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Electrostatic interactions contribute to a folded-back and flexible SCR domain structure of factor H
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Abstracts / Molecular Immunology 45 (2008) 4095–4182
P85 Exploring the mechanism of factor H using FRET Isabell Pechtl a , Henry Hocking a , David Kavanagh b , Paul Barlow a a b
University of Edinburgh, Edinburgh, UK Institute of Human Genetics, Newcastle upon Tyne, UK
The abundant plasma glycoprotein complement factor H (fH, 155 kDa) regulates activation of the alternative pathway. Factor H is monomeric and its 20 CCP modules are arranged in an elongated conformation. The N-terminus of factor H (fH 1–4) acts as a cofactor for proteolytic degradation of C3b, and retains some of the ability of fH to accelerate decay of C3 convertase, C3bBb. Deficiencies in the functions of this part of fH may contribute to diseases associated with inadequate regulation of complement. For example sequence variations in this region may be linked to atypical haemolytic uraemic syndrome and dense deposit disease, which are serious kidney conditions; and age-related macular degeneration, which is a leading cause of blindness in elderly people. The mechanism whereby fH performs its regulatory actions on C3b and C3bBb is as yet unknown. We need this information to fully understand the repercussions of disease-linked sequence variations. Key to progress is to elucidate the relative arrangement of modules and domains within the transient complexes formed between fH and the convertase components. To address this, we have focused on the use of Foerster resonance energy transfer (FRET), which allows measurement of long-range distances between fluorophores attached to proteins, up to 80–100 Å. Using site-directed mutagenesis we have introduced free cysteines into each CCP module of fH 1–4 that serve as attachment points for fluorophores. We have attempted to measure intermolecular distances between these and a fluorophore attached to the thioester of C3b. Some of the fH 1–4 cysteine mutants have been modified to provide a recognition tag for transglutaminase, which can thus be enzymatically modified to bind a second, different, fluorescent label and thus allow intramolecular distance measurements. Combining FRET-derived distances allows triangulation and testing of hypothetical models of some key complexes involved in regulation of the complement system. doi:10.1016/j.molimm.2008.08.085 P86 Structure and architecture of central portions of factor H Christoph Barlow a a b
Schmidt a , Andrew
Herbert a , Arthur
Rowe b , Paul
University of Edinburgh, Edinburgh, UK University of Nottingham, Sutton Bonington, UK
The plasma protein factor H (fH, 155 kDa) is a soluble regulator of the alternative pathway. Polymorphisms and mutations within the 20 complement control protein (CCP) modules that make up the fH molecule are linked to diseases associated with dysfunctional complement activation. A functional remapping exercise revealed only two heparin-binding sites (in CCPs 6–8 and CCPs 19–20) and two dominant C3b-binding sites (in CCPs 1–4 and CCPs 19–20, with a third, low-affinity, site in CCPs 6–8). These new observations raised the question: what is the function of the CCPs 9–18 region that does not carry distinct binding sites for the principal ligands of fH? While we were unable to rule out the possibility that the central segment may contribute weakly to ligand engagement by the more N- and C-terminal binding sites, an attractive hypothesis is
that its main purpose is to act as a hinge allowing the two ligandbinding ends of fH to cooperate. To test this the solution structure of a recombinant version of the module-pair CCP 12–13 was solved at atomic resolution by nuclear magnetic resonance spectrometry to reveal whether these two central modules – which are separated by the longest intermodular linker (eight residues) to be found within fH – are arranged end-to-end (as observed for most other pairs) or side-by-side (consistent with imposing a bend in fH). We next asked to what extent does this arrangement prevail in the context of the rest of the protein. To address this, the recombinant 12–13 construct was extended, by adding one or two CCP modules at either end, yielding CCP 11–14 or CCP 10–15, respectively. The overall dimensions of these longer segments were investigated using analytical ultracentrifugation. These novel biophysical results are considered alongside previous reports based on small angle X-ray scattering and other studies