Solution structure and backbone dynamics of streptopain: insight into diverse substrate specificity

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Streptococcal pyrogenic exotoxin B (SPE B) is a key cysteine protease produced by Streptococcus pyogenes.
• Understanding SPE B's structure-function relationship is vital for targeting its pathogenic activities.

Purpose of the Study:

• To investigate the impact of allelic variants on SPE B protease activity.
• To elucidate the structural and dynamic features of mature SPE B (mSPE B) and their relation to its function.

Main Methods:

• Expression and characterization of SPE B mutant proteins (D9N, G163S, G163S/A172S, G239D).
• Determination of the three-dimensional structure and backbone dynamics of mSPE B using X-ray crystallography and NMR spectroscopy.
• Analysis of mSPE B and inhibitor complexes to understand dynamics and flexibility.

Main Results:

• The G239D mutant exhibited a 12-fold decrease in protease activity compared to other variants.
• Structural comparison revealed distinct C-terminal loop conformations and His-195 orientations between mature SPE B and its zymogen form (proSPE B).
• Dynamics analysis identified the catalytic and C-terminal loops as highly flexible regions in mSPE B, crucial for substrate interaction.

Conclusions:

• The Gly-239 residue and the C-terminal loop significantly influence SPE B protease activity.
• Structural and dynamic flexibility of the C-terminal loop is essential for SPE B's broad substrate specificity.
• His-195 side-chain rotation may be involved in the activation and inactivation mechanisms of SPE B.