scholarly journals Molecular organization of the interferon γ-binding domain in heparan sulphate

1995 ◽  
Vol 310 (2) ◽  
pp. 497-505 ◽  
Author(s):  
H Lortat-Jacob ◽  
J E Turnbull ◽  
J A Grimaud
Keyword(s):  
Binding Site ◽  
Regulatory Element ◽  
Heparan Sulphate ◽  
Interferon Γ ◽  
Binding Domain ◽  
Molecular Organization ◽  
Gamma Activity ◽  
Ifn Gamma ◽  
High Affinity Binding Site ◽  
Protein Binding Sequence

Interferon (IFN)-gamma, in common with a number of cytokines or growth factors, strongly interacts with heparan sulphate (HS). It has been shown previously that one of the C-terminal basic clusters of amino acids (a regulatory element of IFN-gamma activity) is involved in this interaction. The structural organization of the HS domain that binds to human IFN-gamma has been investigated here. IFN-gamma-affinity chromatography of HS oligosaccharides released by either enzymic or chemical cleavage showed that the binding site is not found in a domain that is resistant to either heparinase or heparitinase or exclusively N-sulphated or N-acetylated. This led us to take a ‘footprinting’ approach in which HS was depolymerized in the presence of IFN-gamma and the cytokine-protected sequences were separated from the digested fragments. Using this strategy we consistently isolated an IFN-gamma-protected domain (IPD; approx. 10 kDa) which displayed the same affinity as full-length HS for the cytokine. Treatment of IPD with either heparinase or heparitinase strongly reduced its affinity, confirming that the high-affinity binding site encompassed a mixture of HS structural domains. Patterns of depolymerization with either enzymic or chemical agents were consistent with IPD being composed of an extended internal domain (approx. 7 kDa) which is predominantly N-acetylated and GlcA-rich, flanked by small N-sulphated oligosaccharides (mainly hexa- to octasaccharides). This is the first description of an HS protein-binding sequence with this type of molecular organization. Furthermore, using a cross-linking strategy, we demonstrated that one HS molecule bound to an IFN-gamma dimer. Together these results lead us to propose a novel model for the interaction of HS with a protein, in which two sulphated terminal sequences of the binding domain interact directly with the two IFN-gamma C-termini and bridge the two cytokine monomers through an internal N-acetyl-rich sequence.

scholarly journals The ABC transporter MsbA interacts with lipid A and amphipathic drugs at different sites

2009 ◽  
Vol 419 (2) ◽  
pp. 317-328 ◽  
Author(s):  
Alena Siarheyeva ◽  
Frances J. Sharom
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Lipid A ◽  
Transmembrane Helix ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Nucleotide Binding Domain ◽  
Kd Values ◽  
High Affinity Binding Site ◽  
Substrate Binding Sites

MsbA is an essential ABC (ATP-binding cassette) transporter involved in lipid A transport across the cytoplasmic membrane of Gram-negative bacteria. The protein has also been linked to efflux of amphipathic drugs. Purified wild-type MsbA was labelled stoichiometrically with the fluorescent probe MIANS [2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid] on C315, which is located within the intracellular domain connecting transmembrane helix 6 and the nucleotide-binding domain. MsbA–MIANS displayed high ATPase activity, and its folding and stability were unchanged. The initial rate of MsbA labelling by MIANS was reduced in the presence of amphipathic drugs, suggesting that binding of these compounds alters the protein conformation. The fluorescence of MsbA–MIANS was saturably quenched by nucleotides, lipid A and various drugs, and estimates of the Kd values for binding fell in the range of 0.35–10 μM. Lipid A and daunorubicin were able to bind to MsbA–MIANS simultaneously, implying that they occupy different binding sites. The effects of nucleotide and lipid A/daunorubicin binding were additive, and binding was not ordered. The Kd of MsbA for binding lipid A was substantially decreased when the daunorubicin binding site was occupied first, and prior binding of nucleotide also modulated lipid A binding affinity. These results indicate that MsbA contains two substrate-binding sites that communicate with both the nucleotide-binding domain and with each other. One is a high affinity binding site for the physiological substrate, lipid A, and the other site interacts with drugs with comparable affinity. Thus MsbA may function as both a lipid flippase and a multidrug transporter.

Decreased IFN-gamma activity in anxiety disorders

2010 ◽  
Author(s):  
R. Tukel ◽  
B.A. Arslan ◽  
B.A. Ertekin ◽  
E. Ertekin ◽  
S. Oflaz ◽  
...  
Keyword(s):  
Anxiety Disorders ◽  
Gamma Activity ◽  
Ifn Gamma

Phosphate Binding to Isolated Chloroplast Coupling Factor (CF1)

1988 ◽  
Vol 43 (3-4) ◽  
pp. 213-218 ◽  
Author(s):  
Bernhard Huchzermeyer
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Inorganic Phosphate ◽  
Coupling Factor ◽  
Binding Domain ◽  
Atp Binding ◽  
Phosphate Binding ◽  
Chloroplast Coupling Factor ◽  
Single Binding Site ◽  
Site Binding

A single binding site for phosphate was found on isolated chloroplast coupling factor in the absence of nucleotides. In our experiments the phosphate binding site showed a Kd of 170 μᴍ. We did not observe any differences whether the ATPase activity of CF] had been activated or not. If the enzyme was incubated with [γ-32P]ATP the amount of 32P bound per CF1 depended on the pretreatment of the enzyme: In the presence of ADP no ATP or phosphate was bound to CF,. After activation of ATPase activity one mol of ATP per mol CF, was rapidly bound and hydrolyzed while there was a slowly occurring binding of another phosphate without concomitant nucleotide binding. We conclude that there are two different types of phosphate binding observed in our experiments: 1) Inorganic phosphate can be bound by one catalytic site per mol of CF1 2) The γ-phosphate of ATP is able to bind to an ATP binding domain of the enzyme if this domain can exchange substrates with the incubation medium. This ATP binding domain appears to differ from the site binding inorganic phosphate, because at least a portion of the coupling factor contains more than one labelled phosphate during our ATPase tests.

A Monoclonal Antibody against the Ligand Binding Site of the Receptor for Mouse Interferon-γ

1989 ◽  
Vol 9 (5) ◽  
pp. 551-562 ◽  
Author(s):  
MITALI BASU ◽  
JUDITH L. PACE ◽  
DAVID M. PINSON ◽  
STEPHEN W. RUSSELL
Keyword(s):  
Monoclonal Antibody ◽  
Ligand Binding ◽  
Binding Site ◽  
Interferon Γ ◽  
Ligand Binding Site
Sign in / Sign up

Export Citation Format

Share Document