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.
Highlights
Streptococcus pyogenes (group A Streptococcus (GAS)3), one of the most common human bacterial pathogens, has developed diverse mechanisms that allow the bacteria to evade the immune system [1, 2]
The structural differences between mature SPE B (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 Streptococcal pyrogenic exotoxin B (SPE B) is involved in the His-195 side-chain rotation
Expression and Protease Activity of SPE B Mutants—To identify functional mutation from allelic polymorphism in the SPE B gene, the D9N, G163S, G163S/A172S, and G239D mutant proteins were expressed in E. coli and purified to homogeneity
Summary
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. We found that the Gly-239 residue played an important role in its protease activity, and is located within an undefined S230-G239 loop in the x-ray structure of proSPE B. We used NMR spectroscopy to determine three-dimensional structures and backbone dynamics of the 28-kDa C47S mutant of mSPE B so that the presence of an undefined S230-G239 loop in the protease domain of proSPE B and the possibility of the loop movements can be resolved. Our results provide a structural basis for understanding the mechanism of how the conformation and dynamics properties of the loops of SPE B controls its substrate binding, resulting in its broad substrate specificity
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