Organization of the Aerotaxis Receptor Aer in the Membrane of Escherichia coli

ABSTRACT The Aer receptor guides Escherichia coli to specific oxygen and energy-generating niches. The input sensor in Aer is a flavin adenine dinucleotide-binding PAS domain, which is separated from a HAMP/signaling output domain by two membrane-spanning segments that flank a short (four-amino-acid) periplasmic loop. In this study, we determined the overall membrane organization of Aer by introducing combinations of residues that allowed us to differentiate intradimeric collisions from interdimeric collisions. Collisions between proximal residues in the membrane anchor were exclusively intra- or interdimeric but, with one exception, not both. Cross-linking profiles were consistent, with a rigid rather than flexible periplasmic loop and a tilted TM2 helix that crossed TM2′ at residue V197C, near the center of the lipid bilayer. The periplasmic loop formed a stable neighborhood that (i) included a maximum of three Aer dimers, (ii) did not swap neighbors over time, and (iii) appeared to be constrained by interactions in the cytosolic signaling domain.

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The Aer Protein of Escherichia coli Forms a Homodimer Independent of the Signaling Domain and Flavin Adenine Dinucleotide Binding

Journal of Bacteriology ◽

10.1128/jb.186.21.7456-7459.2004 ◽

2004 ◽

Vol 186 (21) ◽

pp. 7456-7459 ◽

Cited By ~ 15

Author(s):

Qinhong Ma ◽

Francis Roy ◽

Sarah Herrmann ◽

Barry L. Taylor ◽

Mark S. Johnson

Keyword(s):

Escherichia Coli ◽

Flavin Adenine Dinucleotide ◽

Cross Linking ◽

Membrane Anchor ◽

Dimer Formation ◽

Signaling Domain ◽

Aer Receptor

ABSTRACT In vivo cross-linking between native cysteines in the Aer receptor of Escherichia coli showed dimer formation at the membrane anchor and in the putative HAMP domain. Dimers also formed in mutants that did not bind flavin adenine dinucleotide and in truncated peptides without a signaling domain and part of the HAMP domain.

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Interactions between the PAS and HAMP Domains of the Escherichia coli Aerotaxis Receptor Aer

Journal of Bacteriology ◽

10.1128/jb.186.21.7440-7449.2004 ◽

2004 ◽

Vol 186 (21) ◽

pp. 7440-7449 ◽

Cited By ~ 32

Author(s):

Kylie J. Watts ◽

Qinhong Ma ◽

Mark S. Johnson ◽

Barry L. Taylor

Keyword(s):

Escherichia Coli ◽

Conformational Changes ◽

Random Mutagenesis ◽

Pas Domain ◽

Missense Mutations ◽

Membrane Anchor ◽

Functional Interactions ◽

Signaling Domain ◽

Α Helix ◽

Fad Binding

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 α-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.

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Genetic Analysis of the HAMP Domain of the Aer Aerotaxis Sensor Localizes Flavin Adenine Dinucleotide-Binding Determinants to the AS-2 Helix

Journal of Bacteriology ◽

10.1128/jb.187.1.193-201.2005 ◽

2005 ◽

Vol 187 (1) ◽

pp. 193-201 ◽

Cited By ~ 22

Author(s):

Qinhong Ma ◽

Mark S. Johnson ◽

Barry L. Taylor

Keyword(s):

Signal Transduction ◽

Flavin Adenine Dinucleotide ◽

Site Directed Mutagenesis ◽

Pas Domain ◽

E Coli ◽

Signaling Domain ◽

Intracellular Energy ◽

Proximal Signaling ◽

Binding Determinants ◽

Fad Binding

ABSTRACT HAMP domains are signal transduction domains typically located between the membrane anchor and cytoplasmic signaling domain of the proteins in which they occur. The prototypical structure consists of two helical amphipathic sequences (AS-1 and AS-2) connected by a region of undetermined structure. The Escherichia coli aerotaxis receptor, Aer, has a HAMP domain and a PAS domain with a flavin adenine dinucleotide (FAD) cofactor that senses the intracellular energy level. Previous studies reported mutations in the HAMP domain that abolished FAD binding to the PAS domain. In this study, using random and site-directed mutagenesis, we identified the distal helix, AS-2, as the component of the HAMP domain that stabilizes FAD binding. AS-2 in Aer is not amphipathic and is predicted to be buried. Mutations in the sequence coding for the contiguous proximal signaling domain altered signaling by Aer but did not affect FAD binding. The V264M residue replacement in this region resulted in an inverted response in which E. coli cells expressing the mutant Aer protein were repelled by oxygen. Bioinformatics analysis of aligned HAMP domains indicated that the proximal signaling domain is conserved in other HAMP domains that are not involved in chemotaxis or aerotaxis. Only one null mutation was found in the coding sequence for the HAMP AS-1 and connector regions, suggesting that these are not active signal transduction sites. We consider a model in which the signal from FAD is transmitted across a PAS-HAMP interface to AS-2 or the proximal signaling domain.

