Interaction of the S100A6 mutant (C3S) with the V domain of the receptor for advanced glycation end products (RAGE).

S100A6 is involved in several vital biological functions, such as calcium sensing and cell proliferation. It is a homodimeric protein that belongs to the S100 protein family. The receptor for advanced glycation end products (RAGE) has been shown to play a role in the progression of various disease conditions, such as diabetes and immune/inflammatory disorders. Information regarding the association of RAGE with S100 proteins at a molecular level is useful to understand the diversity of the RAGE signaling pathways. In this report, biomolecular NMR techniques were utilized for the resonance assignment of the C3S mutation in human S100A6 and characterizing its interaction with the RAGE V domain. Further binding affinity between S100A6m and the RAGE V domain was determined by isothermal titration calorimetric studies. HADDOCK was used to generate a heterotetramer model of the S100A6m-RAGE V domain complex. This model provides an important insights into the S100-RAGE cellular signaling pathway.

(1)H, (15)N and (13)C assignments of the calcium bound S100P.

To determine the three-dimensional solution structure of the calcium bound S100P protein, the backbone and side chain resonance assignments of the S100P protein have been reported based on triple-resonance experiments using uniformly [(13)C, (15)N]-labeled calcium bound protein.

WITHDRAWN: Interaction of FGF1 with a novel anti-angiogenic drug SSR128129E.

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Molecular level interaction of inositol hexaphosphate with the C2B domain of human synaptotagmin I.

Synaptotagmin I is a synaptic vesicle membrane protein that serves as a multifunctional regulator during the exocytosis of neurotransmitter release. It contains C2A and C2B domains. The binding of Ca(2+) to the C2A domain activates the exocytosis of secretory vesicles, while the binding of inositol polyphosphates (IP4-IP6) to the C2B domain inhibits this process. To understand the IP6-induced inhibition of exocytosis of secretory vesicles, we determined the three-dimensional structure of the C2B-IP6 complex by nuclear magnetic resonance (NMR). In this study, we have determined the binding constant by isothermal titration calorimetry. The circular dichroism measurements demonstrated that IP6 can stabilize the C2B molecule. We identified the binding site using (1)H-(15)N heteronuclear single-quantum coherence spectroscopy titration data and determined the structure of the C2B-IP6 complex using multidimensional NMR studies. This information will aid in the design of better pharmacological treatments for neurological disorders.

1H, 13C and 15N backbone and side chain resonance assignments of human halo S100A1.

As part of our NMR structure determination of the Human S100A1, we report nearly complete NMR chemical shift assignments for the (1)H, (13)C and (15)N nuclei.

The IL1alpha-S100A13 heterotetrameric complex structure: a component in the non-classical pathway for interleukin 1alpha secretion.

Interleukin 1α (IL1α) plays an important role in several key biological functions, such as angiogenesis, cell proliferation, and tumor growth in several types of cancer. IL1α is a potent cytokine that induces a wide spectrum of immunological and inflammatory activities. The biological effects of IL1α are mediated through the activation of transmembrane receptors (IL1Rs) and therefore require the release of the protein into the extracellular space. IL1α is exported through a non-classical release pathway involving the formation of a specific multiprotein complex, which includes IL1α and S100A13. Because IL1α plays an important role in cell proliferation and angiogenesis, inhibiting the formation of the IL1α-S100A13 complex would be an effective strategy to inhibit a wide range of cancers. To understand the molecular events in the IL1α release pathway, we studied the structure of the IL1α-S100A13 tetrameric complex, which is the key complex formed during the non-classical pathway of IL1α release.

Molecular level interaction of the human acidic fibroblast growth factor with the antiangiogenic agent, inositol hexaphosphate .

Acidic fibroblast growth factor (FGF1) regulates a wide array of important biological phenomena such as angiogenesis, cell differentiation, tumor growth, and neurogenesis. Generally, FGFs are known for their strong affinity for the glycosaminoglycan heparin, as a prerequisite for recognition of a specific tyrosine kinase on the cell surface and are responsible for the cell signal transduction cascade. Inositol hexaphosphate (IP6) is a natural antioxidant and is known for its antiangiogenic role, in addition to its ability to control tumor growth. In the present study, we investigated the interaction of IP6 with the acidic fibroblast growth factor (FGF1) using various biophysical techniques including isothermal calorimetry, circular dichroism, and multidimensional NMR spectroscopy. Herein, we have reported the three-dimensional solution structure of the FGF1-IP6 complex. These data show that IP6 binds FGF1 and enhances its thermal stability. In addition, we also demonstrate that IP6 acts as an antagonist to acidic fibroblast growth factor by inhibiting its receptor binding and subsequently decreasing the mitogenic activity. The inhibition likely results in the ability of IP6 to antagonize the angiogenic and mitogenic activity of FGF1.

