POSTERS - BLAST X - University of Utah

BLAST X Poster #69
MAPPING THE SIGNAL TRANSDUCTION PATHWAY WITHIN THE PAS DOMAIN OF THE
Aer RECEPTOR
Asharie J. Campbell, Kylie J. Watts, Mark S. Johnson, and Barry L. Taylor
Dept. Microbiology and Mol. Genetics, Loma Linda University, Loma Linda CA, USA
The E. coli Aer receptor senses redox changes through an FAD-binding PAS domain,
and transmits this redox status to the HAMP and signaling domains. Little is known about the
conformational changes that take place within the PAS domain. In this study, we used errorprone
PCR mutagenesis to find residues critical for PAS FAD-binding, sensing and signal
transduction. We screened 10,000 colonies for function, measured expression for 1,300 clones,
sequenced 400 mutants, and found 84 Aer aerotaxis-defective mutants that had just one amino
acid substitution. Of these, there were 72 substitutions in the PAS domain, 11 in the F1 region
and 1 in the TM region. The swimming behavior of the cells expressing these mutant proteins
included those that 1) were locked in a smooth swimming (CCW), "signal-off" state (60/84), 2)
had increased tumbling (CW) frequency (4/84) or 3) were locked in the CW "signal-on" state
(20/84). Approximately half (49/84) of the Aer mutants were functionally rescued by Tar. Mutant
proteins (11/84) that expressed at levels less than 30% of wild-type Aer showed enhanced
protein degradation rates. These replacements had altered side-chain polarity, mapped to
positions on or near loops and, with one exception, yielded a CCW (signal-off) phenotype. In
contrast, all but one of the CW (signal-on) mutants were stable, and clustered in three localized
regions: 1) in the putative FAD-binding cleft, 2) on the rear surface of the FAD-binding cleft and
3) at or near a loop in the N-terminal Cap region. When expressed at a 1:1 ratio with wild-type
Aer, three CW-locked mutants were dominant, abolishing wild-type mediated aerotaxis. Most
but not all of the FAD-binding lesions resulted in an unstable protein; those that were most
stable were located outside of the putative FAD-binding cleft.
The localization of gain-of-function (CW) lesions to three distinct clusters suggests that
conformational changes in these specific regions mimic the signal-on state of the PAS sensor.
If so, signaling within the Aer-PAS sensor would begin in the FAD-binding cleft and propagate
outward to the N-Cap loop. Previously, we found that removing part of the N-Cap mimics the
signal-on state. Thus, an attractive model is one where FAD reduction in the PAS domain
initiates a conformational change that propagates to the N-Cap loop, which, in turn, acts as a
hinge around which the N-cap moves, unmasking the signal-on state.
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