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Structural investigation of C terminal region of C7
Abstracts / Molecular Immunology 44 (2007) 3909–3994
SCR-1/5 and a monomer–dimer equilibrium for SCR-16/20. The concentration dependences of the Guinier parameters and equilibrium data resulted in a monomer–dimer dissociation constant of 16 M. The constrained scattering modelling of SCR-1/5 and SCR-16/20 based on structural models for the five SCR domains in each showed that partially bent flexible SCR arrangements fit the data better than linear arrangements, and that the dimer can be modelled by the end-to-end association of two SCR20 domains. It is concluded that the N-terminal and C-terminal regions of FH showed different associative properties but similar conformations. The models for SCR-1/5 and SCR-16/20 are consistent with the partially folded back structure for intact wildtype FH, hence providing the first explanation of its appearance. Comparison of the SCR-16/20 model with the crystal structure of C3b clarifies reasons for the distribution of mutations leading to atypical haemolytic uraemic syndrome. doi:10.1016/j.molimm.2007.06.201 P123 Towards a structural understanding of the architecture of complement factor H Isabell C. Pechtl, Christoph Q. Schmidt, Andrew P. Herbert, Paul N. Barlow School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK The plasma protein Factor H (150 kDa) regulates the activity of the alternative pathway. Three C3b-binding sites (CCPs1-4, CCPs12-14 and CCPs19-20), of which one resides in the central part of factor H, have been reported. This central region has been reported to contain a glycosaminoglycan-binding site. Biophysical studies revealed that factor H is monomeric in solution, and suggest that its 20 CCP modules are arranged in a folded-back conformation. The CCP modules of factor H are joined by linking sequences that vary in length, with the longest linkers occurring in the central portion of the molecule. The five factor H linkers of CCP10-CCP14 consist of six, seven or eight residues. The largest numbers of linker residues occur on either side of CCP13, which is the smallest CCP module known. The coincidence of the relatively small CCP module of 13 with the higher number of linkers in the central part of factor H is likely to reflect some unique architectural features. To explore the structural details of this portion of factor H, we are combining fluorescence resonance energy transfer (FRET), electron paramagnetic resonance (EPR) and NMR. FRET and EPR (unlike NMR) measure long-range interatomic distances and thus they support NMR-based structural investigations. By using site-directed mutagenesis, additional cysteine residues are introduced into CCP modules at strategic points. The resultant free thiol groups are used for attachment of fluorescent or paramagnetic probes. Enzymatic modification of a glutamine side chain within a custom-designed N-terminal sequence by transglutaminase (TGase) provides an alternative means of attaching an appropriate probe. Combining these two strategies allows orthogonal attachment of pairs of tags (e.g. a fluorescent donor and acceptor). The distances between pairs
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of probes provide information on the conformational state of factor H and allow hypotheses involving CCP 13 acting as a conformational hinge to be tested. doi:10.1016/j.molimm.2007.06.202 P124 Structural investigation of C terminal region of C7 Marie M. Phelan, Janice Bramham Joseph Black Building, Kings Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK The membrane attack complex (MAC) is a key component at the terminus of the complement pathways; it has the ability to kill cells by puncturing the plasma membrane resulting in cytolysis. If not tightly regulated, membrane attack can damage host as well as foreign cells and deposition of MAC on host cell surfaces has been shown to be responsible for complementmediated tissue damage. The membrane attack complex is assembled by the sequential binding of complement proteins C6, C7, C8 and multiple copies of C9 to C5b (the activated form of C5). Although the MAC forms a stable macromolecular complex, very little is known about the structures of the five MAC proteins. Sequence comparisons of the MAC proteins (C6, C7, C8␣ , C8 and C9) have shown that they are related proteins, sharing several structural domains. We are currently investigating by NMR the structure of the C terminal region of C7, which has been shown to play an essential role in MAC formation. doi:10.1016/j.molimm.2007.06.203 P125 Unravelling the complexities of factor H action on selfsurfaces Christoph Q. Schmidt a,b , Baerbel S. Blaum a,b , Andrew P. Herbert a,b , Viviana Ferreira a,b , Michael K. Pangburn a,b , Dusan Uhrin a,b , Paul N. Barlow a,b a
Schools of Biological Sciences and Chemistry, University of Edinburgh, Edinburgh, EH9 3JJ, UK b Biomedical Research Unit, University of Texas HSC, Tyler, TX 75708, USA The alternative pathway of complement is tightly controlled to avoid inappropriate amplification. Factor H is an abundant plasma glycoprotein that regulates the alternative pathway both in the fluid phase and on self-surfaces. Factor H is composed entirely from a chain of 20 complement control protein (CCP) modules (also known as SCRs) that are connected by short linking sequences. Multiple C3b and polyanion-binding sites have been identified in factor H reflecting the complex mechanism whereby it acts selectively at self-surfaces while allowing unimpeded amplification of complement on microbial or other activating surfaces. The key distinguishing feature exploited by factor H in this respect appears to be the chemical and electrostatic nature of surface-associated carbohydrate groups such as the glycosaminoglycans attached to proteoglycans. In the past
