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Auto-FACE: an NMR based binding site mapping program for fast chemical exchange protein-ligand systems.

Krishnamoorthy J, Yu VC, Mok YK - PLoS ONE (2010)

Bottom Line: This implies that chemical shift perturbation can represent the local binding event much more accurately than the global binding event.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.

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 hBcl(XL) and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of hBcl(XL) 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 10(4)) 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 10(4)). 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.

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Isothermal binding curve for BH3I-1 titrated into .() : Blank experiment where 1 mM of BH3I-1 was titrated into 20 mM phosphate buffer. (▪) : 1mM of BH3I-1 was titrated into 25 M . In all buffer solutions, concentration of DMSO was adjusted to 2.5%.
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pone-0008943-g003: Isothermal binding curve for BH3I-1 titrated into .() : Blank experiment where 1 mM of BH3I-1 was titrated into 20 mM phosphate buffer. (▪) : 1mM of BH3I-1 was titrated into 25 M . In all buffer solutions, concentration of DMSO was adjusted to 2.5%.

Mentions: To confirm the interaction of BH3I-1 with , ITC titration was performed. The isothermal binding curve fitted well to the three sites sequential binding model with good statistics for parameters (Figure 3 and Table 1). A closer look at the equilibrium constants for all three processes revealed that the last event could merely be a non-specific allosteric change rather than an actual binding process. This is evident from its lower value () and much higher value . A recent comparative work on thermodynamics of protein-ligand interaction shows that is more correlated with the binding process than [28], [29]. Considering the possibility that the third process might not be significant, the global interaction mechanism could be primarily dictated by the first two enthalpy dominant processes.


Auto-FACE: an NMR based binding site mapping program for fast chemical exchange protein-ligand systems.

Krishnamoorthy J, Yu VC, Mok YK - PLoS ONE (2010)

Isothermal binding curve for BH3I-1 titrated into .() : Blank experiment where 1 mM of BH3I-1 was titrated into 20 mM phosphate buffer. (▪) : 1mM of BH3I-1 was titrated into 25 M . In all buffer solutions, concentration of DMSO was adjusted to 2.5%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2823773&req=5

pone-0008943-g003: Isothermal binding curve for BH3I-1 titrated into .() : Blank experiment where 1 mM of BH3I-1 was titrated into 20 mM phosphate buffer. (▪) : 1mM of BH3I-1 was titrated into 25 M . In all buffer solutions, concentration of DMSO was adjusted to 2.5%.
Mentions: To confirm the interaction of BH3I-1 with , ITC titration was performed. The isothermal binding curve fitted well to the three sites sequential binding model with good statistics for parameters (Figure 3 and Table 1). A closer look at the equilibrium constants for all three processes revealed that the last event could merely be a non-specific allosteric change rather than an actual binding process. This is evident from its lower value () and much higher value . A recent comparative work on thermodynamics of protein-ligand interaction shows that is more correlated with the binding process than [28], [29]. Considering the possibility that the third process might not be significant, the global interaction mechanism could be primarily dictated by the first two enthalpy dominant processes.

Bottom Line: This implies that chemical shift perturbation can represent the local binding event much more accurately than the global binding event.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.

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 hBcl(XL) and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of hBcl(XL) 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 10(4)) 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 10(4)). 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.

Show MeSH