The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Group A Streptococcus pyogenes employs extracellular DNases to facilitate invasive infections.
• These DNases degrade neutrophil extracellular traps (NETs), aiding bacterial evasion of host immune defenses.
• Spd1 is a nonspecific ββα/metal-dependent nuclease from S. pyogenes, encoded by the SF370.1 prophage.

Purpose of the Study:

• To elucidate the structural basis of Spd1's nucleolytic activity.
• To identify key residues and the active site constellation of Spd1.
• To investigate the multimeric state of Spd1 in solution.

Main Methods:

• X-ray crystallography of wild-type and Asn145Ala mutant Spd1.
• Sequence alignments and mutagenesis studies.
• Size-exclusion chromatography with multi-angle light scattering (SEC-MALLS) in the presence and absence of Mg(2+).

Main Results:

• The X-ray structures of wild-type and mutant Spd1 were determined.
• Key residues His121, Asn145, and Glu164 were identified as crucial for Spd1's nucleolytic activity.
• SEC-MALLS data suggest Spd1 exists as a monomer in solution, despite structural data hinting at a potential dimeric form.

Conclusions:

• Spd1's structure and active site have been characterized, revealing critical residues for its function.
• The findings provide insights into how Spd1 contributes to Streptococcus pyogenes pathogenesis.
• Spd1 functions as a monomer in solution, a finding relevant for understanding its mechanism of action.