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Mapping and characterisation of the C3d binding site on factor H domains 19–20
Abstracts / Molecular Immunology 45 (2008) 4095–4182
P88 Multiple interactions between C3d and the C-terminal SCR-16/20 fragment of complement factor H Azubuike Okemefuna a , Rebecca Ormsby b , Tania Sadlon b , David Gordon b , Stephen Perkins a a
University College London, London, UK Flinders Medical Centre and Flinders University, Bedford Park, Australia b
The activation of C3 to C3b signals the start of the alternative complement pathway. C3d is a 35 kDa fragment of C3b. Factor H (FH) is a major serum regulator of C3b. The C-terminal SCR domains of FH possess binding sites for C3d and heparin. Unbound C3d exists as monomers, dimers and trimers, while the unbound FH fragment SCR-16/20 exists as monomers and dimers. In order to elucidate the interaction between C3d and SCR-16/20 in solution, analytical ultracentrifugation was performed. Weak complex formation occurred in buffers containing 137 mM NaCl. Stronger complex formation occurred in 50 mM NaCl. Size distribution analyses showed the existence of as many as eight different oligomeric species. In addition to peaks assigned to the unbound proteins, other peaks were assigned as complexes of C3d and SCR-16/20 with likely stoichiometries of 1:1, 2:2, 4:4, 6:6 and 8:8. X-ray scattering confirmed complex formation had occurred in 50 mM NaCl. The X-ray distance distribution function P(r) showed a low concentration dependence of the maximum dimension of the complexes that ranged between about 30 and 33 nm. Importantly, this is similar in length to that of unbound SCR-16/20. Molecular modelling explained these observations in terms of a series of compact but indefinite complexes of dimeric C3d and SCR-16/20 binding to each other. As similar complexes were also observed in 137 mM NaCl, although more weakly formed, our results indicate that the physiological interaction between FH and C3d in serum cannot be represented by a simple 1:1 binding stoichiometry between the two proteins. Experiments using intact FH with C3d are in progress to extend these results. This oligomer formation in FH takes place in proximity to the region associated with FH binding to host cell surfaces. Our results have new mechanistic implications for FH regulation of C3b at cell surfaces, and may help rationalise the C-terminal FH mutations that lead to atypical haemolytic uraemic syndrome. doi:10.1016/j.molimm.2008.08.088 P89 Mapping and characterisation of the C3d binding site on factor H domains 19–20 Markus J. Lehtinen, T. Sakari Jokiranta Haartman Institute, University of Helsinki, Helsinki, Finland Complement factor H is the main controller of the activation of the alternative pathway (AP) in plasma and it is an important regulator also on certain surfaces. Function of FH on surfaces is dependent on its C-terminus. This is especially evident in atypical hemolytic uremic syndrome (aHUS), a disease characterized by, e.g. damaged endothelial cells, red blood cells, and platelets. The C-terminal domains 19–20 of FH are the hotspot for aHUSassociated point mutations. This region mediates binding of FH for C3d/C3b and polyanions and defects in these interactions have been associated to some aHUS mutations. Aim of this study was to identify the C3b/C3d binding site on FH19–20. Six new recombinant FH19–20 constructs each with a point mutation in domain 20 (T1184R, L1189R, R1203A, R1206A, R1210A, or R1215Q) were
4125
generated and expressed in Pichia pastoris. Binding of the mutants was analyzed by radioligand assays and surface plasmon resonance technique (Biacore® ). We found that constructs bearing mutations R1203A, R1206A and R1210A had significantly decreased affinity to C3d. Also, it was interesting that in these experiments mutations T1184R, L1189R, and R1215Q, associated with aHUS, did not have significant decrease in affinity of FH19–20 to C3d/C3b. We have previously shown that mutations R1182A, W1183L, K1186A, K1188A, and E1198A lead to decreased binding of FH19–20 to C3d. All the eight mutations that have decreased binding of FH19–20 to C3d are located in the crystal structure of FH19–20 on one side of domain 20. Taken together, using rational mutagenesis we have identified the C3d binding site on one side of FH domain 20. doi:10.1016/j.molimm.2008.08.089 P90 Binding sites on factor H domain 20 for C3d and heparin are partially overlapping Arnab Bhattacharjee a , Markus Lehtinen a , Tommi Adrian Goldman b , T. Sakari Jokiranta a a b
