD is more extended in solution than the immunoglobulin subclass IgA1 (R(G) of 6.1-6.2nm). Its distance distribution function P(r) showed a single peak at 4.7nm and a maximum dimension of 23nm. Velocity experiments gave a sedimentation coefficient of 6.3S, which is similar to that for IgA1 at 6.2S. The complete IgD structure was modelled using molecular dynamics to generate IgD hinge structures, to which homology models for the Fab and Fc fragments were connected. Good scattering curve fits were obtained with 18 semi-extended best fit IgD models that were filtered from 8500 trial models. These best-fit models showed that the IgD hinge does not correspond to an extended polypeptide structure. The averaged solution structure arrangement of the Fab and Fc fragments in IgD is principally T-shaped and flexible, with contribution from Y-shaped and inverted Y-shaped structures. Although the linear sequence of the IgD hinge is much longer, comparison with previous scattering modelling of IgA1 and IgA2(m)1 suggests that the hinge of IgA1 and IgD are more similar than might have been expected, Both possess flexible T-shaped solution structures, probably reflecting the presence of restraining O-linked sugars.
- Published
- 2005
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23. Extended and flexible domain solution structure of the extracellular matrix protein anosmin-1 by X-ray scattering, analytical ultracentrifugation and constrained modelling.
- Author
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Hu Y, Sun Z, Eaton JT, Bouloux PM, and Perkins SJ
- Subjects
- Amino Acid Sequence, Animals, Databases, Protein, Drosophila, Electrophoresis, Polyacrylamide Gel, ErbB Receptors metabolism, Fibronectins chemistry, Humans, Ligands, Milk Proteins chemistry, Models, Biological, Models, Molecular, Molecular Sequence Data, Peptides chemistry, Protein Conformation, Protein Structure, Tertiary, Receptors, Somatomedin metabolism, Recombinant Proteins chemistry, Scattering, Radiation, Ultracentrifugation, Urokinase-Type Plasminogen Activator metabolism, X-Rays, Extracellular Matrix Proteins chemistry, Nerve Tissue Proteins chemistry
- Abstract
Kallmann's syndrome corresponds to a loss of sense of smell and hypogonadotrophic hypogonadism. Defects in anosmin-1 result in the X-linked inherited form of Kallmann's syndrome. Anosmin-1 is an extracellular matrix protein comprised of an N-terminal, cysteine-rich (Cys-box) domain and a whey acidic protein-like (WAP) domain, followed by four fibronectin type III (FnIII) domains. The solution structures of recombinant proteins containing the first three domains (PIWF1) and all six domains (PIWF4) were determined by X-ray scattering and analytical ultracentrifugation. Guinier analyses showed that PIWF1 and PIWF4 have different radii of gyration (R(G)) values of 3.1 nm and 6.7 nm, respectively, but similar cross-sectional radii of gyration (R(XS)) values of 1.5 nm and 1.9 nm, respectively. Distance distribution functions showed that the maximum lengths of PIWF1 and PIWF4 were 11 nm and 23 nm, respectively. Analytical ultracentrifugation gave sedimentation coefficients of 2.52 S and 3.55 S for PIWF1 and PIWF4, respectively. The interpretation of the scattering data by constrained modelling requires homology models for all six domains in anosmin-1. While models were already available for the WAP and FnIII domains, searches suggested the Cys-box domain may resemble the cysteine-rich region of the insulin-like growth factor receptor. Automated constrained molecular modelling based on joining the anosmin-1 domains with structurally randomised linkers resulted in 10,000 models for anosmin-1. A trial-and-error search showed that about 0.1-1.4% of these models fitted the X-ray data. The best models showed that the three domains and six domains in PIWF1 and PIWF4, respectively, were extended. The inter-domain linkers in anosmin-1 could not all be extended at the same time, and there was evidence for inter-domain flexibility. Models with folded-back domain arrangements do not fit the data. These solution structures account for the known biological function of anosmin-1, in particular its ability to interact with its three macromolecular ligands.
