Switching Protein Conformational Substates by Protonation and Mutation

Protein modules that regulate the availability and conformational status of transcription factors determine the rapidity, duration, and magnitude of cellular response to changing conditions. One such system is the single-gene product Cnu, a four-helix bundle transcription co-repressor, which acts as a molecular thermosensor regulating the expression of virulence genes in enterobacteriaceae through modulation of its native conformational ensemble. Cnu and related genes have also been implicated in pH-dependent expression of virulence genes. We hypothesize that protonation of a conserved buried histidine (H45) in Cnu promotes large electrostatic frustration, thus disturbing the H-NS, a transcription factor, binding face. Spectroscopic and calorimetric methods reveal that H45 exhibits a suppressed p K of ∼5.1, the protonation of which switches the conformation to an alternate native ensemble in which the fourth helix is disordered. The population redistribution can also be achieved through a mutation H45V, which does not display any switching behavior at pH values greater than 4. The Wako-Saitô-Muñoz-Eaton (WSME) statistical mechanical model predicts specific differences in the conformations and fluctuations of the fourth and first helices of Cnu determining the observed pH response. We validate these predictions through fluorescence lifetime measurements of a sole tryptophan, highlighting the presence of both native and non-native interactions in the regions adjoining the binding face of Cnu. Our combined experimental-computational study thus shows that Cnu acts both as a thermo- and pH-sensor orchestrated via a subtle but quantifiable balance between the weak packing of a structural element and protonation of a buried histidine that promotes electrostatic frustration.