POSTERS - BLAST X - University of Utah

BLAST X Wed. Morning Session
DISCRETE SIGNAL-ON AND -OFF CONFORMATIONS IN THE AER HAMP DOMAIN
Kylie J. Watts, Mark S. Johnson and Barry L. Taylor
Dept. Microbiology and Mol. Genetics, Loma Linda University, Loma Linda, CA, USA
The PAS-FAD sensor of the aerotaxis receptor, Aer, signals through HAMP and
signaling domains that are similar to these domains in other chemoreceptors. Our previous
crosslinking studies showed that the AS-1 and AS-2 helices of the Aer-HAMP domain might
form a four-helix bundle similar to the Af1503 and Tar HAMP domains. In this study, AS-1
residues were crosslinked to AS-2′ residues in di-cys Aer mutants (using 13 proximal and 4
distal di-cys pairs). The results confirmed a parallel four-helix HAMP bundle for Aer, but one in
which AS-2 is rotated compared to the orientation of AS-2 in Af1503 or Tar.
We extended our HAMP crosslinking studies to probe for structural differences between
the signal-on (CW) and signal-off (CCW) states. In our previous crosslinking studies, we used
the oxidant copper phenanthroline, which maintains Aer in the signal-off state. In order to
generate snapshots of the signal-on state, CW lesions such as PAS-N85S were engineered into
the Aer di-cys and single-cys mutants. When the AS-1 to AS-2′ di-cys crosslinking experiments
were repeated in mutants containing N85S, several di-cys pairs showed significant increases in
dimer formation rates. These di-cys pairs were located at the distal end of the HAMP four-helix
bundle. In contrast, no significant crosslinking changes were observed at the proximal end of
the four-helix bundle. The data supports a model in which the distal ends of the HAMP helices
move closer together during signal transduction. This could be due to an inward lateral
movement of the helices, and may include some element of rotation. However, the entire Aer-
HAMP domain does not appear to rotate as has been proposed for Af1503.
In Aer, HAMP lesions that lock the receptor in the signal-on (CW) state cluster at the
distal end of a HAMP four-helix bundle, indicating a possible site for PAS-HAMP interactions
during signal transduction. We used PEG-maleimide to determine in vivo the solvent-accessible
surface of the HAMP and proximal signaling domains (residues 206-275). Solvent accessibility
was restricted for most AS-2, but not AS-1 or connector, residues in Aer. This indicates that AS-
2 residues that are exposed to solvent in Af1503 and Tar, and are predicted to be exposed in an
Aer-HAMP model, are buried in vivo in Aer. We are currently investigating whether these
residues are buried in a PAS-HAMP contact domain. We are also probing the surface of the
HAMP domain in the signal-on state (with N85S) to determine whether there are differences in
accessibility between the two signaling states.
33