Human POT1 Unfolds G-Quadruplexes by Conformational Selection

ABSTRACTThe reaction mechanism by which shelterin protein POT1 (Protection of Telomeres) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. We show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is fast, with an apparent relaxation time of 80.0 ± 0.4 ms, and strong, with a binding free energy of −10.1 ± 0.3 kcal mol−1. That favourable free energy arises from a large favourable enthalpy contribution of −38.2 ± 0.3 kcal mol−1. In sharp contrast, the binding of POT1 to an initially folded 24 nt G-quadruplex structure is slow, with an average relaxation time of 2000-3000 s. Fluorescence, circular dichroism and analytical ultracentrifugation studies show that POT1 binding is coupled to quadruplex unfolding with a final stoichiometry of 2 POT1 molecules bound per 24 nt DNA. The binding isotherm for the POT1-quadruplex binding interaction is sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provides an excellent quantitative fit to the experimental binding data. The overall favourable free energy of the POT1-quadruplex interaction is −7.1 kcal mol−1, which arises from a balance between unfavourable free energy of +3.4 kcal mol−1 for quadruplex unfolding and a large, favorable free energy of −10.5 kcal mol−1 for POT1 binding. We show that POT1 can unfold and bind to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid or parallel), but will not interact with duplex DNA or with a parallel G-quadruplex structure formed by a c-myc promoter sequence. Finally, molecular dynamics simulations provide a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

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Human POT1 unfolds G-quadruplexes by conformational selection

Nucleic Acids Research ◽

10.1093/nar/gkaa202 ◽

2020 ◽

Vol 48 (9) ◽

pp. 4976-4991 ◽

Cited By ~ 6

Author(s):

Jonathan B Chaires ◽

Robert D Gray ◽

William L Dean ◽

Robert Monsen ◽

Lynn W DeLeeuw ◽

...

Keyword(s):

Structural Model ◽

Analytical Ultracentrifugation ◽

Isothermal Calorimetry ◽

Equilibrium Constants ◽

Kinetic Studies ◽

Single Strand ◽

Conformational Selection ◽

Binding Data ◽

G Quadruplex ◽

Dynamics Simulations

Abstract The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (−10.1 ± 0.3 kcal mol−1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

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Association/dissociation Mechanisms of Intrinsically Disordered Region of Protein Beyond Conformational Selection and Induced Fit

10.26434/chemrxiv.9874379 ◽

2019 ◽

Author(s):

Duy Phuoc Tran ◽

Akio Kitao

Keyword(s):

Free Energy ◽

Transcriptional Activation ◽

Binding Free Energy ◽

Energy Structure ◽

Induced Fit ◽

Hydrogen Bond Formation ◽

Conformational Selection ◽

Intrinsically Disordered ◽

Intrinsically Disordered Region ◽

Dissociation Mechanisms

<p>We investigate association and dissociation mechanisms of a typical intrinsically disordered region (IDR), transcriptional activation subdomain of tumor repressor protein p53 (TAD-p53) with murine double-minute clone 2 protein (MDM2). Using the combination of cycles of association and dissociation parallel cascade molecular dynamics, multiple standard MD, and Markov state model, we are successful in obtaining the lowest free energy structure of MDM2/TAD-p53 complex as the structure very close to that in crystal without prior knowledge. This method also reproduces the experimentally measured standard binding free energy, and association and dissociation rate constants solely with the accumulated MD simulation cost of 11.675 μs, in spite of the fact that actual dissociation occurs in the order of a second. Although there exist a few complex intermediates with similar free energies, TAD-p53 first binds MDM2 as the second lowest free energy intermediate dominantly (> 90% in flux), taking a form similar to one of the intermediate structures in its monomeric state. The mechanism of this step has a feature of conformational selection. In the second step, dehydration of the interface, formation of π-π stackings of the side-chains, and main-chain relaxation/hydrogen bond formation to complete α-helix take place, showing features of induced fit. In addition, dehydration (dewetting) is a key process for the final relaxation around the complex interface. These results demonstrate a more fine-grained view of the IDR association/dissociation beyond classical views of protein conformational change upon binding.</p>

