Complement Inhibition by Staphylococcus aureus: Electrostatics of C3d–EfbC and C3d–Ehp Association

Abstract

Virulence factors EfbC and Ehp from Staphylococcus aureus are potent inhibitors of complement activation. Both are excessively charged and bind to complement protein C3d at an acidic interface. We computationally generated single-alanine mutants of charged residues in the C3d-EfbC and C3d-Ehp complexes, and utilized electrostatic clustering and Poisson-Boltzmann free energy calculations to evaluate the role of electrostatics in association. Our results indicate that both interfacial electrostatic interactions and electrostatic potential distribution are crucial for C3d-EfbC and C3d-Ehp association. The results presented herein serve as a predictive tool in the selection of mutants with desired binding and immunological activity, and will narrow the search for viable candidates for further computational and experimental analyses. This study serves as the foundation for development of an inhibitor of the C3d-EfbC and C3d-Ehp interactions to combat bacterial infection and design of a complement inhibitor using EfbC and Ehp as a model.

The Two Sides of Complement C3d: Evolution of Electrostatics in a Link between Innate and Adaptive Immunity

“…The C3d-CR2 interaction greatly improves the immune response to an antigen, and as a result has been selected by nature as a target for immune evasion [18]–[20]. Structural evidence has shown that virulence factors of Staphylococcus aureus target the electrostatic “hot-spots” of human C3d (Supporting Figure S2).…”

“…Structural evidence has shown that virulence factors of Staphylococcus aureus target the electrostatic “hot-spots” of human C3d (Supporting Figure S2). For example, Staphylococcus aureus secretes the highly cationic virulence factors EfbC and Ehp, which take advantage of the CR2 electrostatic “hot-spot” through the use of long-range, as well as short-range, electrostatic interactions (Supporting Figure S2A) [18]. Additionally, domain IV of the Staphylococcal immunoglobulin-binding protein (Sbi) targets the thioester side electrostatic “hot-spot” of C3d (Supporting Figure S2B), and in conjunction with Sbi domain III results in futile consumption of C3 through the formation of covalent adducts [19].…”

“…The acidic “patch” has been shown to be involved in recognition and binding during the association of C3d to CR2 [8], [10], as well as to bacterial inhibitors of the complement system [18]–[20]. The basic surface however, accelerates the covalent attachment of C3/C3d to pathogenic cell surfaces.…”

Immunophysical Evaluation of the Initiating Step in the Formation of the Membrane Attack Complex

“…We also performed a comparative analysis of the electrostatic properties of C3d, C4d, and C5d. Earlier works have studied the properties of a highly negatively charged concave surface on C3d that is involved in recognition and binding of complement regulators [29] and receptors [28,41] as well as bacterial inhibitors [30,31]. Figure 6A shows the C3d concave surface with the negatively charged patch, surrounded by a neutral rim.…”

“…The viral vaccinia control protein (VCP), that is structurally and functionally homologous to SPICE, is also expected to have a similar binding mode to C3b. In addition, the stand-alone C3d domain, is known to interact with modules CCP1-2 of complement receptor 2 (CR2) [28], modules CCP19-20 of FH [29], in addition to modules CCP1-4 (mentioned above as interacting along C3b), and S. aureus proteins Efb-C, Ecb, and Sbi [30,31]. These structural observations make the C3d domain multifunctional in interacting with complement natural and viral regulators, when C3d is part of C3b, and in attracting CR2, FH (CCP19-20), and bacterial regulators when C3d is stand-alone.…”