Compstatin inhibits complement activation by binding to the beta-chain of complement factor 3.
Compstatin is a peptidic complement inhibitor that prevents the cleavage of complement factor 3 (C3) by C3 convertase. Compstatin differs from other C3-regulatory proteins, such as complement receptor (CR) 1 and decay-accelerating factor (DAF), in that it binds native as well as activated C3 fragments and acts through mechanisms that do not involve the destabilization of the C3 convertase or the accelerated degradation of C3b. Compstatin's activity most likely relies on its affinity for native C3 and the conformational change that results upon binding with C3. Although the intermolecular interactions between compstatin and C3 have been studied, the identity of the targeted region on C3 is still elusive. To address this issue, we synthesized a photo-crosslinking compstatin analog and used it to probe C3 for sites of interaction. We identified a 40-kDa region at the C-terminus of the beta-chain of C3 that included the binding site of the compstatin analog. The specificity of the binding was confirmed by inhibition studies, which showed reduced crosslinking signal after pre-incubation of C3 with compstatin but not with various inactive analogs. Binding studies performed with a recombinant homolog of the 40-kDa region confirmed these findings. Five smaller recombinant proteins corresponding to various overlapping regions of the 40-kDa fragment did not bind compstatin, suggesting that a proper protein conformation, only found in larger fragments, is required for compstatin binding. The identified region on the beta-chain has, thus far, not been implicated in C3 cleavage or interactions with other proteins. Therefore, further research on this part of the C3 molecule may have implications for studies on the regulation of C3 cleavage, as well as for complement-based drug design.
Protein Chemistry Laboratory, Department of Pathology and Laboratory Medicine, 402 Stellar Chance, 422 Curie Blvd., University of Pennsylvania, Philadelphia, PA 19104, United States.
Athena M
AM
Soulika
M Claire H
MC
Holland
Georgia
G
Sfyroera
Arvind
A
Sahu
John D
JD
Lambris
D001665:Binding Sites / Q000276:immunology; D003167:Complement Activation / Q000276:immunology; D003176:Complement C3 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018448:Models, Immunological; D010446:Peptide Fragments / Q000097:blood / Q000276:immunology; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D011994:Recombinant Proteins / Q000378:metabolism
D003176:Complement C3; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; C111828:compstatin