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 Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide

1995 ◽  
Vol 312 (3) ◽  
pp. 839-845 ◽  
Author(s):  
E Elass-Rochard ◽  
A Roseanu ◽  
D Legrand ◽  
M Trif ◽  
V Salmon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Binding Sites ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Tryptic Fragment ◽  
High Affinity ◽  
Loop Region ◽  
High Affinity Binding Site ◽  
Lps Binding

The ability of lactoferrin (Lf), an iron-binding glycoprotein that is also called lactotransferrin, to bind lipopolysaccharide (LPS) may be relevant to some of its biological properties. A knowledge of the LPS-binding site on Lf may help to explain the mechanism of its involvement in host defence. Our report reveals the presence of two Escherichia coli 055B5 LPS-binding sites on human Lf (hLf): a high-affinity binding site (Kd 3.6 +/- 1 nM) and a low-affinity binding site (Kd 390 +/- 20 nM). Bovine Lf (bLf), which shares about 70% amino acid sequence identity with hLf, exhibits the same behaviour towards LPS. Like hLf, bLf also contains a low- and a high-affinity LPS-binding site. The Kd value (4.5 +/- 2 nM) corresponding to the high-affinity binding site is similar to that obtained for hLf. Different LPS-binding sites for human serum transferrin have been suggested, as this protein, which is known to bind bacterial endotoxin, produced only 12% inhibition of hLf-LPS interaction. Binding and competitive binding experiments performed with the N-tryptic fragment (residues 4-283), the C-tryptic fragment (residues 284-692) and the N2-glycopeptide (residues 91-255) isolated from hLf have demonstrated that the high-affinity binding site is located in the N-terminal domain I of hLf, and the low-affinity binding site is present in the C-terminal lobe. The inhibition of hLf-LPS interaction by a synthetic octadecapeptide corresponding to residues 20-37 of hLf and lactoferricin B (residues 17-41), a proteolytic fragment from bLf, revealed the importance of the 28-34 loop region of hLf and the homologous region of bLf for LPS binding. Direct evidence that this amino acid sequence is involved in the high-affinity binding to LPS was demonstrated by assays carried out with EGS-loop hLf, a recombinant hLf mutated at residues 28-34.

scholarly journals Characterization of Ca2+ interactions with matrix metallopeptidase-12: implications for matrix metallopeptidase regulation

2006 ◽  
Vol 398 (3) ◽  
pp. 393-398 ◽  
Author(s):  
Thomas Gossas ◽  
U. Helena Danielson
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Three Dimensional ◽  
Zinc Ion ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Matrix Metallopeptidase

Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca2+ binding for MMP-12 activity and the properties of the different Ca2+ sites, the activity as a function of [Ca2+] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca2+]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the KD was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and >100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The pKa values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate- and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca2+] and pH ranges studied suggest that Ca2+ may be involved in controlling the activity of MMP-12.

scholarly journals The low C5 convertase activity of the C4A6 allotype of human complement component C4

1989 ◽  
Vol 261 (3) ◽  
pp. 743-748 ◽  
Author(s):  
T Kinoshita ◽  
A W Dodds ◽  
S K A Law ◽  
K Inoue
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Alternative Pathway ◽  
Covalent Attachment ◽  
Haemolytic Activity ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Covalently Linked

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.

scholarly journals High-affinity binding site for a specific nuclear protein in the human IgM gene

Nature ◽  
1985 ◽  
Vol 315 (6016) ◽  
pp. 254-254
Author(s):  
L. Hennighausen ◽  
U. Siebenlist ◽  
D. Danner ◽  
P. Leder ◽  
D. Rawlins ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Protein ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Specific Nuclear Protein

Regulatory effect of cholecystokinin on subsequent insulin binding to pancreatic acini

1990 ◽  
Vol 258 (4) ◽  
pp. E562-E568
Author(s):  
Y. Okabayashi ◽  
M. Otsuki ◽  
T. Nakamura ◽  
M. Koide ◽  
H. Hasegawa ◽  
...  
Keyword(s):  
Binding Site ◽  
Insulin Binding ◽  
Pancreatic Acinar Cells ◽  
Affinity Binding ◽  
Regulatory Effect ◽  
Pancreatic Acini ◽  
High Affinity Binding ◽  
Receptor Affinity ◽  
High Affinity ◽  
High Affinity Binding Site

We investigated the regulatory effect of cholecystokinin (CCK) on subsequent insulin binding to pancreatic acinar cells. Rat isolated acini were preincubated with various concentrations of CCK octapeptide (CCK-8) at 37 degrees C. Acini were then washed, resuspended in the binding buffer, and incubated with 8.3 pM 125I-labeled insulin for 60 min at 37 degrees C. Pretreatment with CCK-8 caused inhibition of subsequent 125I-insulin binding that was time and concentration dependent. Significant inhibition was observed with 3 pM CCK-8. Computer analysis of the competition-inhibition study with a nonlinear least-squares curve-fitting program revealed that CCK-8 pretreatment of acini reduced the receptor affinity of the high-affinity binding site. This inhibitory action of CCK-8 was not due to the alteration in degradation or internalization of the tracer. When acini were pretreated with 100 pM CCK-8 for 120 min at 4 degrees C, a reduction in the receptor affinity of the high-affinity binding site was also observed. In pancreatic membrane prepared from acini preincubated with 100 pM CCK-8 for 120 min at 37 degrees C, displacement of 125I-insulin (83 pM) by unlabeled insulin (24 degrees C, 1 h) revealed that CCK-8 inhibited 125I-insulin binding by altering the receptor affinity of the high-affinity binding site. In acinar preparations the inhibitory effect of CCK-8 on 125I-insulin binding was abolished when acini were preincubated with CCK-8 and CCK receptor antagonist L 374718 at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

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