SCR-1/5 and a monomer–dimer equilibrium for SCR-16/20. The concentration dependences of the Guinier parameters and equilibrium data resulted in a monomer–dimer dissociation constant of 16 M. The constrained scattering modelling of SCR-1/5 and SCR-16/20 based on structural models for the five SCR domains in each showed that partially bent flexible SCR arrangements fit the data better than linear arrangements, and that the dimer can be modelled by the end-to-end association of two SCR20 domains. It is concluded that the N-terminal and C-terminal regions of FH showed different associative properties but similar conformations. The models for SCR-1/5 and SCR-16/20 are consistent with the partially folded back structure for intact wildtype FH, hence providing the first explanation of its appearance. Comparison of the SCR-16/20 model with the crystal structure of C3b clarifies reasons for the distribution of mutations leading to atypical haemolytic uraemic syndrome. doi:10.1016/j.molimm.2007.06.201 P123 Towards a structural understanding of the architecture of complement factor H Isabell C. Pechtl, Christoph Q. Schmidt, Andrew P. Herbert, Paul N. Barlow School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK The plasma protein Factor H (150 kDa) regulates the activity of the alternative pathway. Three C3b-binding sites (CCPs1-4, CCPs12-14 and CCPs19-20), of which one resides in the central part of factor H, have been reported. This central region has been reported to contain a glycosaminoglycan-binding site. Biophysical studies revealed that factor H is monomeric in solution, and suggest that its 20 CCP modules are arranged in a folded-back conformation. The CCP modules of factor H are joined by linking sequences that vary in length, with the longest linkers occurring in the central portion of the molecule. The five factor H linkers of CCP10-CCP14 consist of six, seven or eight residues. The largest numbers of linker residues occur on either side of CCP13, which is the smallest CCP module known. The coincidence of the relatively small CCP module of 13 with the higher number of linkers in the central part of factor H is likely to reflect some unique architectural features. To explore the structural details of this portion of factor H, we are combining fluorescence resonance energy transfer (FRET), electron paramagnetic resonance (EPR) and NMR. FRET and EPR (unlike NMR) measure long-range interatomic distances and thus they support NMR-based structural investigations. By using site-directed mutagenesis, additional cysteine residues are introduced into CCP modules at strategic points. The resultant free thiol groups are used for attachment of fluorescent or paramagnetic probes. Enzymatic modification of a glutamine side chain within a custom-designed N-terminal sequence by transglutaminase (TGase) provides an alternative means of attaching an appropriate probe. Combining these two strategies allows orthogonal attachment of pairs of tags (e.g. a fluorescent donor and acceptor). The distances between pairs
3987
of probes provide information on the conformational state of factor H and allow hypotheses involving CCP 13 acting as a conformational hinge to be tested. doi:10.1016/j.molimm.2007.06.202 P124 Structural investigation of C terminal region of C7 Marie M. Phelan, Janice Bramham Joseph Black Building, Kings Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK The membrane attack complex (MAC) is a key component at the terminus of the complement pathways; it has the ability to kill cells by puncturing the plasma membrane resulting in cytolysis. If not tightly regulated, membrane attack can damage host as well as foreign cells and deposition of MAC on host cell surfaces has been shown to be responsible for complementmediated tissue damage. The membrane attack complex is assembled by the sequential binding of complement proteins C6, C7, C8 and multiple copies of C9 to C5b (the activated form of C5). Although the MAC forms a stable macromolecular complex, very little is known about the structures of the five MAC proteins. Sequence comparisons of the MAC proteins (C6, C7, C8␣ , C8 and C9) have shown that they are related proteins, sharing several structural domains. We are currently investigating by NMR the structure of the C terminal region of C7, which has been shown to play an essential role in MAC formation. doi:10.1016/j.molimm.2007.06.203 P125 Unravelling the complexities of factor H action on selfsurfaces Christoph Q. Schmidt a,b , Baerbel S. Blaum a,b , Andrew P. Herbert a,b , Viviana Ferreira a,b , Michael K. Pangburn a,b , Dusan Uhrin a,b , Paul N. Barlow a,b a
Schools of Biological Sciences and Chemistry, University of Edinburgh, Edinburgh, EH9 3JJ, UK b Biomedical Research Unit, University of Texas HSC, Tyler, TX 75708, USA The alternative pathway of complement is tightly controlled to avoid inappropriate amplification. Factor H is an abundant plasma glycoprotein that regulates the alternative pathway both in the fluid phase and on self-surfaces. Factor H is composed entirely from a chain of 20 complement control protein (CCP) modules (also known as SCRs) that are connected by short linking sequences. Multiple C3b and polyanion-binding sites have been identified in factor H reflecting the complex mechanism whereby it acts selectively at self-surfaces while allowing unimpeded amplification of complement on microbial or other activating surfaces. The key distinguishing feature exploited by factor H in this respect appears to be the chemical and electrostatic nature of surface-associated carbohydrate groups such as the glycosaminoglycans attached to proteoglycans. In the past