Exosite binding modulates the specificity of the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease

The C5a peptidase from Streptococcus pyogenes (ScpA) is a highly specific enzyme with potential therapeutic value. ScpA is a good model for studying determinants of specificity in the multidomain immunomodulatory enzymes (IMEs), which comprise a large family of bacterial surface proteases. The surface exposed region of ScpA has 5 main domains which includes 3 C-terminal Fn3-like domains (Fn1, Fn2 and Fn3) (Kagawa et al. 2009). Progressive deletion of the Fn3-like domains from the C-ter resulted in loss of enzyme activity and showed an important role for the Fn2 domain in enzyme function. Functional investigation of specific acidic residues on the Fn2 domain identified 3 residues 30–50 Å from the catalytic site (D783, E864 and D889) which impacted to differing degrees on binding and on catalysis, supporting the presence of an exosite on the Fn2. In particular, residue D783 was observed to impact on both substrate binding affinity and the activity of ScpA. A double mutant cycle analysis showed energetic coupling between the targeted ScpA residues and residues in the core portion (residues 1–67) of the C5a substrate. The data supports the presence of a communication network between the active site and the exosite on Fn2. These findings provide a basis for rational engineering of this important enzyme family to enhance stability, activity and/or specificity.