Dataset Information

NMR chemical exchange as a probe for ligand-binding kinetics in a theophylline-binding RNA aptamer.

ABSTRACT: The apparent on and off rate constants for binding of theophylline to its RNA aptamer in the absence of Mg(2+) were determined here by 2D (1)H-(1)H ZZ-exchange NMR spectroscopy. Analysis of the buildup rate of the exchange cross peaks for several base-paired imino protons in the RNA yielded an apparent k(on) of 600 M(-1) s(-1). This small apparent k(on) results because the free RNA exist as a dynamic equilibrium of inactive states rapidly interconverting with a low population of active species. The data found here indicate that the RNA aptamer employs a conformational selection mechanism for binding theophylline in the absence of Mg(2+). The kinetic data found here also explain a very unusual property of this RNA-theophylline system: slow exchange on the NMR chemical shift time scale for a weakly binding complex. To our knowledge, it is unprecedented to have such a weakly binding complex (K(d) approximately 3.0 mM at 15 degrees C) show slow exchange on the NMR chemical shift time scale, but the results clearly demonstrate that slow exchange and weak binding are readily rationalized by a small k(on). Comparisons with other ligand-receptor interactions are presented.

SUBMITTER: Latham MP

PROVIDER: S-EPMC2752443 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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NMR chemical exchange as a probe for ligand-binding kinetics in a theophylline-binding RNA aptamer.

Latham Michael P MP Zimmermann Grant R GR Pardi Arthur A

Journal of the American Chemical Society 20090401 14

The apparent on and off rate constants for binding of theophylline to its RNA aptamer in the absence of Mg(2+) were determined here by 2D (1)H-(1)H ZZ-exchange NMR spectroscopy. Analysis of the buildup rate of the exchange cross peaks for several base-paired imino protons in the RNA yielded an apparent k(on) of 600 M(-1) s(-1). This small apparent k(on) results because the free RNA exist as a dynamic equilibrium of inactive states rapidly interconverting with a low population of active species. ...[more]

PMID: 19317486

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Project description:BackgroundNuclear 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 findingsWe 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/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.

Project description:RNA aptamers are oligonucleotides that bind with high specificity and affinity to target ligands. In the absence of bound ligand, secondary structures of RNA aptamers are generally stable, but single-stranded and loop regions, including ligand binding sites, lack defined structures and exist as ensembles of conformations. For example, the well-characterized theophylline-binding aptamer forms a highly stable binding site when bound to theophylline, but the binding site is unstable and disordered when theophylline is absent. Experimental methods have not revealed at atomic resolution the conformations that the theophylline aptamer explores in its unbound state. Consequently, in the present study we applied 21 microseconds of molecular dynamics simulations to structurally characterize the ensemble of conformations that the aptamer adopts in the absence of theophylline. Moreover, we apply Markov state modeling to predict the kinetics of transitions between unbound conformational states. Our simulation results agree with experimental observations that the theophylline binding site is found in many distinct binding-incompetent states and show that these states lack a binding pocket that can accommodate theophylline. The binding-incompetent states interconvert with binding-competent states through structural rearrangement of the binding site on the nanosecond to microsecond timescale. Moreover, we have simulated the complete theophylline binding pathway. Our binding simulations supplement prior experimental observations of slow theophylline binding kinetics by showing that the binding site must undergo a large conformational rearrangement after the aptamer and theophylline form an initial complex, most notably, a major rearrangement of the C27 base from a buried to solvent-exposed orientation. Theophylline appears to bind by a combination of conformational selection and induced fit mechanisms. Finally, our modeling indicates that when Mg2+ ions are present the population of binding-competent aptamer states increases more than twofold. This population change, rather than direct interactions between Mg2+ and theophylline, accounts for altered theophylline binding kinetics.

Dataset Information

NMR chemical exchange as a probe for ligand-binding kinetics in a theophylline-binding RNA aptamer.

Publications

NMR chemical exchange as a probe for ligand-binding kinetics in a theophylline-binding RNA aptamer.

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