The DNA-binding domain of the Escherichia coli CpxR two-component response regulator is constitutively active and cannot be fully attenuated by fused adjacent heterologous regulatory domains

Two-component systems (TCS) based on a sensor histidine kinase and a phosphorylated cognate target regulator allow rapid responses to environmental changes. TCS are highly evolutionarily conserved, though in only a few cases are the inducing signals understood. This study focuses on the Escherichia coli CpxR response regulator that responds to periplasmic and outer-membrane stress, N-terminal deletion mutations have been isolated that render the transcription factor constitutively active, indicating that the N terminus functions, in part, to keep the C-terminal winged-helix DNA-binding effector domain in an inactive state. Analysis of truncations spanning the CpxR interdomain region revealed that mutants retaining the alpha 5 helix significantly augment activation. Hybrid proteins obtained by fusing the CpxR effector domain to structurally similar heterologous N-terminal regulatory domains, or even GFP, failed to restore repression to the C-terminal domain. These findings shed light on the mechanism of CpxR effector domain activation and on the investigation of constitutive mutants obtained by truncation in other TCS.