The NMR solution structure of human epidermal growth factor (hEGF) at physiological pH and its interactions with suramin.

Human epidermal growth factor (hEGF) induces the proliferation, differentiation and survival of various cell types including tumor-derived cells. Generally, hEGF performs its biological function by binding to a specific receptor (hEGFR) on the cell surface, thereby inducing signal transduction. Suramin, a polysulfonated naphthylurea that acts as a growth factor blocker, exhibits antiproliferative activity against non-small cell lung cancer (NSCLC) cells that overexpress EGFR on the cell surface. We determined the solution structure of hEGF under physiological conditions and investigated the interaction of suramin with hEGF using isothermal titration calorimetry and NMR spectroscopy techniques. The solution structure of hEGF presented in this paper is different from the bound form of hEGF present in the crystal structure of the 2:2 EGF-EGFR complex because its C-tail contains a hydrophobic core. This conformational difference supports the hypothesis that hEGF undergoes a conformational change when it binds to hEGFR and subsequently induces signal transduction. Based on the docking structure of the hEGF-suramin complex, we demonstrated how suramin blocks hEGF by binding to its receptor binding site (the C-terminal region around Arg45) and inhibits the crucial conformational change.

The heterohexameric complex structure, a component in the non-classical pathway for fibroblast growth factor 1 (FGF1) secretion.

Fibroblast growth factors (FGFs) are key regulators of cell proliferation, tumor-induced angiogenesis, and migration. FGFs are essential for early embryonic development, organ formation, and angiogenesis. FGF1 also plays an important role in inflammation, wound healing, and restenosis. The biological effects of FGF1 are mediated through the activation of the four transmembrane phosphotyrosine kinase fibroblast growth factor receptors in the presence of heparin sulfate proteoglycans and, therefore, require the release of the protein into the extracellular space. FGF1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex. The protein constituents of this complex include FGF1, S100A13, and the p40 form of synaptotagmin 1 (Syt1). Because FGF1 plays an important role in tumor formation, it is clear that preventing the formation of the multiprotein complex would be an effective strategy to inhibit a wide range of cancers. To understand the molecular events in the FGF1 release pathway, we studied the FGF1-S100A13 tetrameric and FGF1-S100A13-C2A hexameric complex structures, which are both complexes possibly formed during the non-classical pathway of FGF1 release.

Molecular level interactions of S100A13 with amlexanox: inhibitor for formation of the multiprotein complex in the nonclassical pathway of acidic fibroblast growth factor.

S100A13 and acidic fibroblast growth factor (FGF1) are involved in a wide array of important biological processes, such as angiogenesis, cell differentiation, neurogenesis, and tumor growth. Generally, the biological function of FGF1 is to recognize a specific tyrosine kinase on the cell surface and initiate the cell signal transduction cascade. Amlexanox (2-amino-7-isopropyl-5-oxo-5H-[1]benzopyrano[2,3-b]pyridine-3-carboxylic acid) is an antiallergic drug that binds S100A13 and FGF1 and inhibits the heat shock induced release of S100A13 and FGF1. In the present study, we investigated the interaction of amlexanox with S100A13 using various biophysical techniques, including isothermal titration calorimetry, fluorescence spectrophotometry, and multidimensional NMR spectroscopy. We report the three-dimensional solution structure of the S100A13-amlexanox complex. These data show that amlexanox binds specifically to the FGF1-S100A13 interface and prevents the formation of the FGF1-releasing complex. In addition, we demonstrate that amlexanox acts as an antagonist of S100A13 by binding to its FGF1 binding site and subsequently inhibiting the nonclassical pathway of these proteins. This inhibition likely results in the ability of amlexanox to antagonize the angiogenic and mitogenic activity of FGF1.

1H, 13C and 15N backbone and side chain resonance assignments of human interleukin 1alpha.

As part of our NMR structure determination of the human Interleukin-1alpha, we report nearly complete NMR chemical shift assignments for the (1)H, (13)C and (15)N nuclei.

S100A13-C2A binary complex structure-a key component in the acidic fibroblast growth factor for the non-classical pathway.

