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|Title:||Auto-FACE: An NMR based binding site mapping program for fast chemical exchange protein-ligand systems||Authors:||Krishnamoorthy, J.
|Issue Date:||18-Feb-2010||Citation:||Krishnamoorthy, J., Yu, V.C.K., Mok, Y.-K. (2010-02-18). Auto-FACE: An NMR based binding site mapping program for fast chemical exchange protein-ligand systems. PLoS ONE 5 (2) : -. ScholarBank@NUS Repository. https://doi.org/10.1371/journal.pone.0008943||Abstract:||Background: Nuclear Magnetic Resonance (NMR) spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, 15N Heteronuclear Single Quantum Correlation (HSQC) experiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of 15N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately. Methodology/Principal Findings: We have automated the mapping of binding sites for fast chemical exchange systems using information obtained from 15N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer) determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues. Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein hBclXL and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of hBclXL were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant (3:0 x 104) calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (12:8 x 104). Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker than the former one. Further NMR based model fitting for individual residues suggest single site model for residues present at these binding sites and two site model for residues present between these sites. This implies that chemical shift perturbation can represent the local binding event much more accurately than the global binding event. Conclusion/Significance: Detail NMR chemical shift perturbation analysis enabled binding site residues to be distinguished from non-binding site residues for accurate mapping of interaction site in complex fast exchange system between small molecule and protein. The methodology is automated and implemented in a program called "Auto-FACE", which also allowed quantitative information of each interaction site and elucidation of binding mechanism. © 2010 Krishnamoorthy et al.||Source Title:||PLoS ONE||URI:||http://scholarbank.nus.edu.sg/handle/10635/100136||ISSN:||19326203||DOI:||10.1371/journal.pone.0008943|
|Appears in Collections:||Staff Publications|
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