Structural basis for complement factor H linked age-related macular degeneration

Abstract

Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to approximately 50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulator's surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance-monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG-FH complex.

Figure 1.

The structure of FH modules 6–8 reveals multiple sulfated sugar-binding sites. (A) The overall structure of FH678_402H is shown in cartoon representation colored from blue at the N terminus (residue 320) to red at the C terminus (residue 506). Electron density for the bound SOS is presented as an F_O–F_C map contoured at 2 σ, which was generated before any modeling of the sugar was attempted. All figures were generated using PyMOL (25). SOS-binding sites are labeled (i–iv) and enlarged views are shown below the main panel. SOS is represented as a ball and stick colored by atom type (C, green; O, red; N, blue; S, yellow). Amino acid sidechains that contact the sugar are shown in stick representation, whereas the rest of the protein molecule is shown as a ribbon. Intermolecular hydrogen bonds are shown as dashed orange lines. The polymorphic residue associated with AMD (His402) is highlighted. (B) Model illustrating the proposed ability of the Tyr/His polymorphism at position 402 to differentially bind sulfated sugars. Position of Tyr402 was taken from the NMR structure of FH7_402Y (Protein Data Bank ID, 2JGX).

Figure 2.

Characterization of GAG-binding sites. (A) Electrostatic surface potential (plotted at ± 5 kT/e) of FH678_402H reveals a positively charged groove that extends between the three major SOS-binding sites. The residues lining the groove that have previously associated with polyanion binding, Arg387, Lys388, and Lys405 (16, 18), are highlighted in blue. The polymorphic residue associated with AMD (His402) is highlighted in red (–5). (B; ¹H, ¹⁵N) HSQC spectrum for free FH78_402Y (50 μM; black) and in the presence of SOS. Protein/ligand ratios are 1:0.6 (red), 1:1.2 (orange), 1:2.5 (green), 1:5 (blue), and 1:10 (purple). The y axis corresponds to ¹⁵N chemical shifts, and the x axis corresponds to ¹H chemical shifts, both in parts per million. Conditions were 20 mM potassium phosphate, pH 7.4, 298 K. (C) Chemical shift perturbations mapped on the surface of FH678_402H. SCR6 is shown in dark gray, as it was not present in the constructs used for these experiments. The sidechains of residues that exhibit the largest combined ¹⁵N and ¹H chemical shift perturbations are highlighted in pink (FH7) and purple (FH78). The data presented in B and C are also shown in Fig. S4 with the molecule rotated in other views. Fig. S4 is available at http://www.jem.org/cgi/content/full/jem.20071069/DC1.

Figure 3.

Proposed model for FH interactions with sulfate-rich domains of GAGs on the cell surface. (A) Analysis of binding of different fragments of FH_402H to a heparin-HiTrap column confirms the binding site in SCR6 and reveals pH dependency of binding by SCR6 and 7. This supports the involvement of histidine sidechains in glycan coordination, as revealed by the structure. (B) Mutation of sidechains seen to coordinate SOS in SCR6 alter the affinity of FH67_402H for a heparin-HiTrap column. (C and D) Model for simultaneous FH binding to C3b (light blue spheres; PDB code, 2I07 [reference 26]) and GAGs (red/green spheres; PDB code, 1HPN [reference 27]) on the retinal epithelium. FH modules for which structural data is available are represented as cartoons and semitransparent surfaces: SCR5 (28), SCR6–8 (PDB code 2UWN; this study), SCR15/16 (Protein Data Bank ID, 1HFH [reference 29]), SCR19/20 (PDB code 2G7I/2BZM [references 30, 31]). Mutational data (dark blue spheres; for review see [reference 32]) mapped onto the structure of C3b suggest FH coils around C3b like a snake.