Fibroblast growth factors (FGFs) are key regulators of cell proliferation, differentiation, tumor-induced angiogenesis and migration. FGFs are essential for early embryonic development, organ formation and angiogenesis. They play important roles in tumor formation, inflammation, wound healing and restenosis. The biological effects of FGFs are mediated through the activation of the four transmembrane phosphotyrosine kinase receptors (FGFRs) in the presence of heparin sulfate proteoglycans (HSPGs) and therefore require the release of FGFs into the extracellular space. However, FGF-1 lacks the signal peptide required for the releasing of these proteins through the classical endoplasmic reticulum (ER)-Golgi secretary pathway. Maciag et al. demonstrated that FGF-1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex [M. Landriscina, R. Soldi, C. Bagala, I. Micucci, S. Bellum, F. Tarantini, I. Prudovsky, T. Maciag, S100A13 participates in the release of fibroblast growth factor 1 in response to heat shock in vitro, J. Biol. Chem. 276 (2001) 22544-22552; C.M. Carreira, T.M. LaVallee, F. Tarantini, A. Jackson, J.T. Lathrop, B. Hampton, W.H. Burgess, T. Maciag, S100A13 is involved in the regulation of fibroblast growth factor-1 and p40 synaptotagmin-1 release in vitro, J. Biol. Chem. 273 (1998) 22224-22231; T.M. LaValle, F. Tarantini, S. Gamble, C.M. Carreira, A. Jackson, T. Maciag, Synaptotagmin-1 is required for fibroblast growth factor-1 release, J. Biol. Chem. 273 (1998) 22217-22223; C. Bagalá, V. Kolev, A. Mandinova, R. Soldi, C. Mouta, I. Graziani, I, Prudovsky, T. Maciag, The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1, Biochem. Biophys. Res. Commun. 310 (2003) 1041-1047]. The protein constituents of this complex include FGF-1, S100A13 (a Ca(2+)-binding protein), and the p40 form of synaptotagmin 1 (Syt1). To understand the molecular events in the FGF-1 releasing pathway, we have studied the interactions of S100A13 with C2A by (1)H-(15)N HSQC titration and 3D-filtered NOESY experiments. We characterized the binary complex structure of S100A13-C2A by using a variety of multi-dimensional NMR experiments. This complex acts as a template for FGF-1 dimerization and multiprotein complex formation.

A new S4-ligated zinc-peptide 1:2 complex for the hydrolytic cleavage of DNA.

A new sulfur-ligated Zn-peptide 1:2 complex, [Zn(II)(Boc-NH-Cys-Gly-Cys-OMe)2]2- (2), was prepared, characterized, and tested for its DNA-binding and -cleavage properties. Complex 2 was found to cleave DNA hydrolytically. The negative charge in 2 reduces the affinity of the complex for DNA, and enhances its binding specificity.

Novel peptide-based copper(II) complexes for total hydrolytic cleavage of DNA.

Stable Cu(II) complexes with histamine- and histidine-containing dipeptides histidylserine and histidylphenylalanine have been developed. Their interaction in solution has been investigated, and the stability of their complexes was determined. The nature of binding in these complexes has been explained with the help of potentiometric pH titrations and 1H-NMR spectroscopy. The geometry of these complexes has been established by electronic spectra. The DNA-binding and -cleavage abilities of these Cu(II) complexes have been probed by the absorption, thermal denaturation, fluorescence, and electrophoresis experiments. The results suggest that these peptide-based Cu(II) complexes effectively bind and efficiently cleave DNA under mild biological conditions. Since Cu(II) complexes are known to play an important role in phosphodiester bond cleavages, these results assume importance.

Ternary zinc(II)-dipeptide complexes for the hydrolytic cleavage of DNA at physiological pH.

A series of Zn(II) complexes with cysteinylglycine (CysGly) and histidylserine (HisSer), and of CysGly and histidylphenylalanine (HisPhe) were investigated. Complex stabilities were determined potentiometrically, and binding geometries were probed by means of 1H-NMR spectroscopy, using Co(II) instead of Zn(II) as a spectroscopic marker. The ternary 1:1:1 complexes [Zn(II)(CysGly)(HisSer)] and [Zn(II)(CysGly)(HisPhe)] were shown by UV experiments, fluorescence titration, and gel electrophoresis to intercalate with DNA, and to hydrolytically cleave supercoiled DNA (form-I), partly also circular (form-II) DNA, under physiological conditions (37 degrees, H2O, pH 7.5).

Copper(II) complexes containing N,N-donor ligands and dipeptides act as hydrolytic DNA-cleavage agents.

Copper(II) complexes are known to play a significant role in both naturally occurring biological systems and pharmaceutical agents. Recently, Cu(II) complexes have gained importance in DNA cleavage essential for the development of anticancer drugs and chemotherapeutic agents. Therefore, we have designed small molecules, consisting of a metal ion, N,N-donor ligands, and dipeptides, to probe their DNA-cleaving potential. Accordingly, the interaction of Cu(II) with ethylenediamine, histamine and the dipeptides histidylglycine, histidylalanine, and histidylleucine has been investigated. The binding modes, stabilities, and geometries of these complexes were determined by various physicochemical techniques. Their DNA-binding abilities were probed by absorption and fluorescence spectroscopy, and their DNA-cleavage potential was tested by electrophoresis.