The C3-inhibitory domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb-C) defines a novel three-helix bundle motif that regulates complement activation. Previous crystallographic studies of Efb-C bound to its cognate subdomain of human C3 (C3d) identified Arg-131 and Asn-138 of Efb-C as key residues for its activity. Our results show that while mutating these residues to Ala do not drastically affect the structure of the Efb-C/C3d recognition complex, they have significant adverse effects on both the thermodynamic and kinetic profiles of the resulting complexes. We also characterized other key interactions along the Efb-C/C3d binding interface and found an intricate network of salt bridges and hydrogen bonds that anchor Efb-C to C3d, resulting in its potent complement inhibitory properties.
This work combined X-ray crystallography, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and Molecular Dynamics (MD) simulations. Detailed computational analysis of pair-wise interactions not only reiterated the importance of Arg-131 and Asn-138, but also allowed us to identify potential contributions of other residues for the interaction between Efb-C and C3d. The majority of these residues were found to form hydrogen bonds and salt bridges, which presumably contribute to the stability of the complex. However, some of the identified interaction pairs are predominantly hydrophobic or involve the backbones of hydrophobic residues. In many cases, these interactions involve residues that at first glance did not appear to be essential for either formation or maintenance of the Efb-C/C3d complex. The approaches and results constitute a valuable extension in our understanding of the Efb-C/C3d interface. In addition to facilitating a systematic mutagenesis of these residues, this work also highlights the highly cooperative framework that drives specific protein-protein interactions.