Abstract
Properdin (FP) is a positive regulator of the immune system stimulating the activity of the proteolytically active C3 convertase C3bBb in the alternative pathway of the complement system. Here we present two crystal structures of FP and two structures of convertase bound FP. A structural core formed by three thrombospondin repeats (TSRs) and a TB domain harbors the convertase binding site in FP that mainly interacts with C3b. Stabilization of the interaction between the C3b C-terminus and the MIDAS bound Mg2+ in the Bb protease by FP TSR5 is proposed to underlie FP convertase stabilization. Intermolecular contacts between FP and the convertase subunits suggested by the structure were confirmed by binding experiments. FP is shown to inhibit C3b degradation by FI due to a direct competition for a common binding site on C3b. FP oligomers are held together by two sets of intermolecular contacts, where the first is formed by the TB domain from one FP molecule and TSR4 from another. The second and largest interface is formed by TSR1 and TSR6 from the same two FP molecules. Flexibility at four hinges between thrombospondin repeats is suggested to enable the oligomeric, polydisperse, and extended architecture of FP. Our structures rationalize the effects of mutations associated with FP deficiencies and provide a structural basis for the analysis of FP function in convertases and its possible role in pattern recognition.
Highlights
The complement system is a tightly regulated proteolytic cascade, central to the innate immune system
Electron microscopy and our first crystallographic study clearly revealed the eye-shaped vertex interacting with the component 3 (C3) convertase, essential questions including which thrombospondin repeats (TSRs) form the vertex, what is the fold of the N-terminal region, how do two FP molecules come together and form the vertex structure, and which structural features allow FP to form a spectrum of oligomers could not be answered [12, 18]
It is clear that the thumb loop in FP TSR5 contributes to a binding pocket for the Cterminus of C3b and that the index finger loop FP TSR6 is likely to interact directly with factor B (FB) and Bb.We conclude based on structural comparisons and molecular dynamics simulations that the specificity for C3b over C3 is primarily due to subtle structural differences and differences in mobility in a small stretch of C3b residues involved in FP binding
Summary
The complement system is a tightly regulated proteolytic cascade, central to the innate immune system. It is involved in the detection, phagocytosis and killing of invading pathogens, as well as clearance of immune complexes. The point of convergence is the cleavage of the 186 kDa complement component 3 (C3) generating the C3b fragment (178 kDa) which becomes covalently associated with the activator surface. C3b can associate with the zymogen form of factor B (FB) forming the AP C3 proconvertase (Figure 1A). C3b bound FB is activated by the serine protease factor D (FD) forming the smaller fragment Ba and the larger fragment Bb (60 kDa), which remains noncovalent associated with C3b. The C3bBb complex is the AP C3 convertase, which catalyzes further C3 cleavage
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