Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS domain by missense and truncation mutagenesis. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23°C was attenuated at 10°C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large multimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers.Motile bacteria migrate to nutrient-rich environments by suppressing directional changes (tumbling) when swimming toward a favorable environment. The receptor responsible for oxygen sensing in Escherichia coli, Aer, is a membrane-bound, homodimeric flavoprotein that is believed to respond to the redox state of the electron transport chain and signal this status to the two-component chemotaxis cascade that ultimately controls flagellar rotation (6, 48). Each monomer of Aer contains a cytosolic PAS domain that is connected by an F1 domain to a membrane anchor consisting of two transmembrane sequences and a short periplasmic loop (3). The second transmembrane sequence is linked to the signaling region of the protein by a HAMP domain (4, 37). The PAS (an acronym of Per-Arnt-Sim) domain is of particular interest because it is a cytoplasmic sensor and a member of the PAS superfamily of sensors found in all kingdoms of life (10, 53, 62; http://smart .embl-heidelberg.de/). PAS domains are sensory input modules that sense light, oxygen, redox potential, energy, and voltage, bind small ligands, and participate in protein-protein interactions (53).Although sequence identity is low in the PAS superfamily (53), the three-dimensional structures of PAS domains are highly conserved (60), suggesting that common mechanisms may be used for signaling. Three-dimensional crystal structures have been resolved for one or more of the PAS domains from the Ectothiorhodospira halophila blue light receptor chromophore 4-hydr...
