Structure of the mature Streptococcal cysteine protease exotoxin mSpeB in its active dimeric form

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Invasive Streptococcus pyogenes infections are driven by the cysteine protease streptococcal pyrogenic exotoxin B (SpeB).
• Previous structural data lacked clarity on the enzyme's active site configuration.
• Understanding SpeB's structure is crucial for targeting bacterial cysteine proteases.

Purpose of the Study:

• To determine the high-resolution crystal structure of the mature streptococcal pyrogenic exotoxin B.
• To elucidate the mechanism of SpeB activation and catalysis through its dimeric structure.
• To provide insights for developing specific inhibitors against bacterial cysteine proteases.

Main Methods:

• X-ray crystallography was used to determine the structure of mature SpeB at 1.55 Å resolution.
• Structural analysis focused on the homodimeric configuration and active site interactions.
• Comparative analysis was performed with other cysteine proteases of clan CA.

Main Results:

• The crystal structure revealed that mature SpeB forms a homodimer, a state unique to the mature enzyme and critical for catalysis.
• A serine residue from one subunit inserts into the active site of the other, facilitating catalysis.
• A tripartite switch system, triggered by dimerization and substrate binding, was identified, involving histidine liberation, substrate pocket unblocking, and tryptophan repositioning.
• The active site of clan CA cysteine proteases was expanded based on this structure, and a deacylation mechanism was proposed.

Conclusions:

• SpeB dimerization is essential for its catalytic activity, involving a unique serine-mediated mechanism.
• The identified tripartite switch system explains the activation and substrate binding of SpeB.
• The structural findings offer a basis for designing targeted protease inhibitors against pathogenic bacteria like Streptococcus pyogenes.