- Published
- 2005
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24. Solution structure of the complex between CR2 SCR 1-2 and C3d of human complement: an X-ray scattering and sedimentation modelling study.
- Author
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Gilbert HE, Eaton JT, Hannan JP, Holers VM, and Perkins SJ
- Subjects
- Humans, In Vitro Techniques, Models, Molecular, Multiprotein Complexes, Protein Conformation, Protein Structure, Tertiary, Recombinant Proteins chemistry, Repetitive Sequences, Amino Acid, Scattering, Radiation, Solutions, Ultracentrifugation, X-Rays, Complement C3d chemistry, Receptors, Complement 3d chemistry
- Abstract
Complement receptor type 2 (CR2, CD21) forms a tight complex with C3d, a fragment of C3, the major complement component. Previous crystal structures of the C3d-CR2 SCR 1-2 complex and free CR2 SCR 1-2 showed that the two SCR domains of CR2 form contact with each other in a closed V-shaped structure. SCR 1 and SCR 2 are connected by an unusually long eight-residue linker peptide. Medium-resolution solution structures for CR2 SCR 1-2, C3d, and their complex were determined by X-ray scattering and analytical ultracentrifugation. CR2 SCR 1-2 is monomeric. For CR2 SCR 1-2, its radius of gyration R(G) of 2.12(+/-0.05) nm, its maximum length of 10nm and its sedimentation coefficient s20,w(o) of 1.40(+/-0.03) S do not agree with those calculated from the crystal structures, and instead suggest an open structure. Computer modelling of the CR2 SCR1-2 solution structure was based on the structural randomisation of the eight-residue linker peptide joining SCR 1 and SCR 2 to give 9950 trial models. Comparisons with the X-ray scattering curve indicated that the most favoured arrangements for the two SCR domains corresponded to an open V-shaped structure with no contacts between the SCR domains. For C3d, X-ray scattering and sedimentation velocity experiments showed that it exists as a monomer-dimer equilibrium with a dissociation constant of 40 microM. The X-ray scattering curve for monomeric C3d gave an R(G) value of 1.95 nm, and this together with its s20,w(o) value of 3.17 S gave good agreement with the monomeric C3d crystal structure. Modelling of the C3d dimer gave good agreements with its scattering and ultracentrifugation parameters. For the complex, scattering and ultracentrifugation experiments showed that there was no dimerisation, indicating that the C3d dimerisation site was located close to the CR2 SCR 1-2 binding site. The R(G) value of 2.44(+/-0.1) nm, its length of 9 nm and its s20,w(o) value of 3.45(+/-0.01) S showed that its structure was not much more elongated than that of C3d. Calculations with 9950 models of CR2 SCR 1-2 bound to C3d through SCR 2 showed that SCR 1 formed an open V-shaped structure with SCR 2 and was capable of interacting with the surface of C3d. We conclude that the open V-shaped structures formed by CR2 SCR 1-2, both when free and when bound to C3d, are optimal for the formation of a tight two-domain interaction with its ligand C3d.
- Published
- 2005
- Full Text
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25. The dimeric and trimeric solution structures of the multidomain complement protein properdin by X-ray scattering, analytical ultracentrifugation and constrained modelling.
