Auto-FACE: An NMR Based Binding Site Mapping Program for Fast Chemical Exchange Protein-Ligand Systems

Abstract

BackgroundNuclear Magnetic Resonance (NMR) spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, N 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 N 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 FindingsWe have automated the mapping of binding sites for fast chemical exchange systems using information obtained from N 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 and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant () 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 (). 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/SignificanceDetail 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.

Open‐ITC: an alternate computational approach to analyze the isothermal titration calorimetry data of complex binding mechanisms

“…They are related to each other by [ L T ] = [ L ] + [ PL ], for a single‐site binding mechanism. Instead of calculating [ L ] from [ L T ] through a polynomial equation obtained from the mass balance equations of protein and ligand (Krishnamoorthy et al ., ), we take an alternative approach in which a system of non‐linear equations corresponding to a particular set of binding mechanics is solved for Δ θ . This approach has the advantage of calculating [ L ], without deriving any analytical expression for [ L ] and is explained here with the help of a single‐site binding mechanism.…”

Fragment-based drug discovery using NMR spectroscopy

“…For example, if the size of the target allows, an X-ray structure of the protein is available, and amino acid backbone assignments are known, then ligand induced changes in chemical shift, e.g., from 15 N- or 13 C-HSQC spectra, can be used in both a qualitative and a quantitative way to map binding sites. The quantitative mapping of chemical shift perturbations on the protein surface is an especially powerful way to identify binding sites, modes, and stoichiometries (McCoy and Wyss 2002, Cioffi et al 2008, Krishnamoorthy et al 2010). In addition to this method, ligand-induced line broadening of protein resonances can be used to characterize binding modes (Reibarkh et al 2006).…”

NMR‐Assisted Molecular Docking Methodologies

“…Auto‐Face (Auto‐FAst Chemical Exchange analyzer) is a tool to define the binding site of a protein using CSP data in an automated manner rather than a molecular docking software 40. The aim of the tool is to distinguish the binding site residues from amino acid perturbed by allosteric structural changes or non‐specific interactions.…”