that have been interpreted in terms of a bent over arrangement of the fH molecule to hypothesize a mode of action for this molecule. doi:10.1016/j.molimm.2008.08.086 P87 Electrostatic interactions contribute to a folded-back and flexible SCR domain structure of factor H Azubuike Okemefuna, Ruodan Nan, Jayesh Gor, Stephen Perkins University College London, London, UK Factor H (FH) is a major serum regulator of C3b in the complement alternative pathway. FH is composed of 20 short complement regulator (SCR) domains. Solution structures for FH and complement receptors types 1 and 2 (which contain between 15–30 SCR domains) showed that, while all three have highly extended structures, FH has the most compact SCR domain arrangement of the three. Up to now, no explanation has been proposed for this. To investigate this, we have performed solution structural studies of FH as a function of ionic strength. Analytical ultracentrifugation was performed in buffers that contain between 50 mM NaCl to 350 mM NaCl in the physiologically relevant concentration range between 0.1 and 1.0 mg/ml FH. Three novel features were identified: (i) the sedimentation coefficient for the FH monomer varied from 5.7 S to 5.3 S, showing that weak electrostatic interactions between the 20 SCR domains are important in determining the overall FH structure. Thus FH monomers become more elongated in high salt conditions. (ii) The concentration-dependent oligomers formed by native FH were consistently reproduced in this ionic strength range, indicating that FH dimers and higher oligomers are not formed through electrostatic interactions. (iii) A small speed dependence of the sedimentation coefficients for the FH oligomers was detected, indicating that the SCR domain arrangement shows conformational flexibility. To show the degree to which FH changes its SCR domain arrangement under these conditions, X-ray scattering and constrained modelling analyses are in progress to generate new FH molecular models. While these results do not correspond to radical alterations of the overall shape of FH, they indicate the importance of electrostatic forces in determining the molecular interactions between FH and its C3b ligands. These results also show clearly that crystal and NMR structural determinations of small FH fragments can give misleading inter-SCR domain conformations. doi:10.1016/j.molimm.2008.08.087
Abstracts / Molecular Immunology 45 (2008) 4095–4182
P85 Exploring the mechanism of factor H using FRET Isabell Pechtl a , Henry Hocking a , David Kavanagh b , Paul Barlow a a b
University of Edinburgh, Edinburgh, UK Institute of Human Genetics, Newcastle upon Tyne, UK
The abundant plasma glycoprotein complement factor H (fH, 155 kDa) regulates activation of the alternative pathway. Factor H is monomeric and its 20 CCP modules are arranged in an elongated conformation. The N-terminus of factor H (fH 1–4) acts as a cofactor for proteolytic degradation of C3b, and retains some of the ability of fH to accelerate decay of C3 convertase, C3bBb. Deficiencies in the functions of this part of fH may contribute to diseases associated with inadequate regulation of complement. For example sequence variations in this region may be linked to atypical haemolytic uraemic syndrome and dense deposit disease, which are serious kidney conditions; and age-related macular degeneration, which is a leading cause of blindness in elderly people. The mechanism whereby fH performs its regulatory actions on C3b and C3bBb is as yet unknown. We need this information to fully understand the repercussions of disease-linked sequence variations. Key to progress is to elucidate the relative arrangement of modules and domains within the transient complexes formed between fH and the convertase components. To address this, we have focused on the use of Foerster resonance energy transfer (FRET), which allows measurement of long-range distances between fluorophores attached to proteins, up to 80–100 Å. Using site-directed mutagenesis we have introduced free cysteines into each CCP module of fH 1–4 that serve as attachment points for fluorophores. We have attempted to measure intermolecular distances between these and a fluorophore attached to the thioester of C3b. Some of the fH 1–4 cysteine mutants have been modified to provide a recognition tag for transglutaminase, which can thus be enzymatically modified to bind a second, different, fluorescent label and thus allow intramolecular distance measurements. Combining FRET-derived distances allows triangulation and testing of hypothetical models of some key complexes involved in regulation of the complement system. doi:10.1016/j.molimm.2008.08.085 P86 Structure and architecture of central portions of factor H Christoph Barlow a a b
