Ligand-Induced Folding of a Two-Component Signaling Receiver Domain

Abstract

To survive and adapt to environmental changes, bacteria commonly use two component signaling systems. Minimally, these pathways use histidine kinases (HKs) to detect environmental signals, harnessing these to control phosphorylation levels of receiver (REC) domains of downstream response regulators that convert this signal into physiological responses. Studies of several prototypical REC domains suggest that phosphorylation shifts these proteins between inactive and active structures that are globally similar and well-folded. However, it is unclear how globally these findings hold within REC domains in general, particularly when considered within full-length proteins. Here we present EL_LovR, a full-length REC-only protein that is phosphorylated in response to blue light in the marine α-proteobacterium Erythrobacter litoralis HTCC2594. Notably, EL_LovR is similar to comparable REC-only proteins used in bacterial general stress responses, where genetic evidence suggests that their potent phosphatase activity is important to shut off such systems. Size exclusion chromatography, light scattering and solution NMR experiments show that EL_LovR is monomeric and unfolded in solution under conditions routinely used for other REC structure determinations. Addition of Mg2+ and phosphorylation induce progressively greater degrees of tertiary structure stabilization, with the solution structure of the fully-activated EL_LovR adopting the canonical receiver domain fold. Parallel functional assays show that EL_LovR has a fast dephosphorylation rate, consistent with its proposed function as a phosphate sink that depletes the HK phosphoryl group, promoting the phosphatase activity of this enzyme. Our findings demonstrate that EL_LovR undergoes substantial ligand-dependent conformational changes that have not been reported for other RRs, expanding the scope of conformational changes and regulation used by REC domains, critical components of bacterial signaling systems.

Basis of Mutual Domain Inhibition in a Bacterial Response Regulator

“…Furthermore, the EL_REC spectrum had more peaks than expected from the sequence, suggesting that the protein underwent slow exchange on the millisecond to second timescale (or slower) among two conformations in a 65%:35% ratio. Titrations with MgCl 2 , supplying the important Mg 2+ cation needed to activate RRs (McDonald et al, 2012; Ocasio et al, 2015), shifted the equilibrium in favor of the less populated of these slow-exchanging states (Figures S2A–S2C). In comparison, 2D 15 N- 1 H NMR spectra of full-length EL_PhyR did not display comparable peaks indicative of conformational exchange and addition of MgCl 2 caused only minor chemical shift displacements.…”

Molecular Mechanisms of Two-Component Signal Transduction

“…Whether these regulatory mechanisms are representative for many RR remains to be seen. The above examples point to a continuous rather than a simple on/off mechanism for the transition from the inactive to the active conformation of the RR [137, 148]. …”

General Stress Signaling in the Alphaproteobacteria

“…Moreover, when combined, EL_LovR promotes dephosphorylation of EL_PhyR~P via EL368 (21). EL_LovR~P has a short half-life, rapidly losing its phosphoryl group to water (82). Rapid dephosphorylation in this system is consistent with a phosphosink model in which the SDRR EL_LovR depletes the kinase of phosphoryl groups and thereby stimulates phosphatase activity of the kinase EL368 and dephosphorylation of PhyR.…”