- Author
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Sun Z, Reid KB, and Perkins SJ
- Subjects
- Amino Acid Sequence, Complement System Proteins metabolism, Dimerization, Humans, Models, Molecular, Molecular Sequence Data, Properdin metabolism, Static Electricity, Ultracentrifugation, X-Ray Diffraction, Properdin chemistry
- Abstract
Properdin regulates the alternative pathway of the complement system of immune defence by stabilising the C3 convertase complex. It contains six thrombospondin repeat type I (TSR-1 to TSR-6) domains and an N-terminal domain. Properdin exists as either a dimer, trimer or tetramer. In order to determine the solution structure of multiple TSR domains, the molecular structures of dimeric and trimeric properdin were studied by X-ray scattering and analytical ultracentrifugation. Guinier analyses showed that the dimer and trimer have radii of gyration R(G) values of 7.5 nm and 10.3 nm, respectively, and cross-sectional radii of gyration R(XS) values of 1.3 nm and 1.5 nm, respectively. Distance distribution functions showed that the maximum lengths of the dimer and trimer were 25 nm and 30 nm, respectively. Analytical ultracentrifugation gave sedimentation coefficients of 5.1S and 5.2S for the dimer and trimer forms, respectively. Homology models for the TSR domains were constructed using the crystal structure of the TSP-2 and TSP-3 domains in human thrombospondin as templates. Properdin could be represented by seven TSR domains, not six as believed, since the crystal structure determined for TSP-2 and TSP-3 showed that the N-terminal domain (TSR-0) could be represented by a truncated TSR domain with the same six conserved Cys residues found in TSR-1 to TSR-6. Automated constrained molecular modelling revealed the solution conformations of multiple TSR domains in properdin at medium resolution. The comparison of 3125 systematically generated conformational models for the trimer with the X-ray data showed that good curve fits could be obtained by assuming that the linker between adjacent TSR domains possessed limited flexibility. Good trimer models correspond to partially collapsed triangular structures, and extended triangular shapes do not fit the data. The corresponding 3125 models for the dimer revealed a similar outcome in which a partially collapsed TSR structure gave good fits. The models account for the effect of mutations that cause properdin deficiencies, and suggest that the biologically active TSR-4, TSR-5 and TSR-6 domains are exposed for protein-protein interactions. The role of the other TSR domains in properdin may be to act as spacers to make TSR-4, TSR-5 and TSR-6 accessible for function.
- Published
- 2004
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26. Characterization and manipulation of the Pseudomonas aeruginosa dimethylarginine dimethylaminohydrolase monomer--dimer equilibrium.
- Author
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Plevin MJ, Magalhães BS, Harris R, Sankar A, Perkins SJ, and Driscoll PC
- Subjects
- Amidohydrolases genetics, Chromatography, Gel, Dimerization, Magnetic Resonance Spectroscopy, Mutagenesis, Site-Directed, Pseudomonas aeruginosa chemistry, Pseudomonas aeruginosa genetics, Amidohydrolases chemistry, Pseudomonas aeruginosa enzymology
- Abstract
In mammals, the enzyme dimethylarginine dimethylaminohydrolase (DDAH) is implicated in the regulation of the cellular levels of asymmetric methylarginines, small molecule metabolites that themselves represent a family of endogenous inhibitors of nitric oxide synthase (NOS). The involvement of DDAH function in the regulation of NOS makes this enzyme a potentially attractive therapeutic target. DDAH from the bacterium Pseudomonas aeruginosa (PaDDAH) is so far the only structurally tractable homologue of mammalian DDAH isoforms. To complement the recent crystal structure of this protein, we show by hydrodynamic measurements that PaDDAH exists in dynamic equilibrium between monomer (ca 29 kDa) and symmetric homodimer (ca 58 kDa) states with a dimer dissociation constant, K(d) approximately 500nM. For the purposes of NMR-based approaches to the study of this enzyme's interactions with substrate and inhibitor ligands, it would be useful to obtain the protein in monomeric form. Through detailed analysis of the homodimer PaDDAH crystal structure we identified key residues involved in the protomer-protomer interface and targeted these for mutation. The hydrodynamic and self-associative properties of a series of PaDDAH interface mutants were analyzed by concentration-dependent analytical size-exclusion chromatography and sedimentation equilibrium analytical ultracentrifugation. The individual substitution of several of the interface residues shifts the equilibrium position towards the monomer, which allowed the design of a double mutant variant (Arg40-->Glu, Arg98-->His) that behaves exclusively as a stable monomer, yet retains greater than 95% catalytic activity compared to wild-type. Comparative two-dimensional (1)H, (15)N heteronuclear NMR spectra indicate that the double mutant remains a monomer even at approximately 1 mM concentration. Accordingly, the double mutant PaDDAH is an attractive template for further NMR-based investigations of the enzyme mechanism and characterization of ligand-binding and inhibitor-binding profiles. These results indicate that dimerization of PaDDAH is not critical for the maintenance of the biological function of the protein. These results are discussed in the context of known modes of self-association between structurally related, but functionally distinct, members of the beta/alpha-propeller fold class.
