Dr. Jing Yuan

Dr. Jing Yuan

Small regulatory proteins in the PhoQ/PhoP two-component system

Max-Planck-Institute of Terrestrial Microbiology, Department of Systems and Synthetic Microbiology, Marburg, Germany

The PhoQ/PhoP two-component system is crucial for survival and virulence of enterobacteria in their mammalian host. Two small membrane proteins, MgrB and SafA, inhibit and activate, respectively, the PhoQ/PhoP two-component system by interacting directly with the membrane located sensor kinase PhoQ. The molecular details of how these small proteins regulate PhoQ functionality are largely unknown and their in vivo interaction dynamics have not been unraveled. In the proposed project, we aim to use both biochemical and biophysical approaches to elucidate the mechanism and the dynamics of MgrB and SafA regulation:

I.) Decipher the nature of MgrB/PhoQ interactions by mutagenesis.

Three residues were identified to be important for MgrB-mediated inhibition of the PhoQ function in Escherichia coli. Mutation of the individual residues to alanine results in a reduction of MgrB activity by approximately 80%. To further specify the functional role of these residues, we will mutate them to other amino acids and test the function of these mutants in vivo. Additionally, we plan to insert a photoactivatable crosslinker in these positions and identify interacting residues in PhoQ through crosslinking and mass spectrometry.

II.) Identify binding surface between the PhoQ periplasmic domain and the C-terminal region of MgrB or SafA in vitro.

The periplasmic domain of PhoQ (PhoQperi) has been shown to interact with the C-terminal region of MgrB (MgrBC-term) or SafA (SafAC-term). We have purified PhoQperi and plan to identify the binding surface using H/D exchange. In addition, we aim to crystallize the PhoQperi/MgrBC-term and PhoQperi/SafAC-term complexes.

III.) Characterization of the interaction dynamics of PhoQ, MgrB and SafA.

E. coli cells encounter various stimuli when entering the mammalian host, which may promote MgrB inhibition, SafA activation, or a combination of both effects. We plan to follow the interaction dynamics of these proteins in vivo via fluorescence resonance energy transfer (FRET). This approach will allow us to understand how PhoQ integrates multiple stimuli with inputs from regulatory small proteins.

  • Jing Yuan, Fan Jin, Timo Glatter and Victor Sourjik (2017) Osmosensing by the bacterial PhoQ/PhoP two-component system. Proc Natl Acad Sci USA. doi:10.1073/pnas.1717272114.