The effect of relaxation on the epitope mapping by saturation transfer difference NMR

Yan, Jiangli; Kline, Allen D.; Mo, Huaping; Shapiro, Michael J.; Zartler, Edward R.

doi:10.1016/s1090-7807(03)00106-x

Cited by 131 publications

(153 citation statements)

References 41 publications

Supporting

Mentioning

147

Contrasting

Order By: Relevance

“…The Trp 25 and His 36 aromatic amino acids of P-␤-Cat 17-48 make contacts with ␤-TrCP as evidenced by STD-NMR experiments. The STD intensities observed for aromatic protons, which classically possess larger T1 relaxation rates, could be overestimated (18). We have shown in our experiments that even when the relaxation delay was increased up to seven times T1 (the average value of T1 was around 500 ms) signals of Trp 25 and essentially those coming from the aromatic chain were always the strongest we observed in STD experiments.…”

Section: Discussioncontrasting

confidence: 50%

“…d ␣N (i,iϩ1), in the NOESY data set. The Arg 18 (residue 2 in the peptide numbering) broad amide signal was not identified for the P-␤-Cat peptide. In a similar way, it could not be observed in the study Binding of ␤-Catenin-phosphorylated Model Peptide to ␤-TrCPof the free peptide (12).…”

Section: Nmr Assignments For the P-␤-catmentioning

confidence: 97%

“…The ligand protons nearest to the protein are most likely to be saturated to the highest degree and therefore have the strongest signal in the STD spectrum; whereas the ligand protons located further away are saturated to a lower degree, and their STD intensities are weaker. Therefore, the degree of saturation of individual ligand protons reflects their proximity to the protein surface and can be used as an epitope method to describe the target-ligand interactions (13,17,18). Thus, STD NMR experiments were performed to define the binding epitopes of the P-␤-Cat 17-48 peptide.…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

View full text Add to dashboard Cite

␤-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...

show abstract

Section: Discussioncontrasting

confidence: 50%

Section: Nmr Assignments For the P-␤-catmentioning

confidence: 97%

mentioning

confidence: 99%

See 1 more Smart Citation

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

View full text Add to dashboard Cite

show abstract

“…In contrast, the N-acetyl methyl protons attached to the C-5 position of Neu5Ac have the strongest interaction with the VCNA, as reflected by the intensity of this signal in the STD spectrum. A quantitative comparison of the STD intensities for the methyl groups of the thiosialoside, that all have similar T 1 values making comparison of their binding epitope valid (25), reveals that when the C-5 acetate methyl intensity (Neu5Ac) is normalized to 100% then the corresponding values for the C-9 acetate, the methyl protons of the GlcNAc acetate, and the ␤1Me aglycon were of the order of 10%.…”

Section: Std Nmr Experiments To Investigate the Binding Of Neu59ac 2mentioning

confidence: 99%

Sialic Acid Recognition by Vibrio cholerae Neuraminidase

Moustafa

Connaris

Taylor

et al. 2004

Journal of Biological Chemistry

135

130

View full text Add to dashboard Cite

Vibrio cholerae neuraminidase (VCNA) plays a significant role in the pathogenesis of cholera by removing sialic acid from higher order gangliosides to unmask GM1, the receptor for cholera toxin. We previously showed that the structure of VCNA is composed of a central ␤-propeller catalytic domain flanked by two lectin-like domains; however the nature of the carbohydrates recognized by these lectin domains has remained unknown. We present here structures of the enzyme in complex with two substrates, ␣-2,3-sialyllactose and ␣-2,6-sialyllactose. Both substrate complexes reveal the ␣-anomer of N-acetylneuraminic acid (Neu5Ac) bound to the N-terminal lectin domain, thereby revealing the role of this domain. The large number of interactions suggest a relatively high binding affinity for sialic acid, which was confirmed by calorimetry, which gave a K d ϳ 30 M. Saturation transfer difference NMR using a non-hydrolyzable substrate, Neu5,9Ac 2 -2-S-(␣-2,6)-GlcNAc␤1Me, was also used to map the ligand interactions at the VCNA lectin binding site. It is well known that VCNA can hydrolyze both ␣-2,3-and ␣-2,6-linked sialic acid substrates. In this study using ␣-2,3-sialyllactose co-crystallized with VCNA it was revealed that the inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) was bound at the catalytic site. This observation supports the notion that VCNA can produce its own inhibitor and has been further confirmed by 1 H NMR analysis. The discovery of the sialic acid binding site in the N-lectin-like domain suggests that this might help target VCNA to sialic acid-rich environments, thereby enhancing the catalytic efficiency of the enzyme.

show abstract

“…Reliable interface mapping requires a relatively high off-rate of the ligand and a large excess of ligand over receptor. Widely different longitudinal relaxation rates of ligand protons [328] or non-uniform saturation of receptor resonances [329] may complicate the quantitative interpretation of differential saturation in terms of binding epitopes.…”

Section: Saturation Transfer Difference (Std) Spectroscopymentioning

confidence: 99%

Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules

Stangler¹,

Hartmann²,

Willbold³

et al. 2006

Zeitschrift für Physikalische Chemie

View full text Add to dashboard Cite

Solution NMR / Structure / Dynamics / InteractionsHigh resolution liquid state nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for in vitro studies of structure and dynamics of soluble biological macromolecules under physiological conditions. The unique combination of atomically resolved structural data with both local and global dynamic features covering the entire range of time scales from picoseconds to seconds makes NMR the method of choice in a very diverse and rapidly growing array of biochemical, biomedical, and pharmaceutical applications. After briefly introducing the basic principles of liquid state NMR we review recent methodological and instrumental advances in the field of biologically focused high resolution NMR. The main emphasis of the second part is on molecular interactions. Such interactions are fundamental for the function of proteins in living systems, e.g. for signal transduction, enzymatic catalysis, and immune defense. The tremendous opportunities of high resolution NMR in the identification and detailed characterization of the sites and modes of molecular interactions will be demonstrated.

show abstract

The effect of relaxation on the epitope mapping by saturation transfer difference NMR

Cited by 131 publications

References 41 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

Sialic Acid Recognition by Vibrio cholerae Neuraminidase

Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules

Contact Info

Product

Resources

About