- Published
- 2004
- Full Text
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27. Solution structure determination of monomeric human IgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1.
- Author
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Furtado PB, Whitty PW, Robertson A, Eaton JT, Almogren A, Kerr MA, Woof JM, and Perkins SJ
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Humans, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Sequence Analysis, Protein, Immunoglobulin A genetics
- Abstract
Immunoglobulin A (IgA), the most abundant human immunoglobulin, mediates immune protection at mucosal surfaces as well as in plasma. It exists as two subclasses IgA1 and IgA2, and IgA2 is found in at least two allotypic forms, IgA2m(1) or IgA2m(2). Compared to IgA1, IgA2 has a much shorter hinge region, which joins the two Fab and one Fc fragments. In order to assess its solution structure, monomeric recombinant IgA2m(1) was studied by X-ray and neutron scattering. Its Guinier X-ray radius of gyration R(G) is 5.18 nm and its neutron R(G) is 5.03 nm, both of which are significantly smaller than those for monomeric IgA1 at 6.1-6.2 nm. The distance distribution function P(r)for IgA2m(1) showed a broad peak with a subpeak and gave a maximum dimension of 17 nm, in contrast to the P(r) curve for IgA1, which showed two distinct peaks and a maximum dimension of 21 nm. The sedimentation coefficients of IgA1 and IgA2m(1) were 6.2S and 6.4S, respectively. These data show that the solution structure of IgA2m(1) is significantly more compact than IgA1. The complete monomeric IgA2m(1) structure was modelled using molecular dynamics to generate random IgA2 hinge structures, to which homology models for the Fab and Fc fragments were connected to generate 10,000 full models. A total of 104 compact best-fit IgA2m(1) models gave good curve fits. These best-fit models were modified by linking the two Fab light chains with a disulphide bridge that is found in IgA2m(1), and subjecting these to energy refinement to optimise this linkage. The averaged solution structure of the arrangement of the Fab and Fc fragments in IgA2m(1) was found to be predominantly T-shaped and flexible, but also included Y-shaped structures. The IgA2 models show full steric access to the two FcalphaRI-binding sites at the Calpha2-Calpha3 interdomain region in the Fc fragment. Since previous scattering modelling had shown that IgA1 also possessed a flexible T-shaped solution structure, such a T-shape may be common to both IgA1 and IgA2. The final models suggest that the combination of the more compact IgA2m(1) and the more extended IgA1 structures will enable human IgA to access a broader range of antigens than either acting alone. The hinges of both IgA subclasses appear to show reduced flexibility when compared to their equivalents in IgG, and this may be important for maintaining an extended IgA structure.
- Published
- 2004
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28. A tetramer-octamer equilibrium in Mycobacterium leprae and Escherichia coli RuvA by analytical ultracentrifugation.
- Author
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Lee YC, Flora R, McCafferty JA, Gor J, Tsaneva IR, and Perkins SJ
- Subjects
- Adenosine Triphosphatases chemistry, Adenosine Triphosphatases metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Buffers, DNA Helicases chemistry, DNA Helicases metabolism, DNA-Binding Proteins metabolism, Escherichia coli Proteins, Sodium Chloride chemistry, Ultracentrifugation, DNA-Binding Proteins chemistry, Escherichia coli chemistry, Mycobacterium leprae chemistry, Protein Structure, Quaternary
- Abstract
In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination. Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer. To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations. The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation. Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl. It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells.
