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Characterisation of the DNA-binding sites of factor H
4126
Abstracts / Molecular Immunology 45 (2008) 4095–4182
morphism. The solution structure of the recombinant SCR-6/8 domains by constrained scattering modelling starting from homology models [Fernando et al., 2007. J. Mol. Biol. 368, 564–581] was published before the crystal structure of the same protein in its complex with SOS [Prosser et al., 2007. J. Exp. Med. 204, 2277–2283]. This prior solution structure was not discussed in the crystallographic study. Here, we have compared the solution and crystal structures. The SCR homology models were reproduced by the crystal structure with similar sidechain positions, in particular that for Tyr402 and His402. The root mean square differences in a-carbon positions were 0.42, 0.30 and 0.35 nm for SCR-6, SCR-7 and SCR-8, respectively. The mean inter-SCR angle was 128–137◦ (±20◦ ) between SCR-6/7, and 126–130◦ (±10◦ ) between SCR-7/8 in the solution structure, which corresponds to the same degree of bend for the free protein and the crystal structure with SOS bound, where these angles were 144 and 134◦ , respectively. However, the goodness-of-agreement R-factor is 4.3% for the scattering modelling, and is worsened at 6.0–6.4% for the crystal structure. This comparison shows that the solution and crystal structures differ from each other. To date, constrained scattering modelling starting from the crystal structure showed that the inter-SCR linkers in the crystal structure require lengthening to give improved fits to the solution data. As for other SCR crystal structures such as b2I and CR2, this comparison show once again that SCR domain arrangements seen by crystallography do not necessarily correspond to their solution structures. Functional mechanisms for complement activity that are proposed from SCR domain arrangements seen by crystallography will require verification by other methods. doi:10.1016/j.molimm.2008.08.091 P92 Solution structure of heparin and its complexes with the factor H SCR-6/8 domains and factor H: Implications for disease Sanaullah Khan a , Jayesh Gor a , Barbara Mulloy b , Stephen Perkins a a b
University College London, London, UK National Institute of Biological Standards and Control, Potters Bar, UK
Factor H (FH) is a major serum regulator of complement, and FH is genetically associated with the development of age-related macular degeneration in the elderly. Binding studies of FH to heparin are used as a model for FH binding to heparin sulphate which is present on host cell surfaces and in the extracellular matrix. Heparin binds to sites found on at least the SCR-6/8 and SCR-19/20 regions of FH. While an NMR structure has been determined using polydisperse heparin, the dependence of the solution structure on molecular weight is not yet understood. Accordingly these structures were determined. Analytical ultracentrifugation (AUC) data for purified dp6 to dp36 fragments of heparin showed that the sedimentation coefficients increased linearly with the size of heparin from 1.0 to 1.8 S. This was confirmed by molecular modelling based on the NMR structure. Synchrotron X-ray scattering on the same dp6 to dp30 fragments gave radii of gyration, RG, that range from 1.32 to 2.83 nm. The maximum length of dp6 to dp30 ranged from 3.0 to 10.4 nm. Molecular modelling of the X-ray data showed that the longest heparin structures exhibit a slight bending. Solution structures of heparin complexes with FH are likewise not known, and were determined. To date, our solution structure for the heparin complex with FH SCR-6/8 shows that FH SCR-6/8 forms aggregates in the presence of heparin. Intact FH forms 5–15% of dimeric and higher oligomer structures in the absence of heparin. When heparin is added to FH, our AUC and X-ray data shows that heparin promotes further strong FH oligomer formation as well as conformational
changes in FH. These conformational changes and self-aggregation results provide the first molecular picture of how FH interacts with host cell surfaces and have a direct relevance for the sub-retinal pigmental endothelial deposits that form in Bruch’s membrane during the development of age-related macular degeneration. doi:10.1016/j.molimm.2008.08.092 P93 Characterisation of the DNA-binding sites of factor H Andrew Herbert, Huw Jenkins, Christoph Schmidt, Dusan Uhrin, Paul Barlow University of Edinburgh, Edinburgh, UK Factor H (fH) has numerous binding partners including DNA. An interaction between this 20-module extracellular complement regulatory protein and nucleic acids might be important in recognition and immune clearance of apoptotic or necrotic cells. We