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

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>

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

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>

scholarly journals Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding

2015 ◽  
Vol 112 (31) ◽  
pp. 9614-9619 ◽  
Author(s):  
Munehito Arai ◽  
Kenji Sugase ◽  
H. Jane Dyson ◽  
Peter E. Wright
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Terminal Region ◽  
Relaxation Dispersion ◽  
Disordered Proteins ◽  
Conformational Ensemble ◽  
Induced Fit ◽  
Conformational Selection ◽  
Intrinsically Disordered ◽  
Factor C ◽  
Binding Mechanisms

Intrinsically disordered proteins (IDPs) frequently function in protein interaction networks that regulate crucial cellular signaling pathways. Many IDPs undergo transitions from disordered conformational ensembles to folded structures upon binding to their cellular targets. Several possible binding mechanisms for coupled folding and binding have been identified: folding of the IDP after association with the target (“induced fit”), or binding of a prefolded state in the conformational ensemble of the IDP to the target protein (“conformational selection”), or some combination of these two extremes. The interaction of the intrinsically disordered phosphorylated kinase-inducible domain (pKID) of the cAMP-response element binding (CREB) protein with the KIX domain of a general transcriptional coactivator CREB-binding protein (CBP) provides an example of the induced-fit mechanism. Here we show by NMR relaxation dispersion experiments that a different intrinsically disordered ligand, the transactivation domain of the transcription factor c-Myb, interacts with KIX at the same site as pKID but via a different binding mechanism that involves elements of conformational selection and induced fit. In contrast to pKID, the c-Myb activation domain has a strong propensity for spontaneous helix formation in its N-terminal region, which binds to KIX in a predominantly folded conformation. The C-terminal region of c-Myb exhibits a much smaller helical propensity and likely folds via an induced-fit process after binding to KIX. We propose that the intrinsic secondary structure propensities of pKID and c-Myb determine their binding mechanisms, consistent with their functions as inducible and constitutive transcriptional activators.

scholarly journals Liquid condensation of reprogramming factor KLF4 with DNA provides a mechanism for chromatin organization

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rajesh Sharma ◽  
Kyoung-Jae Choi ◽  
My Diem Quan ◽  
Sonum Sharma ◽  
Banumathi Sankaran ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Three Dimensional ◽  
Proximal Promoter ◽  
Epigenetic Landscape ◽  
Intrinsically Disordered ◽  
Differentiated Cells ◽  
Epigenetic State ◽  
Local Sequence ◽  
Intrinsically Disordered Region ◽  
Dna Fragment

AbstractExpression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation. Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region. Liquid–liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site. We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state.

scholarly journals Human POT1 Unfolds G-Quadruplexes by Conformational Selection

2019 ◽  
Author(s):  
Jonathan B. Chaires ◽  
Robert D. Gray ◽  
William L. Dean ◽  
Robert Monsen ◽  
Lynn W. DeLeeuw ◽  
...  
Keyword(s):  
Free Energy ◽  
Relaxation Time ◽  
Analytical Ultracentrifugation ◽  
Binding Free Energy ◽  
Equilibrium Constants ◽  
Single Strand ◽  
Binding Interaction ◽  
Conformational Selection ◽  
Quadruplex Structure ◽  
G Quadruplex

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.

scholarly journals Evaluation of the IKKβ Binding of Indicaxanthin by Induced-Fit Docking, Binding Pose Metadynamics, and Molecular Dynamics

2021 ◽  
Vol 12 ◽  
Author(s):  
Mario Allegra ◽  
Marco Tutone ◽  
Luisa Tesoriere ◽  
Alessandro Attanzio ◽  
Giulia Culletta ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Binding Free Energy ◽  
Active Form ◽  
Binding Pocket ◽  
Resting Cells ◽  
Induced Fit Docking ◽  
Induced Fit ◽  
Ikk Complex ◽  
A375 Cells

Background: Indicaxanthin, a betaxanthin belonging to the betalain class of compounds, has been recently demonstrated to exert significant antiproliferative effects inducing apoptosis of human melanoma cells through the inhibition of NF-κB as the predominant pathway. Specifically, Indicaxanthin inhibited IκBα degradation in A375 cells. In resting cells, NF-κB is arrested in the cytoplasm by binding to its inhibitor protein IκBα. Upon stimulation, IκBα is phosphorylated by the IKK complex, and degraded by the proteasome, liberating free NF-κB into the nucleus to initiate target gene transcription. Inhibition of the IKK complex leads to the arrest of the NF-κB pathway.Methods: To acquire details at the molecular level of Indicaxanthin’s inhibitory activity against hIKKβ, molecular modeling and simulation techniques including induced-fit docking (IFD), binding pose metadynamics (BPMD), molecular dynamics simulations, and MM-GBSA (molecular mechanics-generalized Born surface area continuum solvation) have been performed.Results: The computational calculations performed on the active and inactive form, and the allosteric binding site of hIKKβ, revealed that Indicaxanthin inhibits prevalently the active form of the hIKKβ. MM-GBSA computations provide further evidence of Indicaxanthin’s stability inside the active binding pocket with a binding free energy of −22.2 ± 4.3 kcal/mol with respect to the inactive binding pocket with a binding free energy of −20.7 ± 4.7 kcal/mol. BPMD and MD simulation revealed that Indicaxanthin is likely not an allosteric inhibitor of hIKKβ.Conclusion: As a whole, these in silico pieces of evidence show that Indicaxanthin can inhibit the active form of the hIKKβ adding novel mechanistic insights on its recently discovered ability to impair NF-κB signaling in melanoma A375 cells. Moreover, our results suggest the phytochemical as a new lead compound for novel, more potent IKKβ inhibitors to be employed in the treatment of cancer and inflammation-related conditions.

