The Role of a Highly Conserved Loop in Response Regulator Activation in the General Stress Response

Bacteria depend on a diverse array of signaling proteins to sense and respond to their environments. An important class of proteins involved in such signaling cascades are the response regulators, which are controlled by the phosphorylation state of a conserved aspartate residue. Though most response regulators share great structural similarity, the allosteric pathway that governs their activation upon phosphorylation tends to vary. In this study, we examine the response regulator PhyR, a two‐domain protein comprised of a phohsphorylatable receiver domain and a σ‐like domain. This protein functions as an anti‐anti‐σ factor that governs activation of the general stress response (GSR) in alphaproteobacteria. Activated PhyR accomplishes GSR induction by binding to its cognate anti‐σ factor, NepR, through its σ‐like domain. This domain is occluded by the receiver domain in the inactive state. As such, in order for PhyR to carry out its binding activity, its receiver domain must undergo a significant conformational change to detach from the σ‐like domain. While a number of structural studies have focused on PhyR, a full characterization of the active state of the protein remains elusive. In the absence of definitive structural information about the protein's active state, we probed PhyR's activation mechanism through molecular dynamics (MD) simulations. From these simulations, we predicted that, for the protein to activate, a highly conserved loop in the receiver domain must be restructured in a manner which would disrupt a set of strong hydrogen bonds. Mutating the PhyR residues involved in these interactions results in hyperactive binding to NepR. Thus, we show that disruption of a particular interaction interface is sufficient to bias PhyR into the active state and therefore these residues play an important role in its activation dynamics. Support or Funding Information Funding: NIH 5R01Al107159