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

Staphylococcus aureusand many related bacteria encode both anti-sigma factor RsbW and anti-anti-sigma factor RsbV to control stress response by sigma(B), an alternative sigma factor. Our structural and thermodynamic studies of a recombinantS. aureusRsbV (rRsbV) show that the monomeric protein contains five alpha-helices and a mostly parallel but mixed beta-sheet composed of five beta-strands, and interacts with a chimericS. aureusRsbW (rRsbW)in vitro. In addition, rRsbV binds rRsbW with aK(d)of 0.058 mu M using spectroscopy and 0.008 mu M using calorimetry at 25 degrees C. From a gel-shift assay, the affinity of rRsbV to rRsbW was found to be higher than its affinity with a recombinantS. aureus sigma(B)(r sigma(B)). Moreover, the heat generated from the spontaneous rRsbV - rRsbW interaction changed in a compensatory manner with entropy in the 20 degrees-35 degrees 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 Delta Gand 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

Automated summary

The study focused on the interactions between the anti-anti-sigma factor and an anti-sigma factor in Staphylococcus aureus, revealing that the formation of the complex relies on hydrogen bonds and hydrophobic interactions, with a negative heat capacity change observed during the transition to a 2:2 complex formation. Additional experiments showed increased thermodynamic stability of the anti-anti-sigma factor in the presence of the anti-sigma factor, providing valuable insights into the structural and binding thermodynamics of these regulatory proteins.