- Published
- 2003
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29. Structure, stability and dynamics of the central domain of cardiac myosin binding protein C (MyBP-C): implications for multidomain assembly and causes for cardiomyopathy.
- Author
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Idowu SM, Gautel M, Perkins SJ, and Pfuhl M
- Subjects
- Amino Acid Sequence, Binding Sites genetics, Carrier Proteins genetics, Carrier Proteins metabolism, Escherichia coli genetics, Escherichia coli metabolism, Hot Temperature, Humans, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Binding genetics, Protein Conformation, Protein Folding, Protein Structure, Tertiary, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Ultracentrifugation, Cardiomyopathies genetics, Carrier Proteins chemistry, Mutation genetics
- Abstract
The large multidomain muscle protein myosin binding protein C (MyBP-C) has been implicated for some time in cardiac disease while until recently little was known about its structure and function. Here we present a detailed study of the central domain C5 of the cardiac isoform of MyBP-C. This domain is unusual in several aspects. Firstly it contains two sizeable insertions compared to the non-cardiac isoforms. The first insertion comprises the linker between domains cC4 and cC5 that is elongated by ten amino acid residues, the second insertion comprises an elongation of the CD-loop in the middle of the domain by approximately 30 amino acid residues. Secondly two point mutations linked to familial hypertrophic cardiomyopathy (FHC) have been identified in this domain. This work shows that the general fold of cC5 is in agreement with the IgI family of beta-sandwich structures. The long cardiac-specific linker between cC4 and cC5 is not a linker at all but an integral part of the fold of cC5, as evidenced by an unfolded mutant in which this segment was removed. The second insertion is shown to be unstructured, highly dynamic and mostly extended according to NMR relaxation measurements and analytical ultracentrifugation. The loss of several key interactions conserved in the CD-loop of the IgI fold is assumed to be responsible for the low stability of cC5 compared to other IgI domains from titin and MyBP-C itself. The low thermodynamic stability of cC5 is most evident in one of the two FHC-linked mutations, N755K (Asn115 in this construct) which is mainly unfolded with a small proportion of a native-like folded species. In contrast, the second FHC-linked mutation, R654H (Arg14 in this construct) is as well folded and stable as the wild-type. This residue is located in the extended beta-bulge at the N terminus of the protein, pointing towards the surface of the CFGA' beta-sheet. This position is in agreement with recent data pointing to a function of Arg654 in an intermolecular interaction with MyBP-C domain cC8.
- Published
- 2003
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30. The extended multidomain solution structures of the complement protein Crry and its chimeric conjugate Crry-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy.
- Author
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Aslam M, Guthridge JM, Hack BK, Quigg RJ, Holers VM, and Perkins SJ
- Subjects
- Amino Acid Sequence, Animals, Antigens, Surface, Consensus Sequence, Mice, Models, Molecular, Molecular Sequence Data, Neutron Diffraction, Pichia, Protein Structure, Tertiary, Rats, Receptors, Cell Surface, Receptors, Complement 3b, Sequence Homology, Amino Acid, Solutions, Species Specificity, Structure-Activity Relationship, Ultracentrifugation, X-Ray Diffraction, Immunoglobulin G chemistry, Immunoglobulin G metabolism, Receptors, Complement chemistry, Receptors, Complement metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism
- Abstract
Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.
- Published
- 2003
- Full Text
- View/download PDF
31. Autosomal dominant reticuloendothelial iron overload associated with a 3-base pair deletion in the ferroportin 1 gene (SLC11A3).