Schmidt a , Andrew
Herbert a , Arthur
Rowe b , Paul
University of Edinburgh, Edinburgh, UK University of Nottingham, Sutton Bonington, UK
The plasma protein factor H (fH, 155 kDa) is a soluble regulator of the alternative pathway. Polymorphisms and mutations within the 20 complement control protein (CCP) modules that make up the fH molecule are linked to diseases associated with dysfunctional complement activation. A functional remapping exercise revealed only two heparin-binding sites (in CCPs 6–8 and CCPs 19–20) and two dominant C3b-binding sites (in CCPs 1–4 and CCPs 19–20, with a third, low-affinity, site in CCPs 6–8). These new observations raised the question: what is the function of the CCPs 9–18 region that does not carry distinct binding sites for the principal ligands of fH? While we were unable to rule out the possibility that the central segment may contribute weakly to ligand engagement by the more N- and C-terminal binding sites, an attractive hypothesis is
that its main purpose is to act as a hinge allowing the two ligandbinding ends of fH to cooperate. To test this the solution structure of a recombinant version of the module-pair CCP 12–13 was solved at atomic resolution by nuclear magnetic resonance spectrometry to reveal whether these two central modules – which are separated by the longest intermodular linker (eight residues) to be found within fH – are arranged end-to-end (as observed for most other pairs) or side-by-side (consistent with imposing a bend in fH). We next asked to what extent does this arrangement prevail in the context of the rest of the protein. To address this, the recombinant 12–13 construct was extended, by adding one or two CCP modules at either end, yielding CCP 11–14 or CCP 10–15, respectively. The overall dimensions of these longer segments were investigated using analytical ultracentrifugation. These novel biophysical results are considered alongside previous reports based on small angle X-ray scattering and other studies that have been interpreted in terms of a bent over arrangement of the fH molecule to hypothesize a mode of action for this molecule. doi:10.1016/j.molimm.2008.08.086 P87 Electrostatic interactions contribute to a folded-back and flexible SCR domain structure of factor H Azubuike Okemefuna, Ruodan Nan, Jayesh Gor, Stephen Perkins University College London, London, UK Factor H (FH) is a major serum regulator of C3b in the complement alternative pathway. FH is composed of 20 short complement regulator (SCR) domains. Solution structures for FH and complement receptors types 1 and 2 (which contain between 15–30 SCR domains) showed that, while all three have highly extended structures, FH has the most compact SCR domain arrangement of the three. Up to now, no explanation has been proposed for this. To investigate this, we have performed solution structural studies of FH as a function of ionic strength. Analytical ultracentrifugation was performed in buffers that contain between 50 mM NaCl to 350 mM NaCl in the physiologically relevant concentration range between 0.1 and 1.0 mg/ml FH. Three novel features were identified: (i) the sedimentation coefficient for the FH monomer varied from 5.7 S to 5.3 S, showing that weak electrostatic interactions between the 20 SCR domains are important in determining the overall FH structure. Thus FH monomers become more elongated in high salt conditions. (ii) The concentration-dependent oligomers formed by native FH were consistently reproduced in this ionic strength range, indicating that FH dimers and higher oligomers are not formed through electrostatic interactions. (iii) A small speed dependence of the sedimentation coefficients for the FH oligomers was detected, indicating that the SCR domain arrangement shows conformational flexibility. To show the degree to which FH changes its SCR domain arrangement under these conditions, X-ray scattering and constrained modelling analyses are in progress to generate new FH molecular models. While these results do not correspond to radical alterations of the overall shape of FH, they indicate the importance of electrostatic forces in determining the molecular interactions between FH and its C3b ligands. These results also show clearly that crystal and NMR structural determinations of small FH fragments can give misleading inter-SCR domain conformations. doi:10.1016/j.molimm.2008.08.087