Orla M Dunne1,2, Xin Gao1,3, Ruodan Nan1, Jayesh Gor1, Penelope J Adamson4, David L Gordon4, Martine Moulin2, Michael Haertlein2, V Trevor Forsyth2,5, Stephen J Perkins1. 1. Division of Biosciences, Department of Structural and Molecular Biology, University College London, London, United Kingdom. 2. Life Sciences Group, Institut Laue Langevin, Grenoble, France. 3. Division of Medicine, University College London, London, United Kingdom. 4. Department of Microbiology and Infectious Diseases, Flinders Medical Centre and Flinders University, Bedford Park, SA, Australia. 5. Faculty of Natural Sciences, Keele University, Staffordshire, United Kingdom.
Abstract
Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.
n class="Gene">Complement Factor H (n>n class="Gene">CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.
Authors: Tommi Kajander; Markus J Lehtinen; Satu Hyvärinen; Arnab Bhattacharjee; Elisa Leung; David E Isenman; Seppo Meri; Adrian Goldman; T Sakari Jokiranta Journal: Proc Natl Acad Sci U S A Date: 2011-02-01 Impact factor: 11.205
Authors: Petra Pernot; Adam Round; Ray Barrett; Alejandro De Maria Antolinos; Alexandre Gobbo; Elspeth Gordon; Julien Huet; Jerôme Kieffer; Mario Lentini; Muriel Mattenet; Christian Morawe; Christoph Mueller-Dieckmann; Staffan Ohlsson; Werner Schmid; John Surr; Pascal Theveneau; Louiza Zerrad; Sean McSweeney Journal: J Synchrotron Radiat Date: 2013-05-18 Impact factor: 2.616