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Putting a New Spin of G4 Structure and Binding by Analytical Ultracentrifugation

10.1101/359356 ◽

2018 ◽

Cited By ~ 1

Author(s):

William L. Dean ◽

Robert D. Gray ◽

Lynn DeLeeuw ◽

Robert C. Monsen ◽

Jonathan B. Chaires

Keyword(s):

Small Molecule ◽

Analytical Ultracentrifugation ◽

Structure Stability ◽

Quadruplex Structure ◽

G Quadruplex

AbstractAnalytical ultracentrifugation is a powerful biophysical tool that provides information about G-quadruplex structure, stability and binding reactivity. This chapter provides a simplified explanation of the method, along with examples of how it can be used to characterize G4 formation and to monitor small-molecule binding.

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Ligand Selectivity in the Recognition of Protoberberine Alkaloids by Hybrid-2 Human Telomeric G-Quadruplex: Binding Free Energy Calculation, Fluorescence Binding, and NMR Experiments

Molecules ◽

10.3390/molecules24081574 ◽

2019 ◽

Vol 24 (8) ◽

pp. 1574 ◽

Cited By ~ 5

Author(s):

Nanjie Deng ◽

Junchao Xia ◽

Lauren Wickstrom ◽

Clement Lin ◽

Kaibo Wang ◽

...

Keyword(s):

Molecular Dynamics ◽

Free Energy ◽

Binding Free Energy ◽

Nmr Structure ◽

Free Energy Calculation ◽

Energy Calculation ◽

Ligand Selectivity ◽

Protoberberine Alkaloids ◽

Binding Free Energy Calculation ◽

G Quadruplex

The human telomeric G-quadruplex (G4) is an attractive target for developing anticancer drugs. Natural products protoberberine alkaloids are known to bind human telomeric G4 and inhibit telomerase. Among several structurally similar protoberberine alkaloids, epiberberine (EPI) shows the greatest specificity in recognizing the human telomeric G4 over duplex DNA and other G4s. Recently, NMR study revealed that EPI recognizes specifically the hybrid-2 form human telomeric G4 by inducing large rearrangements in the 5′-flanking segment and loop regions to form a highly extensive four-layered binding pocket. Using the NMR structure of the EPI-human telomeric G4 complex, here we perform molecular dynamics free energy calculations to elucidate the ligand selectivity in the recognition of protoberberines by the human telomeric G4. The MM-PB(GB)SA (molecular mechanics-Poisson Boltzmann/Generalized Born) Surface Area) binding free energies calculated using the Amber force fields bsc0 and OL15 correlate well with the NMR titration and binding affinity measurements, with both calculations correctly identifying the EPI as the strongest binder to the hybrid-2 telomeric G4 wtTel26. The results demonstrated that accounting for the conformational flexibility of the DNA-ligand complexes is crucially important for explaining the ligand selectivity of the human telomeric G4. While the MD-simulated (molecular dynamics) structures of the G-quadruplex-alkaloid complexes help rationalize why the EPI-G4 interactions are optimal compared with the other protoberberines, structural deviations from the NMR structure near the binding site are observed in the MD simulations. We have also performed binding free energy calculation using the more rigorous double decoupling method (DDM); however, the results correlate less well with the experimental trend, likely due to the difficulty of adequately sampling the very large conformational reorganization in the G4 induced by the protoberberine binding.

