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

and many related bacteria encode both anti-sigma factor RsbW and anti-anti-sigma factor RsbV to control stress response by σ, an alternative sigma factor. Our structural and thermodynamic studies of a recombinant 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 RsbW (rRsbW) . In addition, rRsbV binds rRsbW with a 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 σ (rσ). 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 Δ 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.