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

Wu J, Wu YQ, Ricklin D, Janssen BJ, Lambris JD, Gros P - Nat. Immunol. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Utrecht, The Netherlands.

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.

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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. 30. 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.
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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. 30. 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.

Mentions: FH supports two, out of three possible, cleavages in the CUB domain of C3b by FI (between R1281-S1282 and R1298-S1299) that yield the inactive iC3b species (Fig. 1e)28. The crystal structure revealed that FH CCP2-3 bound to C3b adjacent to the CUB domain (Fig. 2a, Fig. 5a) with the hypervariable loop of CCP3 directly contacting CUB (Fig. 5a,b)10. Cleavage site R1281-S1282 was well-exposed, whereas R1298-S1299 was occluded in the complex (Fig. 5b), which confirms the notion that R1281-S1282 is cleaved first and suggests that cleavage of R1298-S1299 requires conformational changes. The strong influence of ionicity for the binding of FI indicates that polar interactions are highly important for this interaction29. FH CCP1-3 indeed exhibited several conserved and charged patches on its surface (Fig. 5a and Supplementary Fig. 7 online), which may be involved in the binding of FI. For the FH-homologue vaccinia virus complement control protein (VCP), CCP2 was shown to have a dominant role in complement inhibition. Mutation of four residues in CCP2 markedly enhanced cofactor activity, while only moderately increasing the VCP-C3b affinity30. The equivalent residues in FH (Gln101, Ile106, Asp112 and Asp119) were fully exposed and found adjacent to conserved patches (Fig. 5a), which suggests a role for these four residues in the binding of FI. Furthermore, studies on cobra-venom factor (CVF) indicated that the C345C domain of C3b may contribute to this interaction31. These data suggest that FI binds the C3b-FH complex at the area formed by CCP1-3 of FH and C345C and CUB of C3b (Supplementary Fig. 7 online). Besides providing a platform for FI, FH may have a putative second role in cofactor activity: we hypothesize that the bridge formed by CCP4 between TED and the core of C3b may maintain the position of TED while the connecting CUB domain undergoes further cleavage. This suggested function is supported by disease-related mutations in either FH CCP4 (refs. 32, 33) and in TED of C3b34, which indicates a functional importance for RCA interactions with TED.


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

Wu J, Wu YQ, Ricklin D, Janssen BJ, Lambris JD, Gros P - Nat. Immunol. (2009)

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. 30. 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.
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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. 30. 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.
Mentions: FH supports two, out of three possible, cleavages in the CUB domain of C3b by FI (between R1281-S1282 and R1298-S1299) that yield the inactive iC3b species (Fig. 1e)28. The crystal structure revealed that FH CCP2-3 bound to C3b adjacent to the CUB domain (Fig. 2a, Fig. 5a) with the hypervariable loop of CCP3 directly contacting CUB (Fig. 5a,b)10. Cleavage site R1281-S1282 was well-exposed, whereas R1298-S1299 was occluded in the complex (Fig. 5b), which confirms the notion that R1281-S1282 is cleaved first and suggests that cleavage of R1298-S1299 requires conformational changes. The strong influence of ionicity for the binding of FI indicates that polar interactions are highly important for this interaction29. FH CCP1-3 indeed exhibited several conserved and charged patches on its surface (Fig. 5a and Supplementary Fig. 7 online), which may be involved in the binding of FI. For the FH-homologue vaccinia virus complement control protein (VCP), CCP2 was shown to have a dominant role in complement inhibition. Mutation of four residues in CCP2 markedly enhanced cofactor activity, while only moderately increasing the VCP-C3b affinity30. The equivalent residues in FH (Gln101, Ile106, Asp112 and Asp119) were fully exposed and found adjacent to conserved patches (Fig. 5a), which suggests a role for these four residues in the binding of FI. Furthermore, studies on cobra-venom factor (CVF) indicated that the C345C domain of C3b may contribute to this interaction31. These data suggest that FI binds the C3b-FH complex at the area formed by CCP1-3 of FH and C345C and CUB of C3b (Supplementary Fig. 7 online). Besides providing a platform for FI, FH may have a putative second role in cofactor activity: we hypothesize that the bridge formed by CCP4 between TED and the core of C3b may maintain the position of TED while the connecting CUB domain undergoes further cleavage. This suggested function is supported by disease-related mutations in either FH CCP4 (refs. 32, 33) and in TED of C3b34, which indicates a functional importance for RCA interactions with TED.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Utrecht, The Netherlands.

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.

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