Interactions between the PAS and HAMP Domains of theEscherichia coliAerotaxis Receptor Aer

Abstract

The Escherichia coli energy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 ␣-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer.Escherichia coli responds to its chemical environment by integrating signals from membrane-bound methyl-accepting chemoreceptors (MCPs) Tsr, Tar, Tap, and Trg and navigating bacteria towards nutrient-rich environments. E. coli also has an aerotaxis receptor, Aer, that guides bacteria towards oxygen and favorable environments for energy production (7, 33). The C-terminal signaling domains of Aer and the chemoreceptors are highly conserved (7,33,36), allowing specific interactions between receptors and the cytoplasmic CheA and CheW signaling proteins (8, 21). The MCPs are located in polar clusters in E. coli (25), organized as functional trimers of receptor dimers (2,19,20), where the trimers can include a mixture of MCP homodimer types (2, 37).The N-terminal sensing domain of Aer differs from the periplasmic sensing domain of the other chemoreceptors because it consists of a cytoplasmic PAS domain that binds FAD cofactor (7, 33). The Aer-PAS domain is connected to an F1 domain and transmembrane (TM) anchor, followed by a cytoplasmic HAMP linker domain and the conserved signaling domain (Fig. 1). HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) domains are conserved in many chemoreceptors, sensory histidine kinases, and other sensory proteins in archaea, bacteria, and eukaryotes (5, 23, 35; http://smart.embl-heidelberg.de/). The HAMP domains of the MCPs and Aer consist of approximately 50 amino acids linking the signal input and output domains (5...

Loss- and Gain-of-Function Mutations in the F1-HAMP Region of theEscherichia coliAerotaxis Transducer Aer

“…These findings indicate that the suppressible Aer mutations slow maturation but do not affect function of the mature protein. Thus, we conclude that slow maturation and rapid degradation suffice to account for the functional defects of most Aer null mutants characterized in this study and in previous studies (7,16,26). The severity of the mutant protein instability problem was not apparent in earlier work because steady-state expression levels were assessed at full induction, where degradation capacity is rate limiting (e.g., reference 7).…”

“…The HAMP domain in Aer occupies an analogous position in the molecule, but because stimuli are sensed through the PAS domain, sensory signals in Aer must necessarily follow a different path to the output domain. Genetic evidence suggests that the Aer PAS domain could interact directly with the C terminus of the HAMP AS-2 segment to modulate activity of the adjoining signaling domain (26).…”

“…The HAMP AS-2 segment in MCPs also appears to have a coiled-coil structure (11), and AS-2/AS-2Ј interactions most likely modulate MCP signal domain output (6). Aer signal output might be regulated similarly, through modulation of either AS-2/AS-2Ј or SD/SDЈ interactions (see below) by a direct, perhaps competing, interaction with the PAS sensory domain (25,26). We suggest that CW-biased mutations in PAS (M112I and M112V) and in AS-2 (M252T) augment a PAS interaction responsible for promoting CW signal output.…”

“…The unrescuable lesions, presumably defective in I/O control, lie adjacent to the C terminus of AS-2. Because the AS-2/SD junction appears to be the most likely target for PAS output control interactions (26), the unrescuable lesions may affect determinants or structures important for that PAS interaction.…”

Signaling Interactions between the Aerotaxis Transducer Aer and Heterologous Chemoreceptors in Escherichia coli

“…The extent of rescue seemed to be correlated with the severity of the CW-biased defects, which might reflect residual stimulus sensitivity and output signaling efficiency. CW-biased lesions in PAS (M112V) and HAMP (M252T), which may interact to elicit CW rotation (8,27), were rescued less effectively than a lesion in the HAMP connector (K221E), which has been proposed to play a less direct role in the production of CW output signals in Aer (8). Similarly, a CW-biased lesion in the proximal signaling domain (Q263L), which is downstream of the proposed sensory input control point, was not effectively rescued in any test.…”