Switching Protein Conformational Substates by Protonation and Mutation.

Abhishek Narayan,Athi N Naganathan

doi:10.1021/acs.jpcb.8b05108

Abhishek Narayan, Athi N Naganathan

Open Access

https://doi.org/10.1021/acs.jpcb.8b05108

Copy DOI

Abstract

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 Ka 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.

Highlights

The conformational flexibility of transcription factors (TFs) enables the rapid search of cognate binding sites on DNA
The proteins Hha and Cnu have been implicated in the pH dependent expression of virulence factors in Salmonella pathogenicity island 2 (SPI-2).[23]
We show here that both mutation and protonation of conserved buried histidine alter the native conformational ensemble of Cnu (N) resulting in a higher fraction of the state N*

Summary

Introduction

The conformational flexibility of transcription factors (TFs) enables the rapid search of cognate binding sites on DNA. The availability and transcriptional fecundity of TFs, on the other hand, are determined by multiple variables, including the concentration of small molecules or ligands, proteins, oligomerization, allosteric switches, and environmental conditions.[1−10] Certain protein modules can themselves up- or down-regulate the expression of specific genes. One such example in the enterobacteriaceae is the four-helix Hha-family of proteins that regulates the availability of the transcription factor H-NS.[11−15] Mutational studies[16,17] and spectroscopic experiments provide strong evidence that Cnu (YdgT), a member of the Hha-family, exhibits graded structural polymorphism with temperature[18] that could be critical for the expression of virulence genes in enterobacteriaceae by modulating the binding affinity with H-NS.[19]. What could the sensing mechanism involve? Studies on the effects of pH changes on the protein structure have a long history with protonation predominantly inducing unfolding of proteins at a low pH (

Methods

Results

Conclusion