scholarly journals The Arg-Gly-Asp–Containing, Solvent-Exposed Loop of Ptr ToxA Is Required for Internalization

2008 ◽  
Vol 21 (3) ◽  
pp. 315-325 ◽  
Author(s):  
Viola A. Manning ◽  
Sara M. Hamilton ◽  
P. Andrew Karplus ◽  
Lynda M. Ciuffetti
Keyword(s):  
Phosphorylation Site ◽  
Affinity Binding ◽  
Mesophyll Cells ◽  
High Affinity ◽  
Treatment Zone ◽  
Ptr Toxa ◽  
High Affinity Binding Site ◽  
Toxin Uptake

Internalization of the proteinaceous host-selective toxin, Ptr ToxA (ToxA), into sensitive wheat mesophyll cells is correlated with toxin activity. The solvent-exposed, Arg-Gly-Asp (RGD)-containing loop of ToxA is a candidate for interaction with the plasma membrane, which is a likely prerequisite to toxin internalization. Based on the percentage of cells affected by a given number of ToxA molecules in a treatment zone, the number of ToxA molecules bound to high-affinity sites was estimated at 3 × 106 per cell and the Kd for binding was estimated to be near 1 nM. An improved heterologous expression method of proteins that contain mutations in ToxA, coupled with a newly developed semiquantitative bioassay, revealed that some amino acids in the RGD-containing loop contribute more to toxin activity than others. Protease protection assays that detect internalized protein and inhibition of toxin uptake indicated that, for each ToxA variant tested, the extent of toxin activity correlates with the amount of internalized protein. RGD-containing peptide inhibition of both activity and internalization supported these findings. These data support the hypothesis that ToxA interacts with a high-affinity binding site on wheat mesophyll cells through the RGD-containing, solvent-exposed loop, resulting in toxin internalization and eventual cell death. The inability to detect phosphorylation of ToxA in vitro and in vivo suggests that a putative CKII phosphorylation site in the RGD-containing loop is required for internalization, not phosphorylation.

scholarly journals The TorR High-Affinity Binding Site Plays a Key Role in Both torR Autoregulation and torCADOperon Expression in Escherichia coli

2000 ◽  
Vol 182 (4) ◽  
pp. 961-966 ◽  
Author(s):  
Mireille Ansaldi ◽  
Gwénola Simon ◽  
Michèle Lepelletier ◽  
Vincent Méjean
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Binding Sites ◽  
Response Regulator ◽  
Regulatory System ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

ABSTRACT In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-component regulatory system induces thetorCAD operon, which encodes the TMAO respiratory system ofEscherichia coli. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD. The torRgene and the torCAD operon are divergently transcribed, and the short torR-torC intergenic region contains four direct repeats (the tor boxes) which proved to be TorR binding sites. The tor box 1-box 2 region covers thetorR transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites. By using torR-lacZ operon fusions in different genetic backgrounds, we showed that thetorR gene is negatively autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in atorS mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the torR promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the torCAD operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4.

scholarly journals Modular Structure of PACT: Distinct Domains for Binding and Activating PKR

2001 ◽  
Vol 21 (6) ◽  
pp. 1908-1920 ◽  
Author(s):  
Gregory A. Peters ◽  
Rune Hartmann ◽  
Jun Qin ◽  
Ganes C. Sen
Keyword(s):  
Protein Domain ◽  
Affinity Binding ◽  
Translation Inhibition ◽  
High Affinity ◽  
Cellular Apoptosis ◽  
Domain 3 ◽  
Function Relationship ◽  
Relationship Of

ABSTRACT PACT is a 35-kDa human protein that can directly bind and activate the latent protein kinase, PKR. Here we report that PKR activation by PACT causes cellular apoptosis in addition to PKR autophosphorylation and translation inhibition. We analyzed the structure-function relationship of PACT by measuring its ability to bind and activate PKR in vitro and in vivo. Our studies revealed that among three domains of PACT, the presence of either domain 1 or domain 2 was sufficient for high-affinity binding of PACT to PKR. On the other hand, domain 3, consisting of 66 residues, was absolutely required for PKR activation in vitro and in vivo. When fused to maltose-binding protein, domain 3 was also sufficient for efficiently activating PKR in vitro. However, it bound poorly to PKR at the physiological salt concentration and consequently could not activate it properly in vivo. As anticipated, activation of PKR by domain 3 in vivo could be restored by attaching it to a heterologous PKR-binding domain. These results demonstrated that the structure of PACT is modular: it is composed of a distinct PKR-activation domain and two mutually redundant PKR-interacting domains.

