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Characterization of the active sites in decay-accelerating factor
L A Kuttner-Kondo1, L Mitchell, D E Hourcade
1Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
Insights
Decay-accelerating factor (DAF) regulates complement by dissociating convertases. Key residues in CCP2-4 control its activity, with specific mutations impacting classical or alternative pathways differently.
Area of Science:
- Immunology
- Molecular Biology
- Protein Structure-Function
Background:
- Decay-accelerating factor (DAF) is a crucial complement system regulator.
- DAF prevents self-damage by dissociating C3 convertases on host cell surfaces.
- Its regulatory activity is localized to complement control protein repeats (CCPs) 2-4.
Purpose of the Study:
- To precisely map the residues responsible for DAF's complement regulatory function.
- To investigate the roles of specific CCP domains in DAF's interaction with C3 convertases.
Main Methods:
- Generated and functionally analyzed 31 alanine substitution mutants of DAF.
- Assessed DAF mutant activity against classical (C4b2a) and alternative (C3bBb) pathway C3 convertases.
- Utilized structural modeling based on existing NMR and crystal structures.
Main Results:
- Mutations in the CCP2-3 groove (R69, R96, R100, K127) and CCP3 hydrophobic core (F148, L171) significantly impaired overall DAF function.
- Specific mutations in CCP4 (K126, F169, R206, R212) selectively reduced alternative pathway activity.
- DAF's function on C3 and C5 convertases was comparable, suggesting a similar mechanism of action.
Conclusions:
- Identified critical residues within CCP2, CCP3, and CCP4 essential for DAF's complement regulatory activity.
- Demonstrated distinct roles for CCP4 residues in regulating the alternative pathway.
- Structural modeling suggests these critical residues form a contiguous functional site.
Abstract:
Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2-4). Previous modeling on the nuclear magnetic resonance structure of CCP15-16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R69, R96, R100, and K127 in the positively charged CCP2-3 groove or hydrophobic F148 and L171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K126 and F169 and of R206 and R212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F169, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2-3 of DAF were oriented according to the crystal structure of CCP1-2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.