Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its...

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• σ^B (sigma B) is a key alternative sigma factor in *Staphylococcus aureus* and other Gram-positive bacteria, regulating the general stress response.
• RsbW, an antisigma factor with serine kinase activity, typically inhibits σ^B activity, playing a role in *S. aureus* pathogenesis.

Purpose of the Study:

• To investigate the molecular interactions between recombinant RsbW (rRsbW) and recombinant σ^B (rσ^B) or its domains (rσ^B2, rσ^B3, rσ^B4).
• To elucidate the binding affinities and structural basis of the RsbW-σ^B interaction.
• To explore the functional significance of the RsbW dimerization region and the thermodynamic consequences of complex formation.

Main Methods:

• Recombinant protein expression and purification (rRsbW, rσ^B, rσ^B2, rσ^B3, rσ^B4).
• DNA binding assays with core RNA polymerase.
• Isothermal titration calorimetry (ITC) and other biophysical methods to study protein-protein interactions.
• Structural modeling and genetic studies.

Main Results:

• Only full-length rσ^B, not its individual domains, bound DNA with core RNA polymerase.
• rσ^B3 and rσ^B2 interacted with rRsbW, with binding affinities in the order: rσ^B > rσ^B3 > rσ^B2.
• The interaction between rRsbW and rσ^B or rσ^B3 was spontaneous and exothermic, with rRsbW and rσ^B3 forming a 2:1 complex.
• A structural model suggested rσ^B3 binds at the RsbW dimeric interface, and genetic studies confirmed the dimer-forming region's importance.
• RsbW binding thermodynamically stabilized σ^B3.

Conclusions:

• The σ^B3 domain is critical for RsbW binding and is stabilized by RsbW.
• The dimeric interface of RsbW is essential for its interaction with σ^B, its kinase activity, and dimerization.
• Understanding these interactions provides insights into bacterial stress response regulation and potential avenues for drug discovery targeting *S. aureus* pathogenesis.