Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 97, Issue 11, Pages 5830-5835Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.100118697
Keywords
redox sensing; prosthetic group; PAS domain; membrane topology
Categories
Funding
- NCI NIH HHS [P30 CA042014, CA42014] Funding Source: Medline
- NIGMS NIH HHS [5-R37-GM19559, R37 GM019559] Funding Source: Medline
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Aerotactic responses in Escherichia coli are mediated by the membrane transducer Aer, a recently identified member of the superfamily of PAS domain proteins, which includes sensors of light, oxygen, and redox state. Initial studies of Aer suggested that it might use a flavin adenine dinucleotide (FAD) prosthetic group to monitor cellular redox changes. To test this idea, we purified lauryl maltoside-solubilized Aer protein by His-tag affinity chromatography and showed by high performance liquid chromatography, mass spectrometry, and absorbance spectroscopy that it hound FAD noncovalently, Polypeptide fragments spanning the N-terminal 290 residues of Aer, which contains the PAS motif, were able to bind FAD, Fusion of this portion of Aer to the flagellar signaling domain of Tsr, the serine chemoreceptor, yielded a functional aerotaxis transducer, demonstrating that the FAD-binding portion of Aer is sufficient for aerosensing. Aerotaxis-defective missense mutants identified two regions, in addition to the PAS domain, that play roles in FAD binding. Those regions flank a central hydrophobic segment needed to anchor Aer to the cytoplasmic membrane. They might contact the FAD ligand directly or stabilize the FAD-binding pocket. However, their lack of sequence conservation in Aer homologs of other bacteria suggests that they play less direct roles in FAD binding. One or both regions probably also play important roles in transmitting stimulus-induced conformational changes to the C-terminal flagellar signaling domain to trigger aerotactic behavioral responses.
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