scholarly journals Common and Variable Contributions of Fis Residues to High-Affinity Binding at Different DNA Sequences

2006 ◽  
Vol 188 (6) ◽  
pp. 2081-2095 ◽  
Author(s):  
Leah S. Feldman-Cohen ◽  
Yongping Shao ◽  
Derrick Meinhold ◽  
Charmi Miller ◽  
Wilfredo Colón ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Binding Motif ◽  
Affinity Binding ◽  
Flanking Sequence ◽  
Binding Assays ◽  
High Affinity Binding ◽  
High Affinity

ABSTRACT Fis is a nucleoid-associated protein that interacts with poorly related DNA sequences with a high degree of specificity. A difference of more than 3 orders of magnitude in apparent Kd values was observed between specific (Kd , ∼1 to 4 nM) and nonspecific (Kd , ∼4 μM) DNA binding. To examine the contributions of Fis residues to the high-affinity binding at different DNA sequences, 13 alanine substitutions were generated in or near the Fis helix-turn-helix DNA binding motif, and the resulting proteins were purified. In vitro binding assays at three different Fis sites (fis P II, hin distal, and λ attR) revealed that R85, T87, R89, K90, and K91 played major roles in high-affinity DNA binding and that R85, T87, and K90 were consistently vital for binding to all three sites. Other residues made variable contributions to binding, depending on the binding site. N84 was required only for binding to the λ attR Fis site, and the role of R89 was dramatically altered by the λ attR DNA flanking sequence. The effects of Fis mutations on fis P II or hin distal site binding in vitro generally correlated with their abilities to mediate fis P repression or DNA inversion in vivo, demonstrating that the in vitro DNA-binding effects are relevant in vivo. The results suggest that while Fis is able to recognize a minimal common set of DNA sequence determinants at different binding sites, it is also equipped with a number of residues that contribute to the binding strength, some of which play variable roles.

scholarly journals Mobilization of Chimeric oriT Plasmids by F and R100-1: Role of Relaxosome Formation in Defining Plasmid Specificity

2000 ◽  
Vol 182 (14) ◽  
pp. 4022-4027 ◽  
Author(s):  
Richard A. Fekete ◽  
Laura S. Frost
Keyword(s):  
Binding Sites ◽  
Integration Host Factor ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Negative Dominance ◽  
Transfer Apparatus

ABSTRACT Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage atnic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage atnic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriTallowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).

scholarly journals Interaction of Mycotoxin Alternariol with Serum Albumin

2019 ◽  
Vol 20 (9) ◽  
pp. 2352 ◽  
Author(s):  
Eszter Fliszár-Nyúl ◽  
Beáta Lemli ◽  
Sándor Kunsági-Máté ◽  
Luca Dellafiora ◽  
Chiara Dall’Asta ◽  
...  
Keyword(s):  
Serum Albumin ◽  
Endocrine Disruptor ◽  
Spectroscopic Measurement ◽  
Serum Albumins ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Alternaria Species ◽  
Site Markers

Alternariol (AOH) is a mycotoxin produced by Alternaria species. In vitro studies suggest the genotoxic, mutagenic, and endocrine disruptor effects of AOH, and an increased incidence of esophageal cancer has been reported related to higher AOH exposure. Human serum albumin (HSA) is the most abundant plasma protein in the circulation, it is able to affect toxicokinetic properties of numerous xenobiotics. HSA forms stable complexes with several mycotoxins, however, the interaction of AOH with albumin has not been examined. In this study, the complex formation of AOH with HSA was tested, employing fluorescence spectroscopy, ultrafiltration, and molecular modeling. Each spectroscopic measurement shows the formation of stable AOH-HSA complexes (K = 4 × 105 L/mol). Investigations with site markers (in spectroscopic and ultrafiltration models) as well as modeling studies suggest that AOH occupies Sudlow’s site I as a high-affinity binding site in HSA. The binding affinity of AOH towards bovine, porcine, and rat albumins was also tested, suggesting that AOH binds to rat albumin with considerably higher affinity than other albumins tested. Our results demonstrate the strong interaction of AOH with serum albumins, suggesting the potential in vivo importance of these interactions.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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