Rodriguez, E;
(2015)
Molecular interaction studies of human complement C3 and its regulation.
Doctoral thesis , UCL (University College London).
Abstract
Complement activation of C3 via the alternative pathway involves the generation of C3u and C3b. The inactivation of C3u and C3b by factors H and I results in the inactive fragments C3c and C3d. Several key details of the factors controlling C3b activity are poorly understood. Many pathogeneses are associated with defective complement regulation. Analyses of 317 mutations in four proteins revealed hotspots in the C-terminal domains of factor H and the C3d region of C3, highlighting their importance in dysfunctional protein-protein interactions. Crystal structures of C3b showed the C3c and C3d regions to be in contact with each other. By combining ultracentrifugation and scattering data, this compact crystal structure was confirmed in 50 mM NaCl buffer. In physiological 137 mM NaCl, the C3c and C3d regions in C3b had separated. The compact structures were well explained by the E1032-R102 salt-bridge between C3d and C3c. Its importance was tested by mutagenesis of C3d (A1032), whereupon the interaction was no longer observed. This result explained the different properties between the major C3S and C3F polymorphic forms of C3. The interactions between the different regions of C3 in 50 mM and 137 mM NaCl were explored. C3, C3u and C3b underwent dimerization and the dimerization site was located at their C3d domain. C3b and C3u dimerized the strongest, followed by C3, and these were explained by extended or compact C3d conformations in C3b and C3u. The interactions between C3d and SCR-19/20 of factor H is central for complement regulation. The stoichiometry of this interaction was controversial, being either 2:1 or 1:1. The combination of multiple biophysical techniques showed unequivocally that the correct stoichiometry is 2:1. This outcome clarified a key aspect of how factor H regulates C3b at C3d-decorated host cell surfaces.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | Molecular interaction studies of human complement C3 and its regulation |
| Language: | English |
| UCL classification: | UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/1461042 |
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