Structure of complement fragment C3b-factor H and implications for host protection by complement regulators

Abstract

Factor H (FH) is an abundant regulator of complement activation and protects host cells from self-attack by complement. Here we provide insight into the regulatory activity of FH by solving the crystal structure of the first four domains of FH in complex with its target, complement fragment C3b. FH interacted with multiple domains of C3b, covering a large, extended surface area. The structure indicated that FH destabilizes the C3 convertase by competition and electrostatic repulsion and that FH enables proteolytic degradation of C3b by providing a binding platform for protease factor I while stabilizing the overall domain arrangement of C3b. Our results offer general models for complement regulation and provide structural explanations for disease-related mutations in the genes encoding both FH and C3b.

Figure 1

Characterization of recombinant FH(1–4). (a) Purity of FH(1–4) as assessed by SDS-PAGE (4–15% gradient gel; reducing conditions; Coomassie blue staining). (b–c) Direct binding between soluble FH(1–4) and surface-bound C3b. Injection of the FH fragment (0.08–20 µM) leads to the formation of a short-lived 1:1 complex with C3b, which features a binding affinity of K_D = 11 ± 2 µM. Data are representative of five experiments with different surface densities of C3b (3,000–7,000 RU; resonance units). (d) FH(1–4) features a comparable decay acceleration activity as full-length FH when injected onto surface-based C3 convertase (C3bBb). Additional details and control injections for the decay acceleration assay can be found in Supplementary Fig. 9 online. Data are representative of three independent experiments. (e) The cofactor activity of FH(1–4) is similar to that of FH: incubation of C3b with FI and increasing amounts of either FH(1–4) or FH(1–20) leads to the generation of iC3b as visible by the degradation of the α’ chain to three fragments of 43, 46, and 68 kDa, respectively. Data are representative of more than five individual experiments.

Figure 2

Structure of C3b in complex with FH domains CCP1–4. (a) Overall structure of the C3b-FH(1–4) complex. C3b is shown in ribbon and FH is shown in surface representations; domains are indicated and the thioester is shown by red spheres. (b) On the left-hand side, comparison of the FH CCP1–4 as observed in the complex (orange) with solution structures of CCP1–2 (blue) and CCP2–3 (magenta); and, on the right-hand side, comparison of C3b as observed in complex with FH(1–4) (cyan) and free C3b (grey). Figures were generated by using PyMOL (http://www.pymol.org).

Figure 3

Mapping FH and C3 mutants on complex structure. The molecules are shown in surface representation with FH(1–4) on the left side and C3b on the right side. The four contact regions (I–IV) between FH(1–4) and C3b are highlighted in blue with mutations related to AMD, MPGN-II and aHUS are indicated in red (for FH mutants) and orange (for C3b mutants).

Figure 4

Structural basis of decay acceleration activity. (a) Overlay of the C3b-FH and C3b-Bb complexes (top figure) with C3b (light cyan) and FH CCP1–3 (beige, yellow orange and orange, respectively) in surface representation and Bb in ribbon representation (light green and dark green for VWA and SP domains, respectively); the red surface area indicates the region where FH and Bb overlap (atomic distances less than 2 Å). Bottom figure shows the overlap region (red) in Bb (Bb is rotated by 180° with respect to the top figure). (b) Electrostatic surface potential of FH and ribbon representation of Bb (left side) and vice versa (right side) in the regions facing each other. The potential contours are shown on a scale from −5 (red) to +5 k _b Te _c ⁻¹ (blue). (c) Superposition of DAF CCP2–4 on to FH CCP1–3 shown in the same representation as in panel a. Mutants of DAF are coloured by degree of functional interference, from minor (beige), medium (yellow) and severe (magenta) effect up to complete abortion (red) of decay acceleration by DAF.

Figure 5

Structural implications for cofactor activity. (a) Sites of FI binding and cleavage. FH is shown in surface representation and colour-coded to residue conservation with mutational data from ref. . The conservation scale and corresponding colours are indicated in Supplementary Fig. 4 online. Residues with the conservation scores less than 5 are all coloured in white. Mutations in VCP enhancing FI binding are indicated in orange and the hyper-variable loop (hv-loop) of CCP3 in green. C3b (cyan) is shown in cartoon representation with the CUB (blue), a’NT (yellow) and C345C (dark red) domains highlighted and the first and second scissile bond in the CUB domain indicated by red spheres. (b) Surface representation of the complex from the top view. Domains of C3b are coloured the same as in panel a, and FH domains are in grey. The colouring of domains and FI cleavage sites is consistent with a.