The Aer2 receptor from Pseudomonas
aeruginosa has
an O2-binding PAS-heme domain that stabilizes O2 via a Trp residue in the distal heme pocket. Trp rotates ∼90°
to bond with the ligand and initiate signaling. Although the isolated
PAS domain is monomeric, both in solution and in a cyanide-bound crystal
structure, an unliganded structure forms a dimer. An overlay of the
two structures suggests possible signaling motions but also predicts
implausible clashes at the dimer interface when the ligand is bound.
Moreover, in a full-length Aer2 dimer, PAS is sandwiched between multiple
N- and C-terminal HAMP domains, which would feasibly restrict PAS
motions. To explore the PAS dimer interface and signal-induced motions
in full-length Aer2, we introduced Cys substitutions and used thiol-reactive
probes to examine in vivo accessibility and residue proximities under
both aerobic and anaerobic conditions. In vivo, PAS dimers were retained
in full-length Aer2 in the presence and absence of O2,
and the dimer interface was consistent with the isolated PAS dimer
structure. O2-mediated changes were also consistent with
structural predictions in which the PAS N-terminal caps move apart
and the C-terminal DxT region moves closer together. The DxT motif
links PAS to the C-terminal HAMP domains and was critical for PAS-HAMP
signaling. Removing the N-terminal HAMP domains altered the distal
PAS dimer interface and prevented signaling, even after signal-on
lesions were introduced into PAS. The N-terminal HAMP domains thus
facilitate the O2-dependent shift of PAS to the signal-on
conformation, clarifying their role upstream of the PAS-sensing domain.
P. aeruginosa
is an opportunistic pathogen that interprets environmental stimuli via 26 chemoreceptors that signal through 4 distinct chemosensory systems. The second chemosensory system, Che2, contains a receptor named Aer2 that senses O
2
and mediates stress responses and virulence and tunes chemotactic behavior.
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