show that DNA does not interfere with the fluid-phase cofactor activity of fH. This is consistent with DNA-binding sites that are remote from the N-terminus, which is known to harbour the cofactor and (at least part of) the decay accelerating activities of fH. Fragments of fH (modules 2–3, 7–8, 8–15, 12–13 and 19–20) were expressed in P. pastoris and the purified proteins tested for their ability to bind a 20 mer dsDNA fragment using surface plasmon resonance (SPR). In an analogous fashion to glycosaminoglycan (GAG) binding, only fragments encompassing modules 7 and 19–20 were found to bind to the DNA fragment, with 19–20 being the strongest binder. Nuclear magnetic resonance chemical shift perturbation studies on a 10 mer dsDNA fragment revealed a DNA binding site in module 20 that overlapped with the previously identified GAG-binding site. Knowledge-based molecular docking was performed, using the chemical shift perturbation data to drive the docking, in order to visualise the DNA-binding site. In a related set of experiments wild-type and a panel of 14 recombinantly expressed mutants of fH modules 19–20, some linked to atypical haemolytic uraemic syndrome, were tested using SPR for their ability to bind the 20 mer dsDNA fragment. Some were shown to have differences in their ability to bind DNA as compared to each other and the wildtype protein. Moreover, differences in affinity for DNA were also observed between the age-related macular degeneration associated allotypic variants (402H vs. 402Y) of fH modules 7–8. These results raise the possibility of a significant role for DNA binding of fH in a physiological setting. doi:10.1016/j.molimm.2008.08.093 P94 Uncontrolled zinc- and copper-induced oligomerisation of human complement factor H and its implications for agerelated macular degeneration Ruodan Nan a , Jayesh Gor a , Imre Lengyel b , Stephen Perkins a a b
University College London, London, UK Institute of Ophthalmology, London, UK
Both factor H (FH), a major regulator of complement alternative pathway activation, and the accumulation of high zinc concentrations in the outer retina are risk factors for age-related macular degeneration (AMD). Their involvement in the development of AMD is unclear. Here, zinc-induced FH self-association was quantitatively studied by X-ray scattering and analytical ultracentrifugation to demonstrate uncontrolled FH oligomerisation and FH
Abstracts / Molecular Immunology 45 (2008) 4095–4182
morphism. The solution structure of the recombinant SCR-6/8 domains by constrained scattering modelling starting from homology models [Fernando et al., 2007. J. Mol. Biol. 368, 564–581] was published before the crystal structure of the same protein in its complex with SOS [Prosser et al., 2007. J. Exp. Med. 204, 2277–2283]. This prior solution structure was not discussed in the crystallographic study. Here, we have compared the solution and crystal structures. The SCR homology models were reproduced by the crystal structure with similar sidechain positions, in particular that for Tyr402 and His402. The root mean square differences in a-carbon positions were 0.42, 0.30 and 0.35 nm for SCR-6, SCR-7 and SCR-8, respectively. The mean inter-SCR angle was 128–137◦ (±20◦ ) between SCR-6/7, and 126–130◦ (±10◦ ) between SCR-7/8 in the solution structure, which corresponds to the same degree of bend for the free protein and the crystal structure with SOS bound, where these angles were 144 and 134◦ , respectively. However, the goodness-of-agreement R-factor is 4.3% for the scattering modelling, and is worsened at 6.0–6.4% for the crystal structure. This comparison shows that the solution and crystal structures differ from each other. To date, constrained scattering modelling starting from the crystal structure showed that the inter-SCR linkers in the crystal structure require lengthening to give improved fits to the solution data. As for other SCR crystal structures such as b2I and CR2, this comparison show once again that SCR domain arrangements seen by crystallography do not necessarily correspond to their solution structures. Functional mechanisms for complement activity that are proposed from SCR domain arrangements seen by crystallography will require verification by other methods. doi:10.1016/j.molimm.2008.08.091 P92 Solution structure of heparin and its complexes with the factor H SCR-6/8 domains and factor H: Implications for disease Sanaullah Khan a , Jayesh Gor a , Barbara Mulloy b , Stephen Perkins a a b
University College London, London, UK National Institute of Biological Standards and Control, Potters Bar, UK