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Topology and Boundaries of the Aerotaxis Receptor Aer in the Membrane of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.188.3.894-901.2006 ◽

2006 ◽

Vol 188 (3) ◽

pp. 894-901 ◽

Cited By ~ 25

Author(s):

Divya N. Amin ◽

Barry L. Taylor ◽

Mark S. Johnson

Keyword(s):

Escherichia Coli ◽

Lateral Diffusion ◽

Membrane Vesicles ◽

Membrane Receptors ◽

Disulfonic Acid ◽

Type I ◽

Membrane Anchor ◽

Whole Cells ◽

Membrane Spanning

ABSTRACT Escherichia coli chemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic segment that is thought to form a membrane anchor. Most transmembrane prediction programs predict a single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and to decrease lateral diffusion in the membrane, at 4°C. Membrane boundaries were identified in membrane vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4′-maleimidylstilbene-2, 2′ disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid and peptide replacements.

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Structure-Function Relationships in the HAMP and Proximal Signaling Domains of the Aerotaxis Receptor Aer

Journal of Bacteriology ◽

10.1128/jb.01858-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 2118-2127 ◽

Cited By ~ 42

Author(s):

Kylie J. Watts ◽

Mark S. Johnson ◽

Barry L. Taylor

Keyword(s):

Structure Function ◽

Flavin Adenine Dinucleotide ◽

Cross Linking ◽

Dimer Interface ◽

In Silico Modeling ◽

Signaling Domain ◽

Output Module ◽

Proximal Signaling ◽

Signaling Domains

ABSTRACT Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two α-helices separated by a structured loop. The proximal signaling domain consisted of two α-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the β-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed.

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Loss- and Gain-of-Function Mutations in the F1-HAMP Region of the Escherichia coli Aerotaxis Transducer Aer

Journal of Bacteriology ◽

10.1128/jb.188.10.3477-3486.2006 ◽

2006 ◽

Vol 188 (10) ◽

pp. 3477-3486 ◽

Cited By ~ 38

Author(s):

Maria del Carmen Burón-Barral ◽

Khoosheh K. Gosink ◽

John S. Parkinson

Keyword(s):

Escherichia Coli ◽

Protein Product ◽

Pas Domain ◽

Mutant Proteins ◽

Expression Levels ◽

Swimming Pattern ◽

Normal Expression ◽

Signaling Domain ◽

Proximal Signaling ◽

Fad Binding

ABSTRACT The Escherichia coli Aer protein contains an N-terminal PAS domain that binds flavin adenine dinucleotide (FAD), senses aerotactic stimuli, and communicates with the output signaling domain. To explore the roles of the intervening F1 and HAMP segments in Aer signaling, we isolated plasmid-borne aerotaxis-defective mutations in a host strain lacking all chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family. Under these conditions, Aer alone established the cell's run/tumble swimming pattern and modulated that behavior in response to oxygen gradients. We found two classes of Aer mutants: null and clockwise (CW) biased. Most mutant proteins exhibited the null phenotype: failure to elicit CW flagellar rotation, no aerosensing behavior in MCP-containing hosts, and no apparent FAD-binding ability. However, null mutants had low Aer expression levels caused by rapid degradation of apparently nonnative subunits. Their functional defects probably reflect the absence of a protein product. In contrast, CW-biased mutant proteins exhibited normal expression levels, wild-type FAD binding, and robust aerosensing behavior in MCP-containing hosts. The CW lesions evidently shift unstimulated Aer output to the CW signaling state but do not block the Aer input-output pathway. The distribution and properties of null and CW-biased mutations suggest that the Aer PAS domain may engage in two different interactions with HAMP and the HAMP-proximal signaling domain: one needed for Aer maturation and another for promoting CW output from the Aer signaling domain. Most aerotaxis-defective null mutations in these regions seemed to affect maturation only, indicating that these two interactions involve structurally distinct determinants.

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