Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor

Abstract

Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg(2+)-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics.

Figure 1. SCIN induces formation of dimeric convertases

(a) SPR analysis. Binding of soluble convertase components to surface-immobilized SCIN. Bars indicate responses at the end of injection (all components at 100 nM). Inlay, Sensorgrams of injections with C3bBb: C3b and FB at 10 nM (blue), 30 nM (green), 100 nM (black) or 300 nM (red), FD at 100 nM. (b) Gel filtration analysis and native gel electrophoresis of SCIN-inhibited convertases. (Left) Gel filtration: absorbance peaks of active convertases stabilized by Ni²⁺ (Nickel, black line) and SCIN-inhibited convertases (solid red line). The 178 kDa peak corresponds with free C3b. Dashed red line indicates elution positions of SCIN as determined by ELISA. Right gels, Left panel: native gel electrophoresis of active (top) and SCIN-inhibited convertase (below). Right panel: native gel electrophoresis of fractions eluted from the gel filtration column. (c) Analysis of convertase stability. C3b (500 nM), FB (500 nM), FD (250 nM) and SCIN (1 μM) were incubated at 4 °C, 20 °C or 37 °C for different time periods and subjected to native gel electrophoresis at 4 °C. The SCIN-convertase is most stable at 4 °C (> 20 h). No complexes were detected in the absence of SCIN (−). (d) Native gel electrophoresis of purified SCIN-convertase complexes after 0 to 8 days (left side, coomassie staining) and 25 days (right side, silver staining). Figures a-c are representatives of three independent experiments. Figure d is a representative of two independent experiments.

Figure 1. SCIN induces formation of dimeric convertases

(a) SPR analysis. Binding of soluble convertase components to surface-immobilized SCIN. Bars indicate responses at the end of injection (all components at 100 nM). Inlay, Sensorgrams of injections with C3bBb: C3b and FB at 10 nM (blue), 30 nM (green), 100 nM (black) or 300 nM (red), FD at 100 nM. (b) Gel filtration analysis and native gel electrophoresis of SCIN-inhibited convertases. (Left) Gel filtration: absorbance peaks of active convertases stabilized by Ni²⁺ (Nickel, black line) and SCIN-inhibited convertases (solid red line). The 178 kDa peak corresponds with free C3b. Dashed red line indicates elution positions of SCIN as determined by ELISA. Right gels, Left panel: native gel electrophoresis of active (top) and SCIN-inhibited convertase (below). Right panel: native gel electrophoresis of fractions eluted from the gel filtration column. (c) Analysis of convertase stability. C3b (500 nM), FB (500 nM), FD (250 nM) and SCIN (1 μM) were incubated at 4 °C, 20 °C or 37 °C for different time periods and subjected to native gel electrophoresis at 4 °C. The SCIN-convertase is most stable at 4 °C (> 20 h). No complexes were detected in the absence of SCIN (−). (d) Native gel electrophoresis of purified SCIN-convertase complexes after 0 to 8 days (left side, coomassie staining) and 25 days (right side, silver staining). Figures a-c are representatives of three independent experiments. Figure d is a representative of two independent experiments.

Figure 2. Crystal structure of the C3 convertase C3bBb inhibited by SCIN

(a) Ribbon representation of the C3bBb-SCIN dimeric complex with C3b (blue and turquoise), Bb (green and golden) and SCIN (purple and orange). (b) Stereoview of the monomeric C3bBb-SCIN extracted from the dimer, coloured by protein (SCIN) or protein domain (VWA and SP of Bb; and, all 12 domains of C3b with thioester shown by red spheres); a linear representation of the domain composition is given below (with disulfide bonds, glycosylation sites and thioester marked). (c) Overlay of the four C3bBbSCIN complexes in the asymmetric unit (yellow, orange, green and magenta). The largest variation occurs in the orientation of the C345C domain (see Supplementary Fig. 8).

Figure 3. Inhibition of C3bBb by SCIN

(a) Contact sites of SCIN in the dimeric convertase. The SCIN binding pocket is shown in surface representation (left) with a ribbon representation of SCIN colour coded by molecular contact (right). (b) Amino-acid sequence alignment of SCIN and the non-functional homologue ORF-D. Convertase contact sites in SCIN are underlined. Below a schematic representation of the SCIN chimeras is given (red boxes indicate the exchanged segments). (c) Convertase inhibition by SCIN chimeras: C3 conversion by fluid-phase C3bBb (above) and Bb stabilization on bacterial surfaces (below). (d) Native gel electrophoresis of convertases in the presence of Ni²⁺ (lane 1), SCIN (lane 2), ChN (lane 3) or ChC3b2 (lane 4). Figures c and d are representatives of three independent experiments.

Figure 4. The C3b-Bb structure derived from the C3bBb-SCIN complex

(a) Ribbon representation of the C3bBb complex with C3b (light and dark blue) and Bb (light and dark green). The Mg²⁺-ion is indicated by a pink sphere. (b) The C345C-VWA interface between C3b and Bb. The contact regions are coloured in blue (for C345C domain of C3b) and green (for VWA domain of Bb). The disulfide bond of C1515-C1639 in C345C domain is in stick representation. The residues mutated in FB chimeras are highlighted in spheres (red: in βA-α1 loop; beige, in α3-α4 loop and α4 helix; orange: in βD-α5 loop) based on ref. . (c) Overlay of VWA domains of Bb in complex with C3b, Bb^C428-C435 (pdb code: 1RRK) and C2a (pdb code: 2I6Q) showing the position of helix α7 and the nascent N-terminus. The MIDAS loop βA-α1, α3-α4 and βD-α5 are coloured green. The N-terminus of Bb^C428-C435 is missing and indicated by a dashed line.

Figure 5. The C3b-C3b interface and substrate-binding model

(a) Surface representation of C3b with the C3b-C3b dimer interface shown in yellow, which is formed by domains MG4 (brown) and MG5 (green) of the β-ring of C3b (MG1−6) (cyan). (b) Model of the enzyme-substrate (C3bBb-C3) complex constructed by superimposing C3 (pdb code: 2A73) on the MG4−5 domains of the dimeric C3b molecule in the complex. The convertase C3bBb is colored as in Fig. 4a with the catalytic triad of Bb are indicated by red spheres. The substrate C3 is shown in grey with the anaphylatoxin domain (C3a) highlighted in coral and the scissile bond (S726-R727) indicated by magenta spheres. The dashed line indicates the distance (∼30 Å) between catalytic site and the scissile loop. (c) The relative orientation of C3a domain of C3 (substrate) and the surface loops of the SP domain of Bb forming the substrate-binding groove.