Kajander b ,
Haartman Institute, University of Helsinki, Helsinki, Finland Institute of Biotechnology, University of Helsinki, Helsinki, Finland
Factor H (FH) regulates the alternative pathway (AP) of complement both in plasma and on certain cell surfaces. The two terminal domains of FH, i.e. domains 19 and 20, are essential in recognition of surface-bound C3b as exemplified by clinical cases of atypical haemolytic uremic syndrome (aHUS) with mutations in this region. These domains contain binding sites for C3d (and C3b), heparin, and endothelial cells. The aHUS-associated mutations in FH have been reported to cause impaired binding to C3d/C3b and/or heparin. We have generated a number of point mutations in recombinant constructs consisting of domains 19 and 20 of FH (FH19–20). In functional analyses of these Pichia pastoris expressed mutant constructs we identified one mutant (R1203A) that showed dramatically reduced binding to both heparin and C3d. Since this kind of effect could have been caused by global changes in the structure of FH19–20 we proceeded to look for X-ray crystallographic structure of the mutant. Structure of the FH19–20 (R1203A) was solved at 1.7 Å. The mutation changed the structure and the electrostatic potential of the “heel region” of domain 20 locally without global effects. Since the overall structure did not show major changes it is evident that the functional effects of the mutation R1203A cannot be attributed to any global changes in the structure. Therefore our results show that this region is involved in binding of FH to both C3d/C3b and heparin. We conclude that the C3d and heparin binding sites are partially overlapping and therefore the effects of the aHUS patient mutations for both the heparin and C3d binding functions can be impaired simultaneously. doi:10.1016/j.molimm.2008.08.090 P91 The solution structure of the factor H SCR-6/8 domains differs from its crystal structure: Implications for mechanism Sanaullah Khan, Stephen Perkins University College London, London, UK Factor H (FH) is a major serum regulator of C3b in the complement alternative pathway. The SCR-7 domain in FH is associated with age-related macular degeneration through a Tyr402His poly-
P88 Multiple interactions between C3d and the C-terminal SCR-16/20 fragment of complement factor H Azubuike Okemefuna a , Rebecca Ormsby b , Tania Sadlon b , David Gordon b , Stephen Perkins a a
University College London, London, UK Flinders Medical Centre and Flinders University, Bedford Park, Australia b
The activation of C3 to C3b signals the start of the alternative complement pathway. C3d is a 35 kDa fragment of C3b. Factor H (FH) is a major serum regulator of C3b. The C-terminal SCR domains of FH possess binding sites for C3d and heparin. Unbound C3d exists as monomers, dimers and trimers, while the unbound FH fragment SCR-16/20 exists as monomers and dimers. In order to elucidate the interaction between C3d and SCR-16/20 in solution, analytical ultracentrifugation was performed. Weak complex formation occurred in buffers containing 137 mM NaCl. Stronger complex formation occurred in 50 mM NaCl. Size distribution analyses showed the existence of as many as eight different oligomeric species. In addition to peaks assigned to the unbound proteins, other peaks were assigned as complexes of C3d and SCR-16/20 with likely stoichiometries of 1:1, 2:2, 4:4, 6:6 and 8:8. X-ray scattering confirmed complex formation had occurred in 50 mM NaCl. The X-ray distance distribution function P(r) showed a low concentration dependence of the maximum dimension of the complexes that ranged between about 30 and 33 nm. Importantly, this is similar in length to that of unbound SCR-16/20. Molecular modelling explained these observations in terms of a series of compact but indefinite complexes of dimeric C3d and SCR-16/20 binding to each other. As similar complexes were also observed in 137 mM NaCl, although more weakly formed, our results indicate that the physiological interaction between FH and C3d in serum cannot be represented by a simple 1:1 binding stoichiometry between the two proteins. Experiments using intact FH with C3d are in progress to extend these results. This oligomer formation in FH takes place in proximity to the region associated with FH binding to host cell surfaces. Our results have new mechanistic