Detecting Binding Affinity to Immobilized Receptor Proteins in Compound Libraries by HR-MAS STD NMR

Klein, Jens; Meinecke, Robert; Mayer, Moriz; Meyer, Bernd

doi:10.1021/ja990706x

Cited by 182 publications

(139 citation statements)

References 17 publications

(25 reference statements)

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“…Modifications to the force field for the phosphorylated residues (pSer) were made with CHARMM (30) and reported for molecular dynamics simulations. The residues, except the N-terminal Asp 17 , Gly 34 , Gly 38 , and Pro…”

Section: Reagents-␤-cat Fragments (17-48): Peptides ␤-Catmentioning

confidence: 99%

“…Specific affinity was investigated by STD NMR spectroscopy experiments (38) to study the influence of the serine phosphorylation on binding and to describe the region responsible for the binding interaction of the P-␤-Cat 17-48 peptide with GST-␤-TrCP. Without any GST-␤-TrCP protein present, STD spectra did not contain ligand signals, because saturation transfer does not occur without the protein.…”

Section: Nmr Assignments For the P-␤-catmentioning

confidence: 99%

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STD and TRNOESY NMR Studies on the Conformation of the Oncogenic Protein β-Catenin Containing the Phosphorylated Motif DpSGXXpS Bound to the β-TrCP Protein

Mégy

Bertho

Gharbi-Benarous

et al. 2005

Journal of Biological Chemistry

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␤-TrCP is the F-box protein component of an Skp1/ Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that ␤-TrCP targets the oncogenic protein ␤-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from ␤-catenin containing the phosphorylated motif DpSGXXpS (P-␤-Cat 17-48 ) and ␤-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the ␤-TrCP binding pocket. The amide and aromatic protons of the His 24 and Trp 25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter ␤-catenin fragment with the ␤-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated ␤-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.The ubiquitin-proteasome pathway of protein degradation is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction (1, 2). This work is based on the study of the oncogenic protein ␤-catenin (␤-Cat), 1 which plays an essential role in the Wingless/Wnt signaling pathway and is an important component of cadherin cell-adhesion complexes (Fig. 1A). The abundance of ␤-catenin in the cytoplasm is regulated by ubiquitindependent proteolysis (3), and Wnt signaling is regulated by the presence or absence of the intracellular protein ␤-catenin. When Wnt signal is absent, the signal transduction pathway is off because ␤-catenin is rapidly destroyed. A large multiprotein machine normally facilitates the addition of phosphate groups to ␤-catenin by glycogen synthase kinase-3␤ (GSK3␤). Phosphorylated ␤-catenin binds to a protein called ␤-TrCP and is then modified by the covalent addition of a small protein called ubiquitin. Proteins tagged with ubiquitin are degraded by the 26 S proteasome, the protein-recycling center of the cell. When cells are exposed to the Wnt signal, it binds to cell surface receptors. Receptor activation blocks ␤-catenin phosphorylation, and its subsequent ubiquitination by an unknown mechanism that requires the intervention of the Disheveled protein (Dsh). ␤-Catenin is thus diverted from the proteasome. It accumulates and enters the nucleus, where it finds a par...

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Section: Reagents-␤-cat Fragments (17-48): Peptides ␤-Catmentioning

confidence: 99%

Section: Nmr Assignments For the P-␤-catmentioning

confidence: 99%

STD and TRNOESY NMR Studies on the Conformation of the Oncogenic Protein β-Catenin Containing the Phosphorylated Motif DpSGXXpS Bound to the β-TrCP Protein

Mégy

Bertho

Gharbi-Benarous

et al. 2005

Journal of Biological Chemistry

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“…NMR-STD Analysis-NMR saturation transfer difference (STD) experiments (35,36) were run at 500 MHz in a Varian INOVA-500 instrument. Saturation was achieved by a train of 50-ms Gaussian-shaped pulses with 5-ms intervals, and a total saturation time of ϳ2.5 s. The on-resonance protein irradiation was performed at a chemical shift of Ϫ0.4 ppm, and off-resonance at 34.5 ppm, where no protein signals are present.…”

mentioning

confidence: 99%

Isonicotinic Acid Hydrazide Conversion to Isonicotinyl-NAD by Catalase-peroxidases

