The complement system plays an important role in innate and adaptive immunity. Central to all complement activities is the function of complement component 3 (C3). C3 contains a C-terminal extension of ~150 residues known as the NTR (or C345C) domain. To address the role of the NTR domain in binding and functional activities of C3, a C3/C5 chimera was engineered, in which the NTR domain of C3 was replaced by the homologous domain of the closely related protein C5. Functionally, the C3(C5NTR) was devoid of classical pathway-dependent hemolytic activity and deficient in factor H- and CR1-dependent factor I cleavability. Direct binding SPR assays, using chip bound methylamine treated His6-tagged C3(C5NTR), showed a complete loss of C5 binding while retaining wild type binding with CR1, factor H and factor B. These results present the first evidence for a major C5 binding site within C3 NTR.

The complement system plays an important role in innate and adaptive immunity. Central to all complement activities is the function of complement component 3 (C3). C3 contains a C-terminal extension of ~150 residues known as the NTR (or C345C) domain. To address the role of the NTR domain in binding and functional activities of C3, a C3/C5 chimera was engineered, in which the NTR domain of C3 was replaced by the homologous domain of the closely related protein C5. Functionally, the C3(C5NTR) was devoid of classical pathway-dependent hemolytic activity and deficient in factor H- and CR1-dependent factor I cleavability. Direct binding SPR assays, using chip bound methylamine treated His6-tagged C3(C5NTR), showed a complete loss of C5 binding while retaining wild type binding with CR1, factor H and factor B. These results present the first evidence for a major C5 binding site within C3 NTR.