Understanding the structure, stability, and anti-sigma factor-binding thermodynamics of an anti-anti-sigma factor from Staphylococcus aureus

Abstract

Staphylococcus aureus and many related bacteria encode both anti-sigma factor RsbW and anti-anti-sigma factor RsbV to control stress response by σ^B, an alternative sigma factor. Our structural and thermodynamic studies of a recombinant S. aureus RsbV (rRsbV) show that the monomeric protein contains five α-helices and a mostly parallel but mixed β-sheet composed of five β-strands, and interacts with a chimeric S. aureus RsbW (rRsbW) in vitro. In addition, rRsbV binds rRsbW with a K_d of 0.058 µM using spectroscopy and 0.008 µM using calorimetry at 25 °C. From a gel-shift assay, the affinity of rRsbV to rRsbW was found to be higher than its affinity with a recombinant S. aureus σ^B (rσ^B). Moreover, the heat generated from the spontaneous rRsbV - rRsbW interaction changed in a compensatory manner with entropy in the 20°–35 °C range. The association between rRsbV and rRsbW yielded a negative heat capacity change, suggesting that both hydrogen bonds and hydrophobic interactions participate in the formation of the rRsbV-rRsbW complex. Computational analyses of a homology-based RsbV-RsbW model has mostly supported the formation of a 2: 2 complex verified by gel filtration chromatography, the experimental ΔG and the existence of these non-covalent bonds. Our unfolding experiments show that the thermodynamic stability of rRsbV is significantly increased in the presence of rRsbW. Thus, these studies have provided valuable insights into the structure, stability, and the anti-sigma-binding thermodynamics of an anti-anti-sigma factor.

Communicated by Ramaswamy H. Sarma

Keywords:

• Anti-anti-sigma factor
• anti-sigma factor
• calorimetry
• molecular modeling

Acknowledgements

The authors thank Mr. S. Biswas and Mr. M. Das for their excellent technical support. Ms. D. Sinha and Mr. T. Chakraborty received Junior Research Fellowships from Bose Institute. Mr. D. Sinha received Project Assistantship from Bose Institute.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the financial assistance from Bose Institute.