Wiseman

Carpena

Feliz

et al. 2010

Journal of Biological Chemistry

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Activation of the pro-drug isoniazid (INH) as an anti-tubercular drug in Mycobacterium tuberculosis Isonicotinic acid hydrazide (isoniazid or INH)3 is a widely used anti-tubercular pro-drug that requires activation in a reaction involving the catalase-peroxidase KatG of Mycobacterium tuberculosis (MtKatG) (1) whereby the hydrazine group is removed and the isonicotinyl portion is added to NAD ϩ to generate isonicotinyl-NAD or IN⅐NAD (see Fig. 1). Once formed, IN⅐NAD inhibits the synthesis of mycolic acids, and therefore, the growth of M. tuberculosis, by binding to the longchain enoyl acyl carrier protein reductase (InhA) (2). Despite understanding the role of IN⅐NAD in the inhibition of mycolic acid synthesis and knowing that KatG is required for INH activation in vivo, uncertainties about the mechanism of its formation remain.The involvement of MtKatG in INH activation suggested that the peroxidatic process had a role, and this provided a focus for several studies employing external oxidants such as peroxyacetic acid (3), t-butyl hydroperoxide (4 -6) and low levels of H 2 O 2 (7, 6) to activate the peroxidatic pathway for INH oxidation and activation. In addition, EPR studies have demonstrated that INH can serve as an electron source to reduce peroxidatic intermediates, including specific Trp ⅐ radical species following peroxyacetic acid oxidation of MtKatG (3,8).A multiplicity of methods has been employed to directly and indirectly assay INH activation, including the determination of INH oxidation to isonicotinic acid (9, 10), the HPLC assay of INH disappearance (11), the inactivation of InhA in a mixture of InhA and KatG (7,12,13,14), the HPLC detection of IN⅐NAD (4, 15), and the direct measurement of IN⅐NAD using its characteristic absorbance at 326 nm (6,7,12,15,16). Reports of INH activation in mixtures lacking an external oxidant (4,5,6,9,12,14,15) initially suggested that the peroxidatic process may not be required, but the mixtures of INH, NADH, and KatG would have supported NADH reduction of molecular oxygen to superoxide and low levels of H 2 O 2 (15) to activate the peroxidase reaction.Despite the considerable evidence that a peroxidatic process is involved in IN⅐NAD synthesis, attempts to rationalize the reaction entirely in terms of the peroxidatic pathway generally produced models that were incomplete from the standpoint of electron balance (5,7,10) or that involved hypothetical intermediates not characterized in any other system (5, 6). For example, a common scheme shows the isonicotinyl radical, generated in a peroxidatic reaction, reacting with NAD ϩ to yield IN⅐NAD, when such a reaction would actually yield the IN⅐NAD ϩ ⅐ radical (Fig. 1). The need to reduce this radical in an oxidizing environment suggests that more than a simple peroxidatic pathway is involved in the INH activation process. This rationale leads to superoxide, O 2. , a known reducing agent with

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“…Interestingly, KcsA inactivation is associated to functional uncoupling of channels that exhibited positively coupled gating prior to the addition of the ShB peptide (26). Also, saturation transfer difference (STD) 5 NMR methods (27)(28)(29) have been used to identify interactions at atomic resolution in the KcsA-ShB complex. STD spectra are obtained after selective saturation of resonances of the KcsA protein, whereby the magnetization redistributes within the protein via intramolecular spin diffusion.…”

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confidence: 99%

N-type Inactivation of the Potassium Channel KcsA by the Shaker B “Ball” Peptide

Molina

Barrera

Encinar

et al. 2008

Journal of Biological Chemistry

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Detecting Binding Affinity to Immobilized Receptor Proteins in Compound Libraries by HR-MAS STD NMR

Cited by 182 publications

References 17 publications

STD and TRNOESY NMR Studies on the Conformation of the Oncogenic Protein β-Catenin Containing the Phosphorylated Motif DpSGXXpS Bound to the β-TrCP Protein

STD and TRNOESY NMR Studies on the Conformation of the Oncogenic Protein β-Catenin Containing the Phosphorylated Motif DpSGXXpS Bound to the β-TrCP Protein

Isonicotinic Acid Hydrazide Conversion to Isonicotinyl-NAD by Catalase-peroxidases

N-type Inactivation of the Potassium Channel KcsA by the Shaker B “Ball” Peptide

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