Energetic evaluation of binding modes in the C3d and Factor H (CCP 19-20) complex

Abstract

As a part of innate immunity, the complement system relies on activation of the alternative pathway (AP). While feed-forward amplification generates an immune response towards foreign surfaces, the process requires regulation to prevent an immune response on the surface of host cells. Factor H (FH) is a complement protein secreted by native cells to negatively regulate the AP. In terms of structure, FH is composed of 20 complement-control protein (CCP) modules that are structurally homologous but vary in composition and function. Mutations in these CCPs have been linked to states of autoimmunity. In particular, several mutations in CCP 19-20 are correlated to atypical hemolytic uremic syndrome (aHUS). From crystallographic structures there are three putative binding sites of CCP 19-20 on C3d. Since there has been some controversy over the primary mode of binding from experimental studies, we approach characterization of binding using computational methods. Specifically, we compare each binding mode in terms of electrostatic character, structural stability, dissociative and associative properties, and predicted free energy of binding. After a detailed investigation, we found two of the three binding sites to be similarly stable while varying in the number of contacts to C3d and in the energetic barrier to complex dissociation. These sites are likely physiologically relevant and may facilitate multivalent binding of FH CCP 19-20 to C3b and either C3d or host glycosaminoglycans. We propose thermodynamically stable binding with modules 19 and 20, the latter driven by electrostatics, acting synergistically to increase the apparent affinity of FH for host surfaces.

Keywords: C3d; MM/GBSA binding free energy; Poisson-Boltzmann electrostatics; complement system; factor H; molecular dynamics; steered molecular dynamics.

Figure 1

Comparison of complement response from the AP on surfaces of self (blue) and surfaces of nonself (red). (1, 4) C3b binds to both native and foreign surfaces through nucleophilic attack on an internal thioester bond. (2) On surfaces of self, FH is more likely to interact with attached C3b, preventing association with factor B and limiting the formation of the C3 convertase. In the case that C3 convertase does form, FH acts to accelerate decay of the complex. (3) Additionally, FH serves as a cofactor for conversion of attached C3b to inactive C3b that is eventually cleaved to C3d that remains surface-bound. (5) On surfaces of nonself, factor B associates with bound C3b. (6) Factor D subsequently cleaves a portion of factor B to form the C3 convertase. (7) This enzyme cleaves serum C3 into C3b, an opsonin, and C3a, an anaphylatoxin. (8) C3b generated by the convertase is capable of binding to surfaces once again and results in amplification of immune response.

Figure 2

Molecular graphic representation of the competing binding modes from two crystallographic structures in the Protein Data Bank, 3OXU and 2XQW. Each binding site is described by one or more pairs of interacting chains (separated by a colon) from a specific crystallographic structure (PDB identifier of source noted). C3d is colored dim gray and specific residues associated with the acidic patch are colored in red while residues associated with the thioester domain are colored in yellow. FH is represented by CCP modules 19 and 20. FH CCP 20 is colored orange while CCP 19 is colored purple. At site 1, the binding configuration of C3d and FH varies between models (PDB 3OXU and 2XQW) suggesting apical flexibility.

Figure 3

Change in free energy of binding compared with the wild-type molecule resulting from computational alanine mutagenesis of (A) C3d and (B) FH. The heatmap scale is kept consistent between panels. Black for residue D1119 in panel B indicates no data since there is not an ionizable amino acid in this position within model 2XQW. Mutations boxed in green represent mutations that have been linked to aHUS. Negative values are gain of binding mutations while positive values are loss of binding mutations.

Figure 4

Change in free energy of binding compared with the wild type molecule resulting from computational site-directed mutagenesis for (A) C3d and (B) FH. The heatmap scale is kept consistent between panels. The first amino acid in brackets represents the residue in 3OXU, while the second is the residue in 2XQW. Mutations boxed in green represent mutations that have been linked to aHUS. Negative values are gain of binding mutations.

Figure 5

Interfacial SASA is plotted for each binding mode above and is colored according to binding site. Large, solid lines represent the mean values across a set of three MD trajectories, while smaller, dotted lines represent one standard deviation above or below the mean. Inset next to the key is the Interfacial SASA for site 3's extended MD trajectories from 10 to 20 ns (solid black and red lines here represent the extrapolated mean Interfacial SASA for each associated binding mode and are used to compare with the values for site 3).

Figure 6

Percent occupancies of aliphatic interactions between C3d and FH at (A) binding site 1, (B) binding site 2, and (C) binding site 3. These values represent the percentage of snapshots throughout the triplicate trajectories for each binding site where a pair of functional groups with aliphatic features were observed to be within 4 Å in the C3d:FH complex. Residue labels are colored according to secondary structure. Black represents a coil or turn; red represents an α-helix; blue represents a 3₁₀-helix; green represents a β-sheet; and magenta represents an isolated β-bridge.

Figure 7

Percent occupancies of hydrogen bond interactions between C3d and FH at (A) binding site 1, (B) binding site 2, and (C) binding site 3. These values represent the percentage of snapshots throughout the triplicate trajectories for each binding site where a hydrogen bond is present in the C3d:FH complex. Residue labels are colored according to secondary structure. Black represents a coil or turn; red represents an α-helix; blue represents a 3₁₀-helix; green represents a β-sheet; and magenta represents an isolated β-bridge.

Figure 8

Percent occupancies of salt bridges between C3d and FH at (A) binding site 1, (B) binding site 2, and (C) binding site 3. These values represent the percentage of snapshots throughout the triplicate trajectories for each binding site where a pair of chemical groups capable of forming a salt bridge were observed to be within 5 Å from each other in the C3d:FH complex. Residue labels are colored according to secondary structure. Black represents a coil or turn; red represents an α-helix; blue represents a 3₁₀-helix; and green represents a β-sheet.

Figure 9

Forces required to move FH at a constant velocity along the surface normal vector are plotted over the length of time for each SMD simulation for each binding mode. Large, solid lines represent the mean values across five, independent trajectories for each binding mode. Smaller, dotted lines represent one standard deviation above or below the mean value. Force-time responses are color coded according to the key in the top right corner.

Figure 10

Histograms of energies from MM/GBSA calculations for each binding site are shown above with a bin size of 5 kJ/mol. (A) Distributions for change in the overall free energy of binding. (B) Distributions for change in free energy resulting from electrostatic interactions. (C) Distributions for change in free energy from nonpolar interactions. Energies were calculated for all but the first 100 ps of each MD trajectory, pooling replicates from the same binding modes.