An Alternative Structural Model of Activation for the Anti‐Anti‐σ Factor PhyR and Interaction with the Anti‐σ Factor NepR

Protein phosphorylation is a universally conserved post‐translational modification. In bacteria, aspartyl phosphorylation generally controls transcriptional regulatory proteins, enabling cells to respond to chemical and physical changes in their local environment. In the large and diverse alphaproteobacteria class of bacteria, stress monitoring is primarily regulated by the general stress response (GSR). PhyR is the master regulator of the GSR and is a two‐domain anti‐anti‐σ factor containing a phosphorylatable response regulator receiver domain and a σ‐like output domain. PhyR titrates the anti‐σ factor NepR away from its cognate σ factor in a phosphorylation‐dependent manner to ultimately control global changes in gene expression. The binding site of NepR within PhyR is largely occluded at the domain interface. It has long been hypothesized that phosphorylation of PhyR results in undocking of the two domains to expose the high‐affinity NepR binding site; however, the foundations by which phosphorylation of the PhyR receiver domain controls structure and function remains largely undefined. Small‐angle X‐ray scattering (SAXS) and molecular dynamics (MD) simulations indicate phosphorylation is insufficient to drive PhyR opening. Recent biophysical and biomolecular NMR data indicate intrinsically disordered regions of NepR play a significant role in PhyR activation. These interactions drive an unstructured to structured transition of NepR and are required to facilitate both PhyR phosphorylation and PhyR‐NepR binding. This leads to a model where phosphorylation is necessary but insufficient to expose the high affinity NepR binding site of PhyR and interactions between PhyR and NepR prior to phosphorylation are a necessary component of the structure‐function relationship of these two core regulatory proteins. Support or Funding Information NIH 5R01AI1707159