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Accelerating the Calculation of Protein-Ligand Binding Free Energy and Residence Times using Dynamically Optimized Collective Variables

10.1101/417915 ◽

2018 ◽

Author(s):

Z. Faidon Brotzakis ◽

Vittorio Limongelli ◽

Michele Parrinello

Keyword(s):

Free Energy ◽

Ligand Binding ◽

Degrees Of Freedom ◽

Binding Free Energy ◽

Conformational Dynamics ◽

Computational Cost ◽

Binding Pocket ◽

Binding Mode ◽

Collective Variables ◽

Binding Interaction

AbstractElucidation of the ligand/protein binding interaction is of paramount relevance in pharmacology to increase the success rate of drug design. To this end a number of computational methods have been proposed, however all of them suffer from limitations since the ligand binding/unbinding transitions to the molecular target involve many slow degrees of freedom that hamper a full characterization of the binding process. Being able to express this transition in simple and general slow degrees of freedom, would give a distinctive advantage, since it would require minimal knowledge of the system under study, while in turn it would elucidate its physics and accelerate the convergence speed of enhanced sampling methods relying on collective variables. In this study we pursuit this goal by combining for the first time Variation Approach to Conformational dynamics with Funnel-Metadynamics. In so doing, we predict for the benzamidine/trypsin system the ligand binding mode, and we accurately compute the absolute protein-ligand binding free energy and unbinding rate at unprecedented low computational cost. Finally, our simulation protocol reveals the energetics and structural details of the ligand binding mechanism and shows that water and binding pocket solvation/desolvation are the dominant slow degrees of freedom.

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Putting a New Spin of G-Quadruplex Structure and Binding by Analytical Ultracentrifugation

Methods in Molecular Biology - G-Quadruplex Nucleic Acids ◽

10.1007/978-1-4939-9666-7_5 ◽

2019 ◽

pp. 87-103 ◽

Cited By ~ 1

Author(s):

William L. Dean ◽

Robert D. Gray ◽

Lynn DeLeeuw ◽

Robert C. Monsen ◽

Jonathan B. Chaires

Keyword(s):

Analytical Ultracentrifugation ◽

Quadruplex Structure ◽

G Quadruplex

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Molecular Dynamics Free Energy Simulation study to Investigate Binding Pattern of Isoliquiritigenin as PPAR𝛾 Agonist

10.21203/rs.3.rs-1046506/v1 ◽

2022 ◽

Author(s):

Amit Singh ◽

Abha Mishra

Keyword(s):

Diabetes Mellitus ◽

Free Energy ◽

Binding Energy ◽

Electrostatic Interactions ◽

Binding Free Energy ◽

Solvation Energy ◽

Bioactive Constituents ◽

Binding Interaction ◽

Pparγ Agonist ◽

Polar Solvation

Abstract Phytochemicals are rich source of bioactive constituents and can be used as another alternative to currently used drugs for diseases like Diabetes mellitus. The potential of Isoliquiritigenin (a constituent of Pterocarpus marsupium) as PPAR𝛾 agonist was evaluated by in silico technique. Autodock results showed that Tyr327, and Tyr473 of the PPARγ forms H-bonds with Isoliquiritigenin (binding energy of -7.46 kcal/mol) and Troglitazone (known drug) showed H bond with Tyr327, Ser289, with binding energy of -11.01 kcal/mol. Isoliquiritigenin, binding energy in Extra precision (XP) was -6.74 kcal/mol while Troglitazone docking, gave binding energy in XP mode as -9.59 kcal/mol. The best Induced fit docking (IFD) score of the optimised PPARγ- Isoliquiritigenin complexes was -9.39 Kcal/mol. The important residues in IFD forming H bond were Cys 285, Arg 288, Tyr 327 and Leu 340. The post docking MM/GBSA free energy for PPARγ with Isoliquiritigenin and Troglitazone was -49.29 and -71.48 Kcal/mol respectively. Binding interaction in MD simulation and Principal Component Analysis studies revealed stable binding throughout 100 ns simulation. Post Simulation MM/PBSA free energy was calculated. The results indicated that compound possessed a negative binding free energy with -114.37KJ/mol. It was observed that van der Waals, electrostatic interactions and non-polar solvation energy negatively contributed to the total interaction energy while only polar solvation energy positively contributed to total free binding energy. The Isoliquiritigenin fulfils the criteria of drug-likeness property. Thus, study presents a systematic analysis on molecular mechanism of action of Isoliquiritigenin as PPARγ agonist in controlling Diabetes mellitus.

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