Determination of Redox Sensitivity in Structurally Similar Biological Redox Sensors

Redox stimuli govern a variety of biological processes. The relative sensitivity of redox sensors plays an important role in providing a calibrated response to environmental stimuli and cellular homeostasis. This cellular machinery plays a crucial role in the human pathogen Mycobacterium tuberculosis as it encounters diverse microenvironments in the host. The redox sensory mechanism in M. tuberculosis is governed by two component and one-component systems, alongside a class of transcription factors called the extra cytoplasmic function (ECF) σ factors. ECF σ factors that govern the cellular response to redox stimuli are negatively regulated by forming a complex with proteins called zinc associated anti-σ factors (ZAS). ZAS proteins release their cognate σ factor in response to oxidative stress. The relative sensitivity of the ZAS sensors to redox processes dictate the concentration of free ECF σ factors in the cell. However, factors governing the redox threshold of these sensors remain unclear. We describe here, the molecular characterization of three σ factor/ZAS pairs-σ^L/RslA, σ^E/RseA, and σ^H/RshA-using a combination of biophysical and electrochemical techniques. This study reveals, conclusively, the differences in redox sensitivity in these proteins despite apparent structural similarity and rationalizes the hierarchy in the activation of the cognate ECF σ factors. Put together, the study provides a basis for examining sequence and conformational features that modulate redox sensitivity within the confines of a conserved structural scaffold. The findings provide the guiding principles for the design of intracellular redox sensors with tailored sensitivity and predictable redox thresholds, providing a much needed biochemical tool for understanding host-pathogen interaction.