Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346

Abstract

Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light-sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme's residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.sensor histidine kinase | HDX-MS | ESR spectroscopy | two-component system | photoreceptor

Hydrogen exchange of chemoreceptors in functional complexes suggests protein stabilization mediates long-range allosteric coupling

“…Modulation of a coupled equilibrium between CheA binding and stabilization of a disordered CF is an exciting new idea for explaining the integration of different inputs (ligand-induced piston and methylation) and the long-distance propagation of the signal 200Å from the membrane to the membrane-distal-tip of the chemotaxis receptor. Interestingly, in the related sensor histidine kinases (which contain the sensor and kinase within the same protein), signalingrelated local unfolding of helices has been observed by HDX-MS (33,34). Additional study of the role of folding/stabilization in chemoreceptor signaling is important to provide a detailed understanding of the mechanism and how it compares to observations in other systems in which more localized folding/unfolding is key to signaling mechanisms (35), and in cases where intrinsically disordered domains mediate allosteric interactions (36).…”

Regulation of the Erythrobacter litoralis DSM 8509 general stress response by visible light

“…can encode zero, one, or multiple LOV‐HWE kinases, and the total number of HWE/HisKA2‐family kinases ranges from 2 to 16 (Ulrich and Zhulin, ). Although the three LOV kinases present in strain HTCC 2594 have been biochemically characterized (Correa et al , ; Rivera‐Cancel et al , ; Dikiy et al , ), we anticipated that the potential functional redundancy of these genes could complicate interpretation of genetic and physiological studies in vivo . In contrast, DSM 8509 encodes only one LOV‐HWE kinase.…”

“…Draft genome sequence of DSM 8509 indicated the presence of a single LOV‐HWE kinase gene (Wang et al , ). This distinguishes DSM 8509 from E. litoralis strain HTCC 2594, which encodes multiple LOV‐kinases (Oh et al , ) that have been previously characterized in vitro (Correa et al , ; Rivera‐Cancel et al , ; Dikiy et al , ). HTCC 2594 does not encode the genes required for phototrophy (Oh et al , ; Zheng et al , ), and though DSM 8509 and HTCC 2594 cluster phylogenetically based on their 16S sequences, they do not group based on amino acid sequence of concatenated core genes (Zheng et al , ).…”

Regulation of theErythrobacter litoralisDSM 8509 general stress response by visible light

“…Draft genome sequence of DSM 8509 indicated the presence of a single LOV-HWE kinase gene (Wang et al, 2014). This distinguishes DSM 8509 from E. litoralis strain HTCC 2594, which encodes multiple LOV-kinases (Oh et al, 2009) that have been previously characterized in vitro (Correa et al, 2013, Dikiy et al, 2019, Rivera-Cancel et al, 2014. HTCC 2594 does not encode the genes required for phototrophy (Oh et al, 2009, Zheng et al, 2016, and though DSM 8509 and HTCC 2594 cluster phylogenetically based on their 16S sequences, they do not group based on amino acid sequence of concatenated core genes (Zheng et al, 2016).…”

“…Although the three LOV kinases present in strain HTCC 2594 have been biochemically characterized (Correa et al, 2013, Dikiy et al, 2019, Rivera-Cancel et al, 2014, we anticipated that the potential functional redundancy of these genes could complicate interpretation of genetic and physiological studies in vivo. In contrast, DSM 8509 encodes only one LOV-HWE kinase.…”