Here we illuminate how FH binds to its principal target. This crucial event in regulation of C3b promulgation on self-surfaces is central to protection of host tissues from complement-mediated damage. Building on previous indications that FH19–20 binds to both C3b and the C3b-derived fragment, C3d 32
, we showed that the C3d:FH19–20 interaction is a surrogate for the less experimentally accessible FH:C3b interaction. Our new observations, when combined with published data, show how the FH molecule bends back on itself such that its N terminus performs cofactor and decay accelerating activities while the C terminus recognizes a composite consisting of the TED of C3b and nearby polyanionic carbohydrates. This allows distinction of a self-surface from most bacterial ones.
Mindful of controversy surrounding the physiological validity of a crystal structure of C3d complexed with CR2 CCP1-2 40
, we employed mutagenesis and NMR to select the physiologically relevant intermolecular interface from several possibilities within our crystal structure of C3d:FH19–20. Moreover this interface of C3d is consistent with other observations. (i)
It is accessible in TED (i.e.
within the context of C3b) tethered to a surface; moreover the orientation of FH19–20 means that CCP18 is projected clear of C3b, consistent with our finding that it binds neither C3d nor C3b (Supplementary Fig. 4
and Supplementary Table 3
It does not overlap the FH1–4 interaction site on C3b 14
, consistent with avidity between sites 12
It is directly adjacent to (but non-overlapping with) the Efb-C-binding site 41
(Supplementary Fig. 8
); this is important because Efb-C enhances binding of C3b to FH19–20 42
The C3d and FH residues involved are conserved (Supplementary Fig. 9
We confirmed that previously identified GAG-binding residues of CCP20 are still accessible within this complex. This is compelling structural evidence for the hypothesis that FH19–20 interacts with a composite site consisting of the TED and proximal polyanions. Previous attempts to dock C3d onto the surface of FH19–20 now prove to have been unsuccessful 16,43
. In both of these models of C3d:FH19–20, only CCP20 makes contact with C3d; moreover the interfaces on C3d differ from that identified in our co-crystal structure.
Our new data reveal locations, relative to key interaction surfaces, of disease-linked mutations and sequence variations (). Our structure explains the key role in C3b binding we observed for the disease-linked mutation D1119G, despite suggestions from others that this mutation is functionally null 32
. Our complex also shows that the following aHUS-linked FH mutations 44
affect the C3d:FH19–20 interface and thereby perturb the fine balance of affinities needed for proper FH operation: Y1142D and Y1142C, since Tyr1142 H-bonds to C3d’s Lys178 (Lys1171); Q1143E due to its proximity to Tyr1142; L1189F, L1189R and S1191L, since Tyr1190 H-bonds to Asp122 (Asp1115) of C3d and is partially buried at the interface. Other aHUS-linked FH mutations are in the GAG-binding regions of CCP20 and/or could disrupt putative electrostatic steering events (E1198A, R1203A, R1210C, R1215G, R1182S, W1183R and T1184R). Another subset of aHUS-linked FH mutants likely promotes structural perturbations (W1157R, V1134G, G1194D, V1197A, F1199S and P1226S). Regarding the C3b side of the interaction, the following aHUS-linked mutations likely disrupt the binding interface: P121L (P1114L) 45
and D122N (D1115N) 46
are in the C3d α4-α5 loop occupying the FH19–20 intermodular cleft; C165W (C1158W) and Q168K (Q1161K) 46
are in the C3d α6–α7 loop that, intriguingly, coincides with closest approach of the N-terminal and C-terminal C3b-binding sites of FH ().
Figure 6 The location of mis-sense mutations associated with the development of (aHUS) (and Q1139A) mapped onto the structure of the C3d:FH19–20 complex. The side-chains of C3b mis-sense mutations are shown as blue sticks; side-chains of FH19–20 (more ...)
In a previously solved structure of the FH1–4:C3b complex 14
, the four N-terminal CCPs of FH form an elongated contact with C3b running largely parallel with the long axis of C3b. If C3b in this complex were surface-attached via its thioester, CCP1 would be furthest, and CCP4 closest, to that surface. In other AP regulators, a similar three-CCP or four-CCP C3b-binding segment is connected to a C-terminal transmembrane helix or a GPI anchor, which localizes the regulator upon a cell surface requiring protection from C3b promulgation. FH is not constrained to a membrane but relies on some or all of its C-terminal 16 CCPs for localization, and exploits the preponderance of polyanionic self-surface markers to selectively increase its residency time on self-surfaces. We investigated the path taken by CCPs 5-18, which connect N-terminal and C-terminal C3b-binding sites, to construct an experimentally derived model of the FH:C3b complex.
When the C3d of C3d:FH19–20 is superimposed on the TED of C3b, the FH1–4 C-terminus and FH19–20 N-terminus are close together. A bent-back FH structure would therefore be required were a single molecule to simultaneously occupy both sites on C3b (and interact with self-surface associated polyanions), as in our model (). This is fully consistent with our SAXS-derived shape-envelopes of FH fragments presented here, as well as with existing SAXS, crystal and NMR-derived structures, and agrees with previous work showing that non-liganded FH could adopt a predominantly bent-back conformation 37,47-50
. Our data are incompatible with an alternative model in which FH wraps around the C3b molecule. However, we cannot exclude the existence of a complex in which one FH molecule binds to two neighboring, closely packed, C3b molecules.
Previous studies suggested a weak additional C3b-binding site within FH CCPs 6–8 12,13
. While no such interface exists within our model of the complex, uncertainties in reconstruction of the N-terminal half of FH (lacking structural information for FH4–5/FH5–6) means we cannot exclude CCP6/7-C3b contacts. Nonetheless, our model places CCP7 close to the self-surface where it could recognize additional GAG surface markers; it also places the structurally deviant module, CCP13 37
close to the self-surface, although the nature of any ligand for CCP13 remains undetermined.
In summary, we present an experimentally supported structural model for how a FH molecule engages bivalently with a C3b molecule and simultaneously with self-surface polyanionic markers. The model indicates that effective complement inhibitors, with therapeutic potential, might be constructed through flexible linkage of FH19–20, via a domain with appropriately apposed N and C termini, to FH1–4 or homologous segments of other complement regulators.