Abstract
Streptococcal pyrogenic exotoxin B (SPE B) is a cysteine protease expressed by Streptococcus pyogenes. The D9N, G163S, G163S/A172S, and G239D mutant proteins were expressed to study the effect of the allelic variants on their protease activity. In contrast to other mutants, the G239D mutant was approximately 12-fold less active. The Gly-239 residue is located within the C-terminal S230-G239 region, which cannot be observed in the x-ray structure. The three-dimensional structure and backbone dynamics of the 28-kDa mature SPE B (mSPE B) were determined. Unlike the x-ray structure of the 40-kDa zymogen SPE B (proSPE B), we observed the interactions between the C-terminal loop and the active site residues in mSPE B. The structural differences between mSPE B and proSPE B were the conformation of the C-terminal loop and the orientation of the catalytic His-195 residue, suggesting that activation and inactivation of SPE B is involved in the His-195 side-chain rotation. Dynamics analysis of mSPE B and the mSPE B/inhibitor complexes showed that the catalytic and C-terminal loops were the most flexible regions with low order parameter values of 0.5 to 0.8 and exhibited the motion on the ps/ns timescale. These findings suggest that the flexible C-terminal loop of SPE B may play an important role in controlling the substrate binding, resulting in its broad substrate specificity.