Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors.

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Phosphorylation carries chemical information in biological systems. In two-component systems (TCSs), the sensor histidine kinase and the response regulator are connected through phosphoryl transfer reactions that may be uni- or bidirectional. Directionality enables the construction of complex regulatory networks that optimize signal propagation and ensure the forward flow of information. We combined x-ray crystallography, hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, and systems-integrative kinetic modeling approaches to study phosphoryl flow through the Bacillus subtilis thermosensing TCS DesK-DesR. The allosteric regulation of the histidine kinase DesK was critical to avoid back transfer of phosphoryl groups and futile phosphorylation-dephosphorylation cycles by isolating phosphatase, autokinase, and phosphotransferase activities. Interactions between the kinase’s ATP-binding domain and the regulator’s receiver domain placed the regulator in two distinct positions in the phosphotransferase and phosphatase complexes, thereby determining whether a key glutamine residue in DesK was properly situated to assist in the dephosphorylation reaction. Moreover, an energetically unfavorable phosphotransferase conformation when DesK was not phosphorylated minimized reverse phosphoryl transfer. DesR dimerization and a dissociative phosphoryl transfer reaction also enforced the direction of phosphoryl flow. Shorter or longer distances between the phosphoryl acceptor and donor residues shifted the phosphoryl transfer equilibrium by modulating the stabilizing effect of the Mg 2+ cofactor. These mechanisms control the directionality of signal transmission and show how structure-encoded allostery stores and transmits information in signaling systems.