Saturation Transfer Difference (STD) NMR Spectroscopy Characterization of Dual Binding Mode of a Mannose Disaccharide to DC‐SIGN

Angulo, Jesús; Dı́az, Irene; Reina, José J.; Tabarani, Georges; Fieschi, Franck; Rojo, Javier; Nieto, Pedro M.

doi:10.1002/cbic.200800361

Cited by 62 publications

(57 citation statements)

References 23 publications

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“…3A [see SI Appendix for NMR assignment (SI Appendix, Table S1)]. To precisely map ligand epitopes in close contact with the protein, we acquired STD build up curves by collecting spectra at different saturation times (25)(26)(27)(28) and fitting experimental data with the monoexponential equation: STD = STD max [1 -exp (−k sat t)] (see Materials and Methods, NMR Spectroscopic Analysis for complete details). This method avoids artifacts in the epitope definition, which are consequences of differences in ability to accumulate saturation in the free state, allowing to obtain the real STD effect of each proton, independently of T1 bias, and to prevent any misinterpretation of STD enhancements.…”

Section: Std Nmr Epitope Mapping Of Chitin Oligosaccharides In the Prmentioning

confidence: 99%

Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization

Hayafune

Berisio

Marchetti

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

284

250

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Perception of microbe-associated molecular patterns (MAMPs) through pattern recognition receptors (PRRs) triggers various defense responses in plants. This MAMP-triggered immunity plays a major role in the plant resistance against various pathogens. To clarify the molecular basis of the specific recognition of chitin oligosaccharides by the rice PRR, CEBiP (chitin-elicitor binding protein), as well as the formation and activation of the receptor complex, biochemical, NMR spectroscopic, and computational studies were performed. Deletion and domain-swapping experiments showed that the central lysine motif in the ectodomain of CEBiP is essential for the binding of chitin oligosaccharides. Epitope mapping by NMR spectroscopy indicated the preferential binding of longer-chain chitin oligosaccharides, such as heptamer-octamer, to CEBiP, and also the importance of N-acetyl groups for the binding. Molecular modeling/docking studies clarified the molecular interaction between CEBiP and chitin oligosaccharides and indicated the importance of Ile 122 in the central lysine motif region for ligand binding, a notion supported by site-directed mutagenesis. Based on these results, it was indicated that two CEBiP molecules simultaneously bind to one chitin oligosaccharide from the opposite side, resulting in the dimerization of CEBiP. The model was further supported by the observations that the addition of (GlcNAc) 8 induced dimerization of the ectodomain of CEBiP in vitro, and the dimerization and (GlcNAc) 8 -induced reactive oxygen generation were also inhibited by a unique oligosaccharide, (GlcNβ1,4GlcNAc) 4 , which is supposed to have N-acetyl groups only on one side of the molecule. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and Oryza sativa chitinelicitor receptor kinase-1.plant immunity | MTI/PTI | chitin signaling | receptor-ligand interaction |

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Section: Std Nmr Epitope Mapping Of Chitin Oligosaccharides In the Prmentioning

confidence: 99%

Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization

Hayafune

Berisio

Marchetti

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

284

250

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“…[12] We have shown that such multimodal systems can be quantitatively treated by an appropriate analysis of STD NMR experiments based on STD initial growth rates. Thus, the experimental system can be deconvoluted as a simple sum of the contributions from each mode.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, the experimental system can be deconvoluted as a simple sum of the contributions from each mode. [12] This approach is justified by considering the effects of fast ligand rebinding during the receptor saturation in the STD experiment. If there is a certain probability that a given ligand molecule reenters into the binding site after a preceding binding event (and this rebinding is fast enough related to the relaxation properties), then the ligand spin populations would be partially perturbed due to the previous transfer step, and hence, its capacity to receive more transfer of saturation from the receptor will be different than that from a fresh ligand molecule.…”

Section: Introductionmentioning

confidence: 99%

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

Angulo¹,

Enríquez-Navas²,

Nieto³

2010

Chemistry A European J

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179

213

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The direct evaluation of dissociation constants (K(D)) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor-ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K(D), as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor-ligand dissociation constants (K(D)) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein-ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein-ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein-ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein-ligand systems: the binding of the saccharides GlcNAc and GlcNAcbeta1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental K(D) measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein-ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.

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“…6). We also calculated the STD values at a very low saturation time (100 ms) to assess whether consideration of the initial STD build-up would lead to more reliable predictions [8,9]. The overall profile of this GEM, with low values for all protons except the C6 protons, did fit well with the transferred magnetisation rates, but the ordering of the other protons are again rather poor (Fig.…”

Section: Case A: Jacalinmentioning

confidence: 99%

“…Although the influences are smaller, the observed STD values are still affected by such relaxation processes combined with the exchange kinetics. A more precise and recently used method is to fit the build-up of the STD to an exponential curve and estimate the initial slope [8,9]. This approach is usually done with short saturation times ($0.3 s), meaning the measured STDs are often intrinsically weak and thus more difficult to quantify, so this approach also imposes undesirable experimental consequences and may be time intensive.…”

Section: Theoretical Basismentioning

confidence: 99%

Group epitope mapping considering relaxation of the ligand (GEM-CRL): Including longitudinal relaxation rates in the analysis of saturation transfer difference (STD) experiments

Kemper

Patel

Errey

et al. 2010

Journal of Magnetic Resonance

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Saturation Transfer Difference (STD) NMR Spectroscopy Characterization of Dual Binding Mode of a Mannose Disaccharide to DC‐SIGN

Cited by 62 publications

References 23 publications

Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization

Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

Group epitope mapping considering relaxation of the ligand (GEM-CRL): Including longitudinal relaxation rates in the analysis of saturation transfer difference (STD) experiments

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