SIGNIFICANCE:   All living things must sense and react to their environment. Many single-celled organisms do so by using two-component systems, most simply consisting of a sensor histidine kinase and a response regulator. These systems are involved in pathogenicity pathways and can be targeted by new antibiotics. However, the molecular mechanisms used by histidine kinases to translate sensing into responses are not well understood. To probe this general question, we apply a combination of biophysical techniques to a monomeric histidine kinase that senses blue light to determine the structural changes occurring upon activation. We find these changes to be similar to those predicted for the common dimeric histidine kinases, illustrating that the mechanism of activation is conserved regardless of oligomeric state.
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.