Molecular Dissection of Interactions between Components of the Alternative Pathway of Complement and Decay Accelerating Factor (CD55)

Abstract

The complement regulatory protein decay accelerating factor (DAF; CD55), inhibits the alternative complement pathway by accelerating decay of the convertase enzymes formed by C3b and factor B. We show, using surface plasmon resonance, that in the absence of Mg 2؉ , DAF binds C3b, factor B, and the Bb subunit with low affinity (K D , 14 ؎ 0.1, 44 ؎ 10, and 20 ؎ 7 M, respectively). In the presence of Mg 2؉ , DAF bound Bb or the von Willebrand factor type A subunit of Bb with higher affinities (K D , 1.3 ؎ 0.5 and 2.2 ؎ 0.1 M, respectively). Interaction with the proenzyme C3bB was investigated by flowing factor B across a C3b-coated surface in the absence of factor D. The dissociation rate was dependent on the time of incubation, suggesting that a time-dependent conformational transition stabilized the C3b-factor B interaction. Activation by factor D (forming C3bBb) increased the complex half-life; however, the enzyme became susceptible to rapid decay by DAF, unlike the proenzyme, which was unaffected. A convertase assembled with cobra venom factor and Bb was decayed by DAF, albeit far less efficiently than C3bBb. DAF did not bind cobra venom factor, implying that Bb decay is accelerated, at least in part, through DAF binding of this subunit. It is likely that DAF binds the complex with higher affinity/avidity, promoting a conformational change in either or both subunits accelerating decay. Such analysis of component and regulator interactions will inform our understanding of inhibitory mechanisms and the ways in which regulatory proteins cooperate to control the complement cascade.

Decay-Accelerating Factor Must Bind Both Components of the Complement Alternative Pathway C3 Convertase to Mediate Efficient Decay

“…DAF bound Bb via the von Willebrand factor type A (vWFA) domain rather than the serine protease domain. Functional studies with recombinant C2 and fB mutated within this domain indicated that DAF mediated its regulatory action via interactions with vWFA, and our kinetic analyses demonstrated that the Bb fragment and the vWF domain bind DAF with the same kinetic profiles and affinities (12)(13)(14). The specificity of DAF for the active convertase may be a consequence of its higher affinity for Bb compared with fB, and suggests that the regulatory function of DAF on the cell membrane is focused entirely on inhibition of activated enzymes rather than on binding to inactive proenzymes.…”

“…We have previously used surface plasmon resonance (SPR) to investigate formation and regulation of the AP C3 convertase and have demonstrated that kinetics of formation of the proenzyme and the activated convertase were quite different; activation of the proenzyme produced a more stable enzyme with a slower rate of decay (12). The two complexes also differed in their susceptibility to decay using EDTA and soluble DAF.…”

“…It is clear from work described above that DAF interacts with both subunits of the active C3 convertase and that spacing of DAF above the membrane is crucial to function (12,15,16). Structure/function studies of human DAF using SCR-deletion mutants indicated that the first SCR was not required for regulatory function, whereas SCRs 2 and 3 were required for inhibition of either the CP or AP (15,17).…”

“…Only the activated enzyme was subject to decay acceleration mediated by soluble DAF. Using SPR, we analyzed the way in which DAF interacted with individual components of the AP convertase enzyme (12). DAF bound weakly to C3b (K D ϳ 10 Ϫ5 M), very weakly to fB (K D ϳ 4 ϫ 10 Ϫ5 M), but much more tightly to the cleaved fragment Bb (K D Ͻ 10 Ϫ6 M).…”

Conformational Complexity of Complement Component C3

“…Furthermore, factor H has at least one addi-tional interaction site on C3b, i.e., helices α9-α11 and neighboring loops in the TED domain (1187-1249) 89,90 . For DAF the binding site on C3b is unknown, though it has been shown that DAF can bind to C3b without Bb present 91 . Important sites for interaction with factor H, DAF, and CR1 on fragment Bb have been identified on the VWA domain 60 .…”