Structure-function studies of the staphylococcal complement inhibitor protein family

Abstract

Complement is a primary arm of innate immunity that plays essential roles in clearance of microbial invaders and immune complexes, initiation of inflammation, development, and differentiation and serves as a bridge to the adaptive immune response. Like many successful human pathogens, the Gram-positive bacterium Staphylococcus aureus has evolved a web of intricate strategies to evade complement-mediated immunity. These small (~10 kDa), secreted proteins bind directly to the central opsonin of the complement cascade, called C3b. Among these complement-targeted bacterial inhibitors, are the so-called Staphylococcal Complement Inhibitors (SCINs). SCINs are of particular interest because they stabilize a catalytically-inactive form of the key enzymatic complex of complement, the alternative pathway C3 convertase (C3bBb). A combination of biochemical, biophysical, and structural studies are carried out here that show that SCINs elicit these effects by binding to a functional “hotspot” on C3b. Additionally, experimental evidence shows that SCINs promote formation of inhibited convertase dimers (i.e., (SCIN/C3bBb)2) and inhibitor-enzyme-substrate complexes (i.e., (SCIN/C3bBb)/C3)) that sterically-mask the C3b binding sites of complement receptors expressed on the surface of lymphoid and phagocytic cells. As a consequence, SCINs interfere with opsonophagocytosis not only by inhibiting additional C3b deposition, but also by directly blocking essential C3b/CR interactions as well. Finally, experiments are conducted that demonstrate the functional importance of a sequence divergent and flexible N-terminal domain in SCIN proteins. The development of complement inhibitors has long held great potential for treatment of these conditions. Still, drug discovery efforts in this area have been complicated by the multipronged nature of the complement system, which is predicated upon an integrated network of protein-protein interactions that underlie its initiation, amplification, and regulation. With significant challenges remaining to the development of peptides or other small molecules capable of disrupting these interactions, an unexpected contribution toward complement-directed therapeutics may yet come from thoroughly understanding the naturally occurring inhibitors, such as SCINs. Together, these studies further our understanding of SCIN structure, expand our knowledge of the multi-faceted immune evasion mechanism of SCINs, and provide clues for the potential optimization or synthetic mimicry of SCIN proteins.