Cytoplasmic sensing by the inner membrane histidine kinase EnvZ.
Two-component regulatory systems drive signal transduction in bacteria. The simplest of these employs a membrane sensor kinase and a cytoplasmic response regulator. Environmental sensing is typically coupled to gene regulation. The histidine kinase EnvZ and its cognate response regulator OmpR regulate expression of outer membrane proteins (porins) in response to osmotic stress. We used hydrogen:deuterium exchange mass spectrometry to identify conformational changes in the cytoplasmic domain of EnvZ (EnvZc) that were associated with osmosensing. The osmosensor localized to a seventeen amino acid region of the four-helix bundle of the cytoplasmic domain and flanked the His(243) autophosphorylation site. High osmolality increased autophosphorylation of His(243), suggesting that these two events were linked. The transmembrane domains were not required for osmosensing, but mutants in the transmembrane domains altered EnvZ activity. A photoactivatable fusion protein composed of EnvZc fused to the fluorophore mEos2 (EnvZc-mEos2) was as capable as EnvZc in supporting OmpR-dependent ompF and ompC transcription. Over-expression of EnvZc reduced activity, indicating that the EnvZ/OmpR system is not robust. Our results support a model in which osmolytes stabilize helix one in the four-helix bundle of EnvZ by increased hydrogen bonding of the peptide backbone, increasing autophosphorylation and downstream signaling. The likelihood that additional histidine kinases use similar cytoplasmic sensing mechanisms is discussed.
Mechanobiology Institute, National University of Singapore, Singapore.
Yong Hwee
YH
Foo
Mechanobiology Institute, National University of Singapore, Singapore.
Yunfeng
Y
Gao
Mechanobiology Institute, National University of Singapore, Singapore.
Hongfang
H
Zhang
Mechanobiology Institute, National University of Singapore, Singapore; Jesse Brown Veterans Affairs Medical Center, Chicago, USA; University of Illinois-Chicago, USA. Electronic address: kenneyl@uic.edu.
Linda J
LJ
Kenney
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001696:Biomechanical Phenomena; D002462:Cell Membrane / Q000201:enzymology*; D003593:Cytoplasm / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D064587:Osmoregulation; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; C057843:envZ protein, E coli
Osmoregulation; Robustness; Super-resolution microscopy; Two-component regulatory system EnvZ/OmpR; histidine kinase; hydrogen:deuterium exchange mass spectrometry