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

2004 ◽  
Vol 186 (21) ◽  
pp. 7440-7449 ◽  
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.

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

2007 ◽  
Vol 189 (20) ◽  
pp. 7206-7212 ◽  
Author(s):  
Divya N. Amin ◽  
Barry L. Taylor ◽  
Mark S. Johnson
Keyword(s):  
Escherichia Coli ◽  
Flavin Adenine Dinucleotide ◽  
Cross Linking ◽  
Pas Domain ◽  
Membrane Anchor ◽  
Signaling Domain ◽  
Membrane Spanning ◽  
Aer Receptor ◽  
Over Time ◽  
Output Domain

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.

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

2006 ◽  
Vol 188 (10) ◽  
pp. 3477-3486 ◽  
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.

scholarly journals Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme

2009 ◽  
Vol 191 (24) ◽  
pp. 7614-7619 ◽  
Author(s):  
Rong Shi ◽  
Magda Villarroya ◽  
Rafael Ruiz-Partida ◽  
Yunge Li ◽  
Ariane Proteau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Function Analysis ◽  
Trna Modification ◽  
Conserved Residues ◽  
Limited Trypsinolysis ◽  
Fad Binding ◽  
Trna Binding

ABSTRACT The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

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

2005 ◽  
Vol 187 (1) ◽  
pp. 193-201 ◽  
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.

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

2004 ◽  
Vol 186 (21) ◽  
pp. 7456-7459 ◽  
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.

scholarly journals Isolation and Characterization of Nonchemotactic CheZ Mutants of Escherichia coli

2000 ◽  
Vol 182 (12) ◽  
pp. 3544-3552 ◽  
Author(s):  
Kristin C. Boesch ◽  
Ruth E. Silversmith ◽  
Robert B. Bourret
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Random Mutagenesis ◽  
Signal Transduction Pathway ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Functional Impairments ◽  
Biophysical Characterization ◽  
Mutant Proteins ◽  
Isolation And Characterization

ABSTRACT The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ1–189 peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.

scholarly journals Dynamic architecture of the Escherichia coli Structural Maintenance of Chromosomes (SMC) complex, MukBEF

2019 ◽  
Author(s):  
Karthik V. Rajasekar ◽  
Minzhe Tang ◽  
Rachel Baker ◽  
Katarzyna Zawadzka ◽  
Oliwia Koczy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chromosome Segregation ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Dynamic Architecture ◽  
Functional Interactions ◽  
Protein Conformational Changes ◽  
C And N ◽  
Atp Binding And Hydrolysis ◽  
Structural Maintenance Of Chromosomes

AbstractStructural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organise chromosomes, thereby facilitating chromosome segregation. They utilise cycles of ATP binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contacts. We have analysed changes in the architecture of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis. This builds upon previous work showing that MukF kleisin directs formation of a MukBEF tripartite ring as a consequence of functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively (Zawadzka et al., 2018). Using both model truncated substrates and complexes containing full length MukB, we now demonstrate formation of MukBEF ‘dimers of dimers’, dependent on MukF dimerization, MukB head-engagement and MukE, which plays an essential role in organizing MukBEF complexes.

scholarly journals Different Signaling Roles of Two Conserved Residues in the Cytoplasmic Hairpin Tip of Tsr, the Escherichia coli Serine Chemoreceptor

2008 ◽  
Vol 190 (24) ◽  
pp. 8065-8074 ◽  
Author(s):  
Patricia Mowery ◽  
Jeffery B. Ostler ◽  
John S. Parkinson
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Contact Region ◽  
Ternary Complexes ◽  
State Transitions ◽  
Receptor Complexes ◽  
Signaling Domain ◽  
And Function ◽  
Mutant Receptors

ABSTRACT Bacterial chemoreceptors form ternary signaling complexes with the histidine kinase CheA through the coupling protein CheW. Receptor complexes in turn cluster into cellular arrays that produce highly sensitive responses to chemical stimuli. In Escherichia coli, receptors of different types form mixed trimer-of-dimers signaling teams through the tips of their highly conserved cytoplasmic domains. To explore the possibility that the hairpin loop at the tip of the trimer contact region might promote interactions with CheA or CheW, we constructed and characterized mutant receptors with amino acid replacements at the two nearly invariant hairpin charged residues of Tsr: R388, the most tip-proximal trimer contact residue, and E391, the apex residue of the hairpin turn. Mutant receptors were subjected to in vivo tests for the assembly and function of trimers, ternary complexes, and clusters. All R388 replacements impaired or destroyed Tsr function, apparently through changes in trimer stability or geometry. Large-residue replacements locked R388 mutant ternary complexes in the kinase-off (F, H) or kinase-on (W, Y) signaling state, suggesting that R388 contributes to signaling-related conformational changes in the trimer. In contrast, most E391 mutants retained function and all formed ternary signaling complexes efficiently. Hydrophobic replacements of any size (G, A, P, V, I, L, F, W) caused a novel phenotype in which the mutant receptors produced rapid switching between kinase-on and -off states, indicating that hairpin tip flexibility plays an important role in signal state transitions. These findings demonstrate that the receptor determinants for CheA and CheW binding probably lie outside the hairpin tip of the receptor signaling domain.

scholarly journals Alteration of Substrate-Induced Conformational Changes of Escherichia coli Melibiose Permease by Mutating Arg149

2020 ◽  
Author(s):  
Yibin Lin
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Structural Changes ◽  
Spectroscopic Techniques ◽  
Difference Spectroscopy ◽  
Difference Spectra ◽  
Binding Properties ◽  
Structure Changes ◽  
Β Sheets ◽  
Α Helix

AbstractFourier transform infrared difference spectroscopy and fluorescence spectroscopic techniques have been used to obtain information about substrate-induced structural changes of the melibiose permease mutant R149C, compared with the Cys-less, which were reconstituted into liposomes. ATR-FTIR evidences show that Na+-induced difference spectra of R149C and Cys-less are similar. However, Na+ induces some new peaks for R149C mutant permease. This means that replacement of Arg-149 by Cys may affect the structure of MelB, and then affect the binding of Na+. Melibiose-induced difference spectra of R149C in the presence of Na+ show some peaks in the amide I region not seen in Cys-less, corresponding to turns, β-sheets, α-helix changes. This suggests that R149C mutant permease undergo some different secondary structure changes compared to Cys-less mutant permease, when binding melibiose. Comparison of the permease intrinsic fluorescence variations of R149C and Cys-less indicate that there are similar substrate binding properties between R149C and Cys-less. When analyzing the effects of different sugars it appears that the R149C mutant is more sensitive to the sugar. All these data indicate that replacement of Arg-149 by Cys will affect Na+ and sugar binding, and enhance the selectivity and sensitivity to sugars.

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