Factor H (FH) is a major serum regulator of complement, and FH is genetically associated with the development of age-related macular degeneration in the elderly. Binding studies of FH to heparin are used as a model for FH binding to heparin sulphate which is present on host cell surfaces and in the extracellular matrix. Heparin binds to sites found on at least the SCR-6/8 and SCR-19/20 regions of FH. While an NMR structure has been determined using polydisperse heparin, the dependence of the solution structure on molecular weight is not yet understood. Accordingly these structures were determined. Analytical ultracentrifugation (AUC) data for purified dp6 to dp36 fragments of heparin showed that the sedimentation coefficients increased linearly with the size of heparin from 1.0 to 1.8 S. This was confirmed by molecular modelling based on the NMR structure. Synchrotron X-ray scattering on the same dp6 to dp30 fragments gave radii of gyration, RG, that range from 1.32 to 2.83 nm. The maximum length of dp6 to dp30 ranged from 3.0 to 10.4 nm. Molecular modelling of the X-ray data showed that the longest heparin structures exhibit a slight bending. Solution structures of heparin complexes with FH are likewise not known, and were determined. To date, our solution structure for the heparin complex with FH SCR-6/8 shows that FH SCR-6/8 forms aggregates in the presence of heparin. Intact FH forms 5–15% of dimeric and higher oligomer structures in the absence of heparin. When heparin is added to FH, our AUC and X-ray data shows that heparin promotes further strong FH oligomer formation as well as conformational
changes in FH. These conformational changes and self-aggregation results provide the first molecular picture of how FH interacts with host cell surfaces and have a direct relevance for the sub-retinal pigmental endothelial deposits that form in Bruch’s membrane during the development of age-related macular degeneration. doi:10.1016/j.molimm.2008.08.092 P93 Characterisation of the DNA-binding sites of factor H Andrew Herbert, Huw Jenkins, Christoph Schmidt, Dusan Uhrin, Paul Barlow University of Edinburgh, Edinburgh, UK Factor H (fH) has numerous binding partners including DNA. An interaction between this 20-module extracellular complement regulatory protein and nucleic acids might be important in recognition and immune clearance of apoptotic or necrotic cells. We show that DNA does not interfere with the fluid-phase cofactor activity of fH. This is consistent with DNA-binding sites that are remote from the N-terminus, which is known to harbour the cofactor and (at least part of) the decay accelerating activities of fH. Fragments of fH (modules 2–3, 7–8, 8–15, 12–13 and 19–20) were expressed in P. pastoris and the purified proteins tested for their ability to bind a 20 mer dsDNA fragment using surface plasmon resonance (SPR). In an analogous fashion to glycosaminoglycan (GAG) binding, only fragments encompassing modules 7 and 19–20 were found to bind to the DNA fragment, with 19–20 being the strongest binder. Nuclear magnetic resonance chemical shift perturbation studies on a 10 mer dsDNA fragment revealed a DNA binding site in module 20 that overlapped with the previously identified GAG-binding site. Knowledge-based molecular docking was performed, using the chemical shift perturbation data to drive the docking, in order to visualise the DNA-binding site. In a related set of experiments wild-type and a panel of 14 recombinantly expressed mutants of fH modules 19–20, some linked to atypical haemolytic uraemic syndrome, were tested using SPR for their ability to bind the 20 mer dsDNA fragment. Some were shown to have differences in their ability to bind DNA as compared to each other and the wildtype protein. Moreover, differences in affinity for DNA were also observed between the age-related macular degeneration associated allotypic variants (402H vs. 402Y) of fH modules 7–8. These results raise the possibility of a significant role for DNA binding of fH in a physiological setting. doi:10.1016/j.molimm.2008.08.093 P94 Uncontrolled zinc- and copper-induced oligomerisation of human complement factor H and its implications for agerelated macular degeneration Ruodan Nan a , Jayesh Gor a , Imre Lengyel b , Stephen Perkins a a b
University College London, London, UK Institute of Ophthalmology, London, UK
Both factor H (FH), a major regulator of complement alternative pathway activation, and the accumulation of high zinc concentrations in the outer retina are risk factors for age-related macular degeneration (AMD). Their involvement in the development of AMD is unclear. Here, zinc-induced FH self-association was quantitatively studied by X-ray scattering and analytical ultracentrifugation to demonstrate uncontrolled FH oligomerisation and FH