Oxygen-Induced Conformational Changes in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor

The Aer2 receptor from Pseudomonas aeruginosa has an O-2-binding PAS-heme domain that stabilizes O-2 via a Trp residue in the distal heme pocket. Tip rotates similar to 90 degrees 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 RAMP 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 O-2, and the dimer interface was consistent with the isolated PAS dimer structure. O-2-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 RAMP domains and was critical for PAS-RAMP signaling. Removing the N-terminal RAMP domains altered the distal PAS dimer interface and prevented signaling, even after signal-on lesions were introduced into PAS. The N-terminal RAMP domains thus facilitate the O-2-dependent shift of PAS to the signal-on conformation, clarifying their role upstream of the PAS-sensing domain.