Kinetic Analysis of the Interactions between Vaccinia Virus Complement Control Protein and Human Complement Proteins C3b and C4b

Abstract

The vaccinia virus complement control protein (VCP) is an immune evasion protein of vaccinia virus. Previously, VCP has been shown to bind and support inactivation of host complement proteins C3b and C4b and to protect the vaccinia virions from antibody-dependent complement-enhanced neutralization. However, the molecular mechanisms involved in the interaction of VCP with its target proteins C3b and C4b have not yet been elucidated. We have utilized surface plasmon resonance technology to study the interaction of VCP with C3b and C4b. We measured the kinetics of binding of the viral protein to its target proteins and compared it with human complement regulators factor H and sCR1, assessed the influence of immobilization of ligand on the binding kinetics, examined the effect of ionic contacts on these interactions, and sublocalized the binding site on C3b and C4b. Our results indicate that (i) the orientation of the ligand is important for accurate determination of the binding constants, as well as the mechanism of binding; (ii) in contrast to factor H and sCR1, the binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions as predicted earlier; (iii) VCP has a 4.6-fold higher affinity for C4b than that for C3b, which is also reflected in its factor I cofactor activity; (iv) ionic interactions are important for VCP-C3b and VCP-C4b complex formation; (v) VCP does not bind simultaneously to C3b and C4b; and (vi) the binding site of VCP on C3b and C4b is located in the C3dg and C4c regions, respectively.

Species Selectivity in Poxviral Complement Regulators Is Dictated by the Charge Reversal in the Central Complement Control Protein Modules

“…The binding abilities of SPICE, VCP, and the mutants to bovine C3b were determined using surface plasmon resonance (SPR)-based biosensor Biacore 2000 (Biacore, Uppsala, Sweden) (36). Bovine C3b (∼5000 response units, RU) labeled through its free thiol group with biotin (Supplemental Fig.…”

Functional Characterization of the Complement Control Protein Homolog of Herpesvirus Saimiri

“…Sequence alignment of sCCPH with SPICE showed that sCCPH has Leu (Leu-106) and Arg (Arg-118) in the corresponding positions. Because earlier studies have shown that ionic interactions play a critical role in CCP-C3b/C4b interactions (34,(42)(43)(44) we suspected that Arg-118 might be responsible for the enhanced C3b cofactor activity. To further probe this possibility, we built a three-dimensional model of sCCPH by homology modeling using the crystal structure of VCP (45) as the template.…”

“…The experiments were performed in phosphate-buffered saline-Tween (10 mM sodium phosphate, 145 mM NaCl, pH 7.4, containing 0.05% Tween 20) at 25°C. For proper orientation of these proteins, the free SH groups of both C3b and C4b were biotinylated and then immobilized on the streptavidin chip (Sensor Chip SA, Biacore AB) (34). FC-2 was immobilized with C3b (1592 RU), FC-3 was immobilized with C4b (1197 RUs), and FC-1 (blank flow cell) served as the control flow cell.…”

“…Measurement of Factor I Cofactor Activity-Analysis of factor I cofactor activities of sCCPH and the mutant was essentially performed as described (34). These assays were performed in physiological ionic strength buffer (phosphate-buffered saline).…”

Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism

“…Complement C4b was immobilised on the NeutrAvidin-coated sensor surface in its physiological orientation by labelling its free exposed thiol group with biotin ( Figure 1A) [38]. The T M and T E response data for the C4b injection onto the NeutrAvidin layer occurred at 12 500 s (Figure 3).…”