Large-Scale Assessment of Binding Free Energy Calculations in Active Drug Discovery Projects

2020 ◽  
Author(s):  
Christina Schindler ◽  
Hannah Baumann ◽  
Andreas Blum ◽  
Dietrich Böse ◽  
Hans-Peter Buchstaller ◽  
...  
Keyword(s):  
Free Energy ◽  
Drug Discovery ◽  
Large Scale ◽  
Active Drug ◽  
Binding Free Energy ◽  
Free Energy Calculations ◽  
Energy Calculation ◽  
Large Scale Assessment ◽  
New Public ◽  
Binding Free Energy Calculations

Here we present an evaluation of the binding affinity prediction accuracy of the free energy calculation method FEP+ on internal active drug discovery projects and on a large new public benchmark set.<br>

Automated, Accurate, and Scalable Relative Protein-Ligand Binding Free Energy Calculations using Lambda Dynamics

2020 ◽  
Author(s):  
E. Prabhu Raman ◽  
Thomas J. Paul ◽  
Ryan L. Hayes ◽  
Charles L. Brooks III
Keyword(s):  
Free Energy ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Binding Free Energy ◽  
Computational Cost ◽  
Combinatorial Libraries ◽  
Free Energy Calculations ◽  
Lead Optimization ◽  
Efficient Estimation ◽  
Lead Compound

<p>Accurate predictions of changes to protein-ligand binding affinity in response to chemical modifications are of utility in small molecule lead optimization. Relative free energy perturbation (FEP) approaches are one of the most widely utilized for this goal, but involve significant computational cost, thus limiting their application to small sets of compounds. Lambda dynamics, also rigorously based on the principles of statistical mechanics, provides a more efficient alternative. In this paper, we describe the development of a workflow to setup, execute, and analyze Multi-Site Lambda Dynamics (MSLD) calculations run on GPUs with CHARMm implemented in BIOVIA Discovery Studio and Pipeline Pilot. The workflow establishes a framework for setting up simulation systems for exploratory screening of modifications to a lead compound, enabling the calculation of relative binding affinities of combinatorial libraries. To validate the workflow, a diverse dataset of congeneric ligands for seven proteins with experimental binding affinity data is examined. A protocol to automatically tailor fit biasing potentials iteratively to flatten the free energy landscape of any MSLD system is developed that enhances sampling and allows for efficient estimation of free energy differences. The protocol is first validated on a large number of ligand subsets that model diverse substituents, which shows accurate and reliable performance. The scalability of the workflow is also tested to screen more than a hundred ligands modeled in a single system, which also resulted in accurate predictions. With a cumulative sampling time of 150ns or less, the method results in average unsigned errors of under 1 kcal/mol in most cases for both small and large combinatorial libraries. For the multi-site systems examined, the method is estimated to be more than an order of magnitude more efficient than contemporary FEP applications. The results thus demonstrate the utility of the presented MSLD workflow to efficiently screen combinatorial libraries and explore chemical space around a lead compound, and thus are of utility in lead optimization.</p>

Application of the ESMACS Binding Free Energy Protocol to a Highly Varied Ligand Dataset: Lactate Dehydogenase A

2019 ◽  
Author(s):  
David Wright ◽  
Fouad Husseini ◽  
Shunzhou Wan ◽  
Christophe Meyer ◽  
Herman Van Vlijmen ◽  
...  
Keyword(s):  
Free Energy ◽  
Surface Area ◽  
Binding Free Energy ◽  
Normal Mode Analysis ◽  
Binding Mode ◽  
Accessible Surface Area ◽  
Solvent Accessible Surface Area ◽  
Mode Analysis ◽  
Energy Calculation ◽  
Accessible Surface

<div>Here, we evaluate the performance of our range of ensemble simulation based binding free energy calculation protocols, called ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) for use in fragment based drug design scenarios. ESMACS is designed to generate reproducible binding affinity predictions from the widely used molecular mechanics Poisson-Boltzmann surface area (MMPBSA) approach. We study ligands designed to target two binding pockets in the lactate dehydogenase A target protein, which vary in size, charge and binding mode. When comparing to experimental results, we obtain excellent statistical rankings across this highly diverse set of ligands. In addition, we investigate three approaches to account for entropic contributions not captured by standard MMPBSA calculations: (1) normal mode analysis, (2) weighted solvent accessible surface area (WSAS) and (3) variational entropy. </div>

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