- Author
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Devalia V, Carter K, Walker AP, Perkins SJ, Worwood M, May A, and Dooley JS
- Subjects
- Adult, Amino Acid Sequence, Cation Transport Proteins chemistry, Family Health, Female, Ferritins blood, Genes, Dominant, Humans, Kupffer Cells metabolism, Kupffer Cells pathology, Molecular Sequence Data, Mononuclear Phagocyte System metabolism, Protein Structure, Tertiary, Cation Transport Proteins genetics, Iron Overload genetics, Mononuclear Phagocyte System pathology, Sequence Deletion
- Abstract
We describe a family with autosomal dominant inheritance of increased body iron stores characterized by raised serum ferritin concentration and normal transferrin saturation. Liver biopsy showed iron deposition in Kupffer cells without fibrosis. The clinical features of HFE-related hemochromatosis were absent, as were the Cys282Tyr and His63Asp mutations. Venesection therapy was poorly tolerated, suggesting a defect in iron release from reticuloendothelial stores. A 3-base pair deletion in exon 5 of the ferroportin 1 gene (SLC11A3) predicting Val162 deletion was found in affected members, but not in unaffected individuals or in 100 control subjects. Consensus structural predictions of the transmembrane helices showed that the deletion is in the extracellular loop between the third and fourth predicted transmembrane helices and lies within a spatial cluster of other known ferroportin 1 mutations. These results indicate that this extracellular cluster is functionally important for iron transport, and its disruption leads to iron overload.
- Published
- 2002
- Full Text
- View/download PDF
32. Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy.
- Author
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Lee YC, Boehm MK, Chester KA, Begent RH, and Perkins SJ
- Subjects
- Amino Acid Sequence, Antineoplastic Agents chemistry, Dimerization, Humans, Immunoglobulin Fragments chemistry, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Neutrons, Protein Structure, Quaternary, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Sequence Alignment, Spectroscopy, Fourier Transform Infrared, Temperature, Ultracentrifugation, Antineoplastic Agents metabolism, Immunoglobulin Fragments metabolism
- Abstract
MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V(H) and V(L) domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V(H)-V(L)-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9x10(3)-2.2x10(3) M(-1). Sedimentation velocity experiments using the time derivative g(s(*)) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V(H) and V(L) domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for seven other scFv molecules have shown that, while the contact residues for symmetric back-to-back dimer formation in MFE-23 are not fully conserved, in principle, back-to-back contacts can be formed in these too. This offers possibilities for the creation of other forms of scFv molecules., ((c) 2002 Elsevier Science Ltd.)
- Published
- 2002
- Full Text
- View/download PDF
33. Molecular modelling of the C-terminal domains of factor H of human complement: a correlation between haemolytic uraemic syndrome and a predicted heparin binding site.
- Author
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Perkins SJ and Goodship TH
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Complement Factor H genetics, Crystallography, X-Ray, Humans, Models, Molecular, Molecular Sequence Data, Mutation, Missense genetics, Protein Structure, Tertiary, Repetitive Sequences, Amino Acid genetics, Sequence Alignment, Structure-Activity Relationship, Complement Factor H chemistry, Complement Factor H metabolism, Hemolytic-Uremic Syndrome genetics, Heparin metabolism
- Abstract
Factor H (FH) of the complement system acts as a regulatory cofactor for the factor I-mediated cleavage of C3b and binds to polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains. A set of 12 missense mutations in the C-terminal domains between SCR-16 to SCR-20 is associated with haemolytic uraemic syndrome. Recent structural models for intact FH permit the molecular interpretation of these amino acid substitutions. As all nine SCR-20 substitutions correspond to normal amounts of FH in plasma, and were localised in mostly surface-exposed positions, these are inferred to lead to a functional defect in FH. The nine substitutions occur in the same spatial region of SCR-20. As this surface coincides with conserved basic residues in the C-terminal SCR-20 domain, the substitutions provide direct evidence for a polyanionic binding surface. The positions of these conserved basic residues coincide with those of heparin-binding residues in the crystal structure of the acidic fibroblast growth factor-heparin complex. A tenth substitution and another conserved basic residue in SCR-19 are proximate to this binding site. As the remaining FH substitutions could also be correlated with their proximity to conserved basic residues, haemolytic uraemic syndrome may result from a failure of FH to interact with polyanions at cell surfaces in the kidney., (Copyright 2002 Academic Press.)
- Published
- 2002
- Full Text
- View/download PDF
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