Biochemical analysis of ScpA-C5a interactions: a model system for studies of substrate specificity in cell envelope proteases from gram positive bacteria

The kinetic and thermodynamic parameters for the ScpA-C5a complex formation were measured by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). These studies showed that C5a was bound with high affinity (34 nM) to ScpA, that the majority of interactions was provided by C5a core, and that the C-terminal R74 contributed the majority of the energy provided by the tail. The association of the complex was dominated by favourable enthalpy with a small contribution of favourable entropy. Furthermore, it was shown that the long-range electrostatic interactions were important for bringing the substrate to the surface of the enzyme.

The functional role of the ScpA domain in the proteolytic activity and in the substrate binding was investigated. This study demonstrated that the ScpA C-terminal fibronectin type III-like domains were essential for the proper folding and stability of the N-terminal domains. All domains were required for maximal proteolytic activity of ScpA towards human C5a. Progressive truncation at the C-terminus led to the complete loss of the enzymatic activity. The Fn2 domain was essential for the hydrolysis activity of the enzyme, which could be due to its exo-site role in binding the substrate. The PA domain was not essential for substrate cleavage but needed for maximal performance of the enzyme. This domain contributed to the specificity of ScpA.

C5a core residues involved in the salt bridges with ScpA in the published model were tested. Alanine substitution and SPR analysis revealed that the residues located in the N-terminal region of C5a core and helix IV were not involved in ionic interactions with ScpA. These results showed inconsistences with the published model of the complex. Therefore, a new model was proposed, which accounted for the new experimental information. This model placed C5a core on the Fn2 domain with the tail bound in an extended form in the active site and making contacts with Cat and PA domains. Heilx II and III of the core were facing down on the Fn2 with helix I creating weak hydrophobic interactions.

The ability of ScpA to bind and cleave mouse C5a was tested. These tests showed that mouse C5a bound to ScpA with 50-fold lower affinity than human C5a, and that the enzyme was not able to hydrolyse this protein. The studies suggest that the role of ScpA in the streptococcal virulence should not be investigated in a murine model.