Electrostatic modeling predicts the activities of orthopoxvirus complement control proteins

Abstract

Regulation of complement activation by pathogens and the host are critical for survival. Using two highly related orthopoxvirus proteins, the vaccinia and variola (smallpox) virus complement control proteins, which differ by only 11 aa, but differ 1000-fold in their ability to regulate complement activation, we investigated the role of electrostatic potential in predicting functional activity. Electrostatic modeling of the two proteins predicted that altering the vaccinia virus protein to contain the amino acids present in the second short consensus repeat domain of the smallpox protein would result in a vaccinia virus protein with increased complement regulatory activity. Mutagenesis of the vaccinia virus protein confirmed that changing the electrostatic potential of specific regions of the molecule influences its activity and identifies critical residues that result in enhanced function as measured by binding to C3b, inhibition of the alternative pathway of complement activation, and cofactor activity. In addition, we also demonstrate that despite the enhanced activity of the variola virus protein, its cofactor activity in the factor I-mediated degradation of C3b does not result in the cleavage of the alpha' chain of C3b between residues 954-955. Our data have important implications in our understanding of how regulators of complement activation interact with complement, the regulation of the innate immune system, and the rational design of potent complement inhibitors that might be used as therapeutic agents.

Figure 1

Residue differences between VCP and SPICE A, Ribbon representation of the modeled structure of SPICE with stick representation of the 11 mutations of SPICE relative to VCP. The structure of SPICE was modeled using chain A of the VCP structure with PDB code 1g40. The SCR modules and the mutations are marked in the figure B, VCP-mutants expressed in E. coli. The first letter represents the VCP amino acid; the number indicates the amino acid position; and the second letter indicates the SPICE amino acid.

Figure 2

Biochemical characterization of VCP and SPICE. A, Mass spectrometric analysis of VCP (left panel) and SPICE (right panel) B, Coomassie-stained 10% SDS-PAGE gel showing VCP and SPICE under reducing conditions. Numbers on the left indicate the molecular mass in kilodaltons of mass markers C, CD analysis of the proteins. Secondary structure composition calculated by deconvoluting the spectra using the CDSSTR algorithm, which is a part of the web based CD analysis tool DICHROWEB (28, 29).

Figure 3

Complement modulating activity of VCP and SPICE. A, Binding of SPICE and VCP to human C3b by SPR. SPR experiments were conducted using an SA chip where biotinylated C3b was immobilized. Solutions of SPICE and VCP (each 50 nM) were passed through with a flow of 10 μl/min. After injection of the protein (360 s), SPR was followed for an additional 200 s. The response during the time is expressed in resonance response units (RU). B, Inhibition of complement activation. The deposition of human C3b in the presence of various amounts of SPICE and VCP on an immunocomplex (classical pathway) or on LPS (alternative pathway) was detected using a polyclonal anti-C3b Ab. The results are expressed as the percent inhibition of complement activation. The solid lines indicate the classical pathway, and the dashed lines indicate the alternative pathway C, Factor I cofactor activity of VCP and SPICE. VCP and SPICE (300 ng) were incubated with C3b (500 ng) in the presence of factor I (fI, 10 ng). Samples were collected at indicated time points and were subjected to Western blot analysis. For the detection of the degradation products of C3b, a polyclonal anti-C3b Ab was used. The appearance of the 46 kDa and the 43 kDa indicate the generation of the iC3b₁ and iC3b₂, respectively.

Figure 4

Iso-potential contour surfaces for native VCP and several theoretical mutants of VCP including SPICE. Blue and red denote positive and negative electrostatic potential, respectively. The structure of VCP with PDB Code 1g40 (19) was used to construct the structures of SPICE and theoretical mutants. The coordinates of all mutants were fit to the coordinates of native VCP using the backbone Cα atoms. The orientation of the structures and the electrostatic potential calculations were performed using the same parameters for all panels. Modeling was performed using the program Swiss-PdbViewer (26).

Figure 5

Mapping of SPICE SCRs involved in the enhanced interaction of SPICE with C3b A, Binding of VCP-SPICE mutants to immobilized C3b. The experiment was conducted as described in Fig. 3A. B, Inhibition of alternative pathway by VCP-SPICE variants. The experiment was conducted as described in Fig. 3B. C, factor I (fI) cofactor activity of VCP-SPICE mutants. Recombinant proteins were incubated with C3b in the presence of fI and analyzed as described in Fig. 3C.

Figure 6

Mapping of the activity of SPICE by single mutations in SCR-2. A, Analysis of the binding of VCP-SPICE variants to immobilized C3b. The letters and numbers above each arrow represent the individual residue changes in VCP summarized in Fig. 1B. The experiment was conducted as it is described in Fig. 3A. B, Inhibition of alternative pathway complement activation by VCP-SPICE mutants. The experiment was conducted as described previously in Fig. 3B.

Figure 7

Mapping of the activity of SPICE by mutating positions E108K and E120K on VCP. A, Binding of VCP variants to immobilized C3b. The experiment was conducted as it is described in Fig. 3A. B, factor I cofactor activity of VCP-E108K/E120K. The experiment was performed as described in Fig. 3C.