The central portion of factor H (modules 10-15) is compact and contains a structurally deviant CCP module

Abstract

The first eight and the last two of 20 complement control protein (CCP) modules within complement factor H (fH) encompass binding sites for C3b and polyanionic carbohydrates. These binding sites cooperate self-surface selectively to prevent C3b amplification, thus minimising complement-mediated damage to host. Intervening fH CCPs, apparently devoid of such recognition sites, are proposed to play a structural role. One suggestion is that the generally small CCPs 10-15, connected by longer-than-average linkers, act as a flexible tether between the two functional ends of fH; another is that the long linkers induce a 180 degrees bend in the middle of fH. To test these hypotheses, we determined the NMR-derived structure of fH12-13 consisting of module 12, shown here to have an archetypal CCP structure, and module 13, which is uniquely short and features a laterally protruding helix-like insertion that contributes to a prominent electropositive patch. The unusually long fH12-13 linker is not flexible. It packs between the two CCPs that are not folded back on each other but form a shallow vee shape; analytical ultracentrifugation and X-ray scattering supported this finding. These two techniques additionally indicate that flanking modules (within fH11-14 and fH10-15) are at least as rigid and tilted relative to neighbours as are CCPs 12 and 13 with respect to one another. Tilts between successive modules are not unidirectional; their principal axes trace a zigzag path. In one of two arrangements for CCPs 10-15 that fit well with scattering data, CCP 14 is folded back onto CCP 13. In conclusion, fH10-15 forms neither a flexible tether nor a smooth bend. Rather, it is compact and has embedded within it a CCP module (CCP 13) that appears to be highly specialised given both its deviant structure and its striking surface charge distribution. A passive, purely structural role for this central portion of fH is unlikely.

Fig. 1

Summary of human fH and its orthologues. (a) CCPs are represented by ovals sized to reflect the number of residues that each contains (51–62 residues); intermodular linker lengths (from three to eight residues) and ligand-binding regions are also summarised. Modules 10–15 are highlighted; the blow-up displays the number of residues in each CCP and the aligned sequences of linkers within orthologues. Also shown is an equivalent region (CCPs 3–7 out of nine CCPs) in human fH-related protein 5 (labelled fH-r5). (b) Sequence of CCPs 12 and 13 aligned based on their structures. β-Strands are indicated by arrows and by the first and the last residue numbers. Shaded boxes highlight identities. Underscored residues are exposed, an overscore identifies a buried residue, and a double overscore indicates a residue buried at the intermodular interface.

Fig. 2

Assigned ¹H,¹⁵N HSQC spectrum of fH12–13. See the text for sample conditions and data collection parameters. Alphanumeric characters indicate assignments. Spectrally folded peaks are shown in red. Paired asparagine and glutamine NH₂ resonances are joined by dotted lines.

Fig. 3

Ensemble of NMR-derived structures. Backbone overlays (and RMSDs) of the 20 lowest-energy structures selected from the 100 calculated. (a) Overlaid on module 12. (b) Overlaid on module 13. (c) Overlaid on both CCPs (RMSD per residue plotted in Fig. 4). (d) Summary of intermodular angles for ensemble. (e) Cartoon (PyMOL: http://www.pymol.org) of the nearest-to-mean structure; disulphides are highlighted by sphere representations of sulphur atoms. β-Strands (from STRIDE⁴¹) are labelled on the basis of alignment (data not shown) with other CCP structures and convention based on the occurrence of a maximum of eight strands (A–H) in any given CCP.

Fig. 4

Relaxation data for fH12–13. T₁, T₂, and ¹H,¹⁵N NOE values plotted against residue number. The central panel incorporates, in addition to T₂ values (left-hand y-axis), the per-residue RMSD (right-hand y-axis) for the ensemble (overlaid on the bimodule). On each panel, β-strands (numbered according to the convention used in Fig. 3) are summarised; a curvy line represents the intermodular linker. Along the x-axes, circles indicate residues giving rise to broad or weak NH signals from which no measurements could be made; triangles correspond to prolines; and (^⁎) labels the amide that was not found in the HSQC.

Fig. 5

Comparisons of CCPs 12 and 13 with a set of known CCP structures. Cartoon representations (PyMOL) of CCPs 12 and 13 flank a C^α trace overlay (generated using the program MAMMOTH-mult¹⁹) of all CCPs with experimentally derived three-dimensional structures from the complement system. Highlighted within the overlay are CCP 12 (red) and CCP 13 (blue).

Fig. 6

Illustrations of the electrostatic surface and intermodular interface of fH12–13. (a) Electrostatic surface (top; same view as in Fig. 3e); CCP 12 is predominantly electronegative, whilst the linker and CCP 13 display an electropositive patch that includes the helix-like hypervariable region. (b) In this cartoon (PyMOL), disulphides are drawn as sticks (yellow sulphur atoms), CCP 12 is shown in red, CCP 13 is shown in blue, and linker is shown in yellow/orange. Different shades of these colours are used to distinguish side chains (drawn as spheres; heavy atoms only) contributing to the intermodular interface. Side chains are labelled in the blow-up (bottom).

Fig. 7

Overview of SAXS data and analysis. (a) Scattering curves for fH12–13 (red), fH11–14 (blue), and fH10–15 (yellow). Continuous lines represent fits obtained by CRYSOL for the best fH12–13 NMR model, or by rigid-body modelling (BUNCH) for fH11–14 and fH10–15; curves have been arbitrarily displaced along the logarithmic axis for clarity. (b) p(r) functions (arbitrary units) for fH12–13 (red), fH11–14 (blue), and fH10–15 (yellow), computed from X-ray scattering patterns using GNOM. (c) Radius-of-gyration distributions of pools (red lines) and selected structures (black) for fH12–13, fH11–14, and fH10–15 using EOM. Integral of area defined by histograms = 1.

Fig. 8

Modelling from SAXS data. (a) Overlay of DAMMIF-derived ab initio shape envelope (left) and GASBOR-derived dummy residue model (right) with the NMR-derived ensemble of fH12–13. (b) Overlay of the DAMMIF-derived ab initio shape envelope with the most typical BUNCH-derived model of fH11–14 (left); ensemble of 10 BUNCH models for fH11–14 (right). (c) Overlay of the DAMMIF-derived ab initio shape envelopes with the two most typical BUNCH models of fH10–15 (I and II). (d) The two most typical BUNCH models of fH10–15 (I and II). In (a)–(c), lower views are rotated 90° clockwise about the horizontal axis. The CCPs are shown in green (CCP 10), cyan (CCP 11), red (CCP 12), blue (CCP 13), yellow (CCP 14), and pink (CCP 15), with orange spheres representing linker residues modelled by BUNCH as a chain of dummy atoms.