implications for FH regulation of C3b at cell surfaces, and may help rationalise the C-terminal FH mutations that lead to atypical haemolytic uraemic syndrome. doi:10.1016/j.molimm.2008.08.088 P89 Mapping and characterisation of the C3d binding site on factor H domains 19–20 Markus J. Lehtinen, T. Sakari Jokiranta Haartman Institute, University of Helsinki, Helsinki, Finland Complement factor H is the main controller of the activation of the alternative pathway (AP) in plasma and it is an important regulator also on certain surfaces. Function of FH on surfaces is dependent on its C-terminus. This is especially evident in atypical hemolytic uremic syndrome (aHUS), a disease characterized by, e.g. damaged endothelial cells, red blood cells, and platelets. The C-terminal domains 19–20 of FH are the hotspot for aHUSassociated point mutations. This region mediates binding of FH for C3d/C3b and polyanions and defects in these interactions have been associated to some aHUS mutations. Aim of this study was to identify the C3b/C3d binding site on FH19–20. Six new recombinant FH19–20 constructs each with a point mutation in domain 20 (T1184R, L1189R, R1203A, R1206A, R1210A, or R1215Q) were
4125
generated and expressed in Pichia pastoris. Binding of the mutants was analyzed by radioligand assays and surface plasmon resonance technique (Biacore® ). We found that constructs bearing mutations R1203A, R1206A and R1210A had significantly decreased affinity to C3d. Also, it was interesting that in these experiments mutations T1184R, L1189R, and R1215Q, associated with aHUS, did not have significant decrease in affinity of FH19–20 to C3d/C3b. We have previously shown that mutations R1182A, W1183L, K1186A, K1188A, and E1198A lead to decreased binding of FH19–20 to C3d. All the eight mutations that have decreased binding of FH19–20 to C3d are located in the crystal structure of FH19–20 on one side of domain 20. Taken together, using rational mutagenesis we have identified the C3d binding site on one side of FH domain 20. doi:10.1016/j.molimm.2008.08.089 P90 Binding sites on factor H domain 20 for C3d and heparin are partially overlapping Arnab Bhattacharjee a , Markus Lehtinen a , Tommi Adrian Goldman b , T. Sakari Jokiranta a a b
Kajander b ,
Haartman Institute, University of Helsinki, Helsinki, Finland Institute of Biotechnology, University of Helsinki, Helsinki, Finland
Factor H (FH) regulates the alternative pathway (AP) of complement both in plasma and on certain cell surfaces. The two terminal domains of FH, i.e. domains 19 and 20, are essential in recognition of surface-bound C3b as exemplified by clinical cases of atypical haemolytic uremic syndrome (aHUS) with mutations in this region. These domains contain binding sites for C3d (and C3b), heparin, and endothelial cells. The aHUS-associated mutations in FH have been reported to cause impaired binding to C3d/C3b and/or heparin. We have generated a number of point mutations in recombinant constructs consisting of domains 19 and 20 of FH (FH19–20). In functional analyses of these Pichia pastoris expressed mutant constructs we identified one mutant (R1203A) that showed dramatically reduced binding to both heparin and C3d. Since this kind of effect could have been caused by global changes in the structure of FH19–20 we proceeded to look for X-ray crystallographic structure of the mutant. Structure of the FH19–20 (R1203A) was solved at 1.7 Å. The mutation changed the structure and the electrostatic potential of the “heel region” of domain 20 locally without global effects. Since the overall structure did not show major changes it is evident that the functional effects of the mutation R1203A cannot be attributed to any global changes in the structure. Therefore our results show that this region is involved in binding of FH to both C3d/C3b and heparin. We conclude that the C3d and heparin binding sites are partially overlapping and therefore the effects of the aHUS patient mutations for both the heparin and C3d binding functions can be impaired simultaneously. doi:10.1016/j.molimm.2008.08.090 P91 The solution structure of the factor H SCR-6/8 domains differs from its crystal structure: Implications for mechanism Sanaullah Khan, Stephen Perkins University College London, London, UK Factor H (FH) is a major serum regulator of C3b in the complement alternative pathway. The SCR-7 domain in FH is associated with age-related macular degeneration through a Tyr402His poly-