Gain-of-function mutations cluster in distinct regions associated with the signalling pathway in the PAS domain of the aerotaxis receptor, Aer.
The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD-containing PAS domain. Here, we randomly mutagenized the region encoding residues 14-119 of the PAS domain and found 72 aerotaxis-defective mutants, 24 of which were gain-of-function, signal-on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS-FAD domain in NifL from Azotobacter vinlandii. Signal-on lesions clustered in the FAD binding pocket, the beta-scaffolding and in the N-cap loop. We suggest that the signal-on lesions mimic the 'signal-on' state of the PAS domain, and therefore may be markers for the signal-in and signal-out regions of this domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the beta-scaffolding and the N-cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS beta-scaffold and residue Q248C in the HAMP AS-2 helix.
Campbell
Asharie J
AJ
Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, CA 92350, USA.
Watts
Kylie J
KJ
Johnson
Mark S
MS
Taylor
Barry L
BL
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
C108679:Aer protein, E coli; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D036341:Intercellular Signaling Peptides and Proteins; D005182:Flavin-Adenine Dinucleotide