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

Abstract

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.

The Aer2 chemoreceptor from Vibrio vulnificus is a tri‐PAS‐heme oxygen sensor

“…In Pa Aer2, PAS signals are initiated after O 2 enters the distal heme cleft, the Hβ‐Leu moves out of the ligand‐binding site, and the Iβ‐Trp rotates to bond with heme‐bound O 2 (Airola et al, 2013 ; Garcia et al, 2017 ; Sawai et al, 2012 ). PAS signals are ultimately relayed to the distal HAMP domains via a conserved “DxT” motif at the PAS C‐terminus, that in Pa Aer2, has been shown to undergo significant O 2 ‐induced repositioning (Airola et al, 2013 ; Orillard et al, 2021 ). Ala substitutions of the Hβ‐Leu and the Thr of the DxT motif both lock Pa Aer2 into the signal‐on state (Garcia et al, 2017 ).…”

“…Removing tri‐HAMP alters the PAS dimer interface and causes the distal PAS interface to splay apart. This abolishes signaling, even in the presence of PAS signal‐on lesions (Anaya et al, 2022 ; Orillard et al, 2021 ). Thus, unlike Pa Aer2 and Vc Aer2, Vv Aer2 and Li Aer2 can apparently maintain a physiologically‐appropriate PAS dimer interface in the absence of any N‐terminal domains.…”

“…All three PAS domains bound b ‐type heme and O 2 , although PAS1 readily oxidized in vitro. Possible intramolecular dynamics are shown based on O 2 ‐induced changes known to occur in Pa PAS (Orillard et al, 2021 ). Inward motions at the PAS C‐terminal DxT motifs are shown as red arrows and concomitant outward motions at the PAS N‐terminus (N‐cap) are shown as green arrows.…”

“…Ala substitutions at each of the DxT threonines in Vv Aer2 impacted behavior (Figure 8 ). The DxT motif at the C‐terminus of Pa PAS has been shown to undergo significant O 2 ‐induced rearrangements and is critical for downstream signaling (Orillard et al, 2021 ). O 2 ‐binding causes the Pa PAS N‐caps to move apart and the DxT motif to move inward towards the dimer interface (Airola et al, 2013 ; Orillard et al, 2021 ).…”

“…Much of what is known about Aer2 comes from studies on the Aer2 receptors of P. aeruginosa ( Pa Aer2) and V. cholerae ( Vc Aer2). Both are soluble receptors that coordinate b ‐type heme in their PAS (Per‐ARNT‐Sim) domains and signal in response to O 2 binding (Garcia et al, 2017; Greer‐Phillips et al, 2018; Orillard et al, 2021; Watts et al, 2011). However, Aer2 receptors are incongruent in their number of PAS domains.…”