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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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Related in: MedlinePlus

Mapping of the unique residues identified from ‘3D’ plot onto the structure of  and comparison with J-surface mapping.(A) Two distinct regions are shown which are colored differently (red, yellow); (B) & (C) are the J-surface mapping of BH3I-1 at lower (P∶L::1∶0.229) and higher (P∶L::1∶0.918) ligand concentrations, respectively. Each red dot represents the possible location of the centroid of the aromatic ring of BH3I-1. The collection of dots suggests that the aromatic ring could be anywhere in that mapped region. The initial map appears diffused covering G94, G196, G148 residues but slowly converges near F143 and F146 as the concentration of ligand increases. J-surface map were calculated using JSURF program considering perturbations  ppm. Other parameters like  (standard deviation for data spread),  (number of random points to fill the sphere) and  (an offset in  added to radius of sphere) were set at 3, 2000 and 1, respectively. All the figures were made using the software Chimera [54].
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pone-0008943-g007: Mapping of the unique residues identified from ‘3D’ plot onto the structure of and comparison with J-surface mapping.(A) Two distinct regions are shown which are colored differently (red, yellow); (B) & (C) are the J-surface mapping of BH3I-1 at lower (P∶L::1∶0.229) and higher (P∶L::1∶0.918) ligand concentrations, respectively. Each red dot represents the possible location of the centroid of the aromatic ring of BH3I-1. The collection of dots suggests that the aromatic ring could be anywhere in that mapped region. The initial map appears diffused covering G94, G196, G148 residues but slowly converges near F143 and F146 as the concentration of ligand increases. J-surface map were calculated using JSURF program considering perturbations ppm. Other parameters like (standard deviation for data spread), (number of random points to fill the sphere) and (an offset in added to radius of sphere) were set at 3, 2000 and 1, respectively. All the figures were made using the software Chimera [54].

Mentions: Residues like G94, E96, Q111, L112, V126, E129, F143, F146, G147, G148, V192 and G196 from the plot and residues like L90, L99, Q111 and I114 from the plot were mapped onto the structure of (Figure 7A , Figure S1 & Table 2 ). Two distinct regions that are adjacent to each other but separated by a minimum distance of 10 were identified. The first site () is located at the edge of the extended hydrophobic BH3 groove near the ‘C’ terminal region. Residues like G94, E96, L99, V192 and G196 that constitute this site are part of the BH3 domain. The second site () is located at the middle of the highly conserved but less exposed hydrophobic groove. Residues like Q111, L112, V126, E129, F143, F146, G147 and G148 that spans the BH3 binding groove are proximally distributed within BH1 and BH2 domain. As mentioned above, the perturbation at saturation limit may or may not be directly related to the binding process. This is evident from residues like F27 and K157 that are not at the binding site, as confirmed by the slope values of 0.035 and 0.014 ppm/mM, but have high perturbation values of 0.211 and 0.346 ppm. This implies that mapping binding site using perturbation alone could be misleading in complex protein-ligand interactions.


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)

Mapping of the unique residues identified from ‘3D’ plot onto the structure of  and comparison with J-surface mapping.(A) Two distinct regions are shown which are colored differently (red, yellow); (B) & (C) are the J-surface mapping of BH3I-1 at lower (P∶L::1∶0.229) and higher (P∶L::1∶0.918) ligand concentrations, respectively. Each red dot represents the possible location of the centroid of the aromatic ring of BH3I-1. The collection of dots suggests that the aromatic ring could be anywhere in that mapped region. The initial map appears diffused covering G94, G196, G148 residues but slowly converges near F143 and F146 as the concentration of ligand increases. J-surface map were calculated using JSURF program considering perturbations  ppm. Other parameters like  (standard deviation for data spread),  (number of random points to fill the sphere) and  (an offset in  added to radius of sphere) were set at 3, 2000 and 1, respectively. All the figures were made using the software Chimera [54].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0008943-g007: Mapping of the unique residues identified from ‘3D’ plot onto the structure of and comparison with J-surface mapping.(A) Two distinct regions are shown which are colored differently (red, yellow); (B) & (C) are the J-surface mapping of BH3I-1 at lower (P∶L::1∶0.229) and higher (P∶L::1∶0.918) ligand concentrations, respectively. Each red dot represents the possible location of the centroid of the aromatic ring of BH3I-1. The collection of dots suggests that the aromatic ring could be anywhere in that mapped region. The initial map appears diffused covering G94, G196, G148 residues but slowly converges near F143 and F146 as the concentration of ligand increases. J-surface map were calculated using JSURF program considering perturbations ppm. Other parameters like (standard deviation for data spread), (number of random points to fill the sphere) and (an offset in added to radius of sphere) were set at 3, 2000 and 1, respectively. All the figures were made using the software Chimera [54].
Mentions: Residues like G94, E96, Q111, L112, V126, E129, F143, F146, G147, G148, V192 and G196 from the plot and residues like L90, L99, Q111 and I114 from the plot were mapped onto the structure of (Figure 7A , Figure S1 & Table 2 ). Two distinct regions that are adjacent to each other but separated by a minimum distance of 10 were identified. The first site () is located at the edge of the extended hydrophobic BH3 groove near the ‘C’ terminal region. Residues like G94, E96, L99, V192 and G196 that constitute this site are part of the BH3 domain. The second site () is located at the middle of the highly conserved but less exposed hydrophobic groove. Residues like Q111, L112, V126, E129, F143, F146, G147 and G148 that spans the BH3 binding groove are proximally distributed within BH1 and BH2 domain. As mentioned above, the perturbation at saturation limit may or may not be directly related to the binding process. This is evident from residues like F27 and K157 that are not at the binding site, as confirmed by the slope values of 0.035 and 0.014 ppm/mM, but have high perturbation values of 0.211 and 0.346 ppm. This implies that mapping binding site using perturbation alone could be misleading in complex protein-ligand interactions.

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
Related in: MedlinePlus