Rational design and molecular characterization of a chimaeric response regulator protein

Area of Science:

• Molecular biology
• Microbiology
• Protein biochemistry

Background:

• BvgA (Bordetella pertussis) and EvgA (Escherichia coli) are homologous response regulators involved in bacterial gene regulation.
• Understanding their domain structure and function is crucial for deciphering bacterial signaling pathways.

Purpose of the Study:

• To delineate the domain borders and linker sequences of BvgA and EvgA.
• To construct and characterize a chimeric protein (HA) combining EvgA receiver and BvgA output domains.
• To investigate the role of protein domains and phosphorylation in DNA binding and transcriptional activation.

Main Methods:

• Limited proteolysis coupled with MALDI-TOF-MS to identify protein domain boundaries.
• Construction and characterization of a chimeric protein (HA).
• In vitro and in vivo assays to assess phosphorylation, dimerization, DNA binding, and transcriptional activation.

Main Results:

• Thermolysin-sensitive linker regions were identified in BvgA (Leu130-Thr144) and EvgA (Leu127-Ser133).
• The chimeric protein HA, containing EvgA receiver and BvgA output domains, exhibited intact folding, phosphorylation, and dimerization.
• Phosphorylation enhanced BvgA DNA binding affinity and specificity, whereas HA's DNA binding was unaffected by phosphorylation.
• The chimeric protein HA failed to activate transcription of the BvgA-dependent fha promoter.

Conclusions:

• Specific interactions between the receiver and output domains of BvgA are essential for phosphorylation-induced transcriptional activation.
• Alterations in these interactions within the chimeric protein HA disrupt its ability to activate transcription.
• This study highlights the importance of domain communication in the function of bacterial response regulators.