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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.
Journal of Immunology (Baltimore, Md. : 1950)
|August 8, 2001
Summary
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.