The connector SafA interacts with the multi-sensing domain of PhoQ in Escherichia coli.
Sensor histidine kinases of two-component signal transduction systems (TCSs) respond to various environmental signals and transduce the external stimuli across the cell membrane to their cognate response regulators. Recently, membrane proteins that modulate sensory systems have been discovered. Among such proteins is SafA, which activates the PhoQ/PhoP TCS by direct interaction with the sensor PhoQ. SafA is directly induced by the EvgS/EvgA TCS, thus connecting the two TCSs, EvgS/EvgA and PhoQ/PhoP. We investigated how SafA interacted with PhoQ. Bacterial two-hybrid and reporter assays revealed that the C-terminal region (41-65 aa) of SafA activated PhoQ at the periplasm. Adding synthetic SafA(41-65) peptide to the cell culture also activated PhoQ/PhoP. Furthermore, direct interaction between SafA(41-65) and the sensor domain of PhoQ was observed by means of surface plasmon resonance. NMR spectroscopy of (15) N-labelled PhoQ sensor domain confirmed that SafA and Mg(2+)  provoked a different conformational change of PhoQ. Site-directed mutagenesis studies revealed that R53, within SafA(41-65), was important for the activation of PhoQ, and D179 of the PhoQ sensor domain was required for its activation by SafA. SafA activated PhoQ by a different mechanism from cationic antimicrobial peptides and acidic pH, and independent of divalent cations and MgrB.
Department of Bioscience, Graduate School of Agriculture, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan.
Yoko
Y
Eguchi
Eiji
E
Ishii
Masatake
M
Yamane
Ryutaro
R
Utsumi
D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C576029:PhoQ protein, E coli; C576028:SafA protein, E coli