doi: 10.1016/j.jmb.2012.01.057. Epub 2012 Feb 7.
Enthalpy-entropy compensation and cooperativity as thermodynamic epiphenomena of structural flexibility in ligand-receptor interactions
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- PMID:22342886
- PMCID: PMC3349339
- DOI: 10.1016/j.jmb.2012.01.057
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Enthalpy-entropy compensation and cooperativity as thermodynamic epiphenomena of structural flexibility in ligand-receptor interactions
Andrea Ferrante et al. J Mol Biol..
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Abstract
Ligand binding is a thermodynamically cooperative process in many biochemical systems characterized by the conformational flexibility of the reactants. However, the contribution of conformational entropy to cooperativity of ligation needs to be elucidated. Here, we perform kinetic and thermodynamic analyses on a panel of cycle-mutated peptides, derived from influenza H3 HA(306-319), interacting with wild type and a mutant HLA-DR. We observe that, within a certain range of peptide affinity, this system shows isothermal entropy-enthalpy compensation (iEEC). The incremental increases in conformational entropy measured as disruptive mutations are added in the ligand or receptor are more than sufficient in magnitude to account for the experimentally observed lack of free-energy decrease cooperativity. Beyond this affinity range, compensation is not observed, and therefore, the ability of the residual interactions to form a stable complex decreases in an exponential fashion. Taken together, our results indicate that cooperativity and iEEC constitute the thermodynamic epiphenomena of the structural fluctuation that accompanies ligand-receptor complex formation in flexible systems. Therefore, ligand binding affinity prediction needs to consider how each source of binding energy contributes synergistically to the folding and kinetic stability of the complex in a process based on the trade-off between structural tightening and restraint of conformational mobility.
Copyright © 2012 Elsevier Ltd. All rights reserved.
Figures
Natural log (ln) plot of cooperativity vs.KD for each pMHCII complex tested at 37 °C. Because we defined as the ratio of the observed to expected values forKD, andKD is inversely proportional to affinity, positive cooperativity in affinity is indicated on the y-axis by values <0 and negative cooperativity by values >0. Horizontal error bars represent the SD of theKD measurement. Vertical error bars represent the error of cooperativity as calculated through SE propagation. The line indicates the fit of the data to a linear regression. White dots represent P1P6P9 substituted peptides, whereas black dots show β81P2P3P10 substituted complexes.
(A) Binding affinity and kinetics of DR1 interaction with a range of peptides generated by cycle mutation from the sequence of HA306-319 at P1, P6 and P9 position was measured at 37 °C by equilibrium based-competitive binding assay and FP. Effect of peptide substitution on binding affinity and kinetics is presented as a fold difference compared with the wtHA. Substitutions that negatively affect binding have relative affinities and kinetics lower than 1. Values shown are the mean ± SD of three independent experiments performed at least in triplicate. (B) Binding affinity and kinetics of wtDR1 and β81mut interaction with a range of peptides generated by cycle mutation from the sequence of HA306-319 at P2, P3, and P10 positions was measured at 37 °C by equilibrium based-competitive binding assay and FP. Bar graph as in (A). (C) - (D) Natural log (ln) plot of cooperativity vs. association (C) and dissociation rates (D) for each multiple substituted pMHCII complex whose peptide release was measurable. White dots represent P1P6P9 substituted peptides, whereas β81/P2P3P10 data are indicated as black dots. The lines indicate the fit of the data to a linear regression. Error bars as in Figure 1.
Plot of cooperativity vs.KD for each pMHCII complex tested at 37 °C. Because we defined as the ratio of the observed to expected values forKD, andKD is inversely proportional to affinity, the cooperative effect is positive if, while if 0 the cooperative effect is negative. The line indicates the fit of the data to a single exponent power law. White dots represent P1P6P9 substituted peptides, whereas black dots show β81P2P3P10 substituted complexes.
(A) Comparison of binding thermodynamics of wtDR1 interaction with P1P6P9 substituted peptides. Energetic profile of interaction is described by change in binding energy, and its enthalpic and entropic component. (B) Comparison of binding thermodynamics of wtDR1 and β81mut interaction with P2P3P10 substituted peptides. Energetic profile of interaction is described as in (A). (C) Values of ΔH° are plotted against TΔS° where each data point represents values calculated from the van’t Hoff data for the actual temperature at which equilibrium binding measurements were made. The predicted ΔH° and TΔS° as a function of temperature were calculated from ΔH =ΔC°p(T − TH) andTΔS =TΔC°pln(T/TS). (D) The enthalpic (ΔH°) and entropic (−TΔS°) contributions to binding free energy are shown at 300.15 K for all the complexes reported in Table I. The strong apparent correlation is related to the clustering of the ΔG°bind.
(a) Plot of theKd variation upon loss of the β81 H-bond (expressed as the ratio of peptide affinity for β81mut over wtDR1) versus the logarithmicKd to wtDR1 for all peptides tested. The broken line (ratio = 1) identifies theKd value (205 nM) above which peptide binding to wtDR1 is energetically favored if compared with β81mut. Blue dots indicate peptides with greater affinity for β81mut than for wtDR1. Brown dots represent peptides with greater affinity for wtDR1. (b) PeptideKd variation upon loss of the β81 H-bond (expressed as the ratio of peptide affinity for β81mut over wtDR1) is analyzed as variation of on and off rates (here indicated as pMHCII half-lives). Color code as in panel (a). (c) Analysis of the modifications of the entropic (top panel) and the enthalpic (middle panel) contributions to free-energy decrease for group 1 (brown bars) and group 2 (blue bars) peptides in binding to either wtDR1 (dark bars) or β81mut (light bars). Bottom panel shows the extent of compensation between enthalpy and entropy consequent to the loss of the b81 H-bond. Negative bars indicate a productive compensation, resulting in increased or unchanged ΔG. Positive bars indicate undercompensation, as the loss of enthalpy is not balanced by increased entropy. The consequent reduced ΔG indicates less favorable binding to DR1 as compared with β81mut.
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