Phenol sensing in nature is modulated via a conformational switch governed by dynamic allostery

Abstract

The NtrC family of proteins senses external stimuli and accordingly stimulates stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. However, the structural determinants that mediate this activation are not well understood. Here, we establish using computational, structural, biochemical and biophysical studies that MopR, an NtrC protein, harbors a dynamic bi-directional electrostatic network that connects the phenol pocket to two distal regions, namely the "G-hinge" and the "allosteric-linker". While the G-hinge influences the entry of phenol into the pocket, the allosteric-linker passes the signal to the downstream ATPase domain. We show that phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery, and demonstrate that perturbation of the core relay residues results in a complete loss of ATPase stimulation. Furthermore, we found a mutation of the G-hinge, ∼20Å from the phenol pocket, promotes altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. Finally, we conducted a global analysis which illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species. Taken together, these results provide insights into the mechanisms of dynamic allostery-mediated conformational changes in NtrC sensor proteins.