Abstract
Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring.
Highlights
Perception of environmental and intracellular cues is an essential feature of life
Symmetric organizations are a hallmark of histidine kinase (HK) in the ‘auto-kinase off / phosphatase on’ states (Albanesi et al, 2009; Casino et al, 2009; Yamada et al, 2009)
The 2:2 HK:RR stoichiometry was confirmed in solution by isothermal titration calorimetry (ITC) (Figure 1B, Table 2), revealing an entropy-driven, endothermic association reaction
Summary
Perception of environmental and intracellular cues is an essential feature of life. Signaling pathways enable cells to regulate genetic and biochemical programs for adaptation and survival. The simplest TCSs comprise a sensory histidine kinase (HK) and a response regulator (RR) component (Gao and Stock, 2009). The HK is turned on or off after signal-dependent allosteric rearrangements, which control autophosphorylation on a conserved histidine residue. The phosphoryl group is transferred from the phosphorylated kinase to a conserved aspartate on the RR receiver domain, activating it to effect a specific output response. When auto-kinase activity is turned off, HKs often act as a phosphatase of their cognate phosphorylated RR (P~RR) (Goulian, 2010), contributing to shutting down the pathway. Despite being paradoxical for a protein kinase, the phosphatase activity of HKs is physiologically relevant ensuring robust homeostatic responses (Schramke et al, 2016)
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