Biophysical and Biochemical Characterization of a REC Domain: Unfolded to Folded Transition of EL_LovR




Ocasio, Victor J.

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Prokaryotes frequently use two component systems to couple environmental stimuli to adaptive responses. These pathways use histidine kinases to detect environmental cues, harnessing these to control phosphorylation of the receiver domain of the response regulator, which convert this signal into a physiological response. Knowledge of how phosphorylation shifts receiver domains between their inactive and active states is limited, chiefly assembled from several prototypical receiver domains that switch between two similar and well-folded structures. However, it remains unclear how general these observations apply to other receiver domains, particularly for full-length proteins. Here we present a blue light-regulated two-component system from the marine α-proteobacterium Erythrobacter litoralis HTCC2594. The sensor domain of the 3 histidine kinases found in E. litoralis contain a LOV (Light-Oxygen-Voltage) domain, part of the widely used PAS (Per-ARNT-Sim) family of environmental sensors. Interestingly, one of the histidine kinases (EL362) contains a naturally occurring glycine to arginine mutation in the LOV domain that prevents chromophore binding, resulting in a "blind" histidine kinase. Reverting the arginine to a glycine residue allows blue light to trigger the autophosphorylation of EL362 and subsequent phosphotransfer towards the cognate response regulator EL_LovR. This arrangement of RRs is reminiscent of similar systems used in other bacterial general stress responses, most of which have been characterized entirely with genetic methods. Notably, EL_LovR is a single domain response regulator proposed to play a critical role in shutting off such systems via a potent phosphatase activity. Size exclusion chromatography, light scattering and NMR experiments show that phosphorylation and Mg(II) transitions EL_LovR between unfolded and folded monomeric states. Parallel functional assays show that EL_LovR has a fast dephosphorylation rate, consistent with its proposed function as a phosphate sink. Taken together, our findings provide evidence that EL_LovR undergoes drastic conformational changes that have not been seen in other response regulators, likely with effects on its autophosphatase activity. In conclusion, our work expands the kinds of conformational changes and regulation used by receiver domains, critical components of bacterial signaling systems.

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