The intrinsically disordered N-terminal domain of galectin-3 dynamically mediates multisite self-association of the protein through fuzzy interactions

Intrinsically disordered regions (IDRs) are common and important functional domains in many proteins. However, IDRs are difficult to target for drug development due to the lack of defined structures which would facilitate the identification of possible drug-binding pockets. Galectin-3 is a carbohydrate-binding protein of which overexpression has been implicated in a wide variety of disorders including cancer and inflammation. Apart from its carbohydrate recognition/binding domain (CRD), Galectin-3 also contains a functionally important disordered N-terminal domain (NTD) that contacts the C-terminal domain (CTD) and could be a target for drug development. To overcome challenges involved in inhibitor design due to lack of structure and the highly dynamic nature of the NTD, we used a novel protocol combining nuclear magnetic resonance data from recombinant Galectin-3 with accelerated molecular dynamics (MD) simulations to identify a shallow pocket in the CTD with which the NTD makes frequent contact. In accordance with this model, a Galectin-3 double mutant of residues L131 and L203 in the CTD lost agglutination ability. In-silico design was used to narrow down candidate inhibitory peptides and experimental testing of only 3 of these yielded one peptide that inhibits the agglutination promoted by wild type Galectin-3. NMR experiments further confirmed that this peptide makes contacts with a non-carbohydrate binding moiety of the CTD. Our results show that it is possible to apply a combination of MD simulations and NMR experiments to precisely predict the binding interface of a disordered domain with a structured domain, and furthermore use this predicted interface for designing inhibitors. This procedure can thus be potentially extended to many other targets in which similar IDR interactions play a vital functional role.

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EGCG binds intrinsically disordered N-terminal domain of p53 and disrupts p53-MDM2 interaction

Nature Communications ◽

10.1038/s41467-021-21258-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jing Zhao ◽

Alan Blayney ◽

Xiaorong Liu ◽

Lauren Gandy ◽

Weihua Jin ◽

...

Keyword(s):

Large Scale ◽

Molecular Mechanisms ◽

Epigallocatechin Gallate ◽

Cancer Drug ◽

Dynamic Interactions ◽

Cancer Drug Discovery ◽

Intrinsically Disordered ◽

Terminal Domain ◽

Cancerous Cells

AbstractEpigallocatechin gallate (EGCG) from green tea can induce apoptosis in cancerous cells, but the underlying molecular mechanisms remain poorly understood. Using SPR and NMR, here we report a direct, μM interaction between EGCG and the tumor suppressor p53 (KD = 1.6 ± 1.4 μM), with the disordered N-terminal domain (NTD) identified as the major binding site (KD = 4 ± 2 μM). Large scale atomistic simulations (>100 μs), SAXS and AUC demonstrate that EGCG-NTD interaction is dynamic and EGCG causes the emergence of a subpopulation of compact bound conformations. The EGCG-p53 interaction disrupts p53 interaction with its regulatory E3 ligase MDM2 and inhibits ubiquitination of p53 by MDM2 in an in vitro ubiquitination assay, likely stabilizing p53 for anti-tumor activity. Our work provides insights into the mechanisms for EGCG’s anticancer activity and identifies p53 NTD as a target for cancer drug discovery through dynamic interactions with small molecules.

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Structural heterogeneity in the intrinsically disordered RNA polymerase II C-terminal domain

Nature Communications ◽

10.1038/ncomms15231 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 28

Author(s):

Bede Portz ◽

Feiyue Lu ◽

Eric B. Gibbs ◽

Joshua E. Mayfield ◽

M. Rachel Mehaffey ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Structural Heterogeneity ◽

Intrinsically Disordered ◽

Terminal Domain

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Assessing induced folding within the intrinsically disordered C-terminal domain of theHenipavirusnucleoproteins by site-directed spin labeling EPR spectroscopy

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2012.706068 ◽

2013 ◽

Vol 31 (5) ◽

pp. 453-471 ◽

Cited By ~ 29

Author(s):

Marlène Martinho ◽

Johnny Habchi ◽

Zeina El Habre ◽

Léo Nesme ◽

Bruno Guigliarelli ◽

...

Keyword(s):

Epr Spectroscopy ◽

Spin Labeling ◽

Intrinsically Disordered ◽

Terminal Domain ◽

Induced Folding ◽

Site Directed Spin Labeling

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Improving the Performance of Simulations of the Intrinsically Disordered N-Terminal Domain from P53

Biophysical Journal ◽

10.1016/j.bpj.2016.11.1146 ◽

2017 ◽

Vol 112 (3) ◽

pp. 207a-208a

Author(s):

Nic A. Ezzell ◽

Yue Zhang ◽

Steven T. Whitten ◽

Nicholas C. Fitzkee

Keyword(s):

Intrinsically Disordered ◽

Terminal Domain

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The C Terminus of the Ribosomal-Associated Protein LrtA is an Intrinsically Disordered Oligomer

International Journal of Molecular Sciences ◽

10.3390/ijms19123902 ◽

2018 ◽

Vol 19 (12) ◽

pp. 3902 ◽

Cited By ~ 2

Author(s):

José L. Neira ◽

A. Marcela Giudici ◽

Felipe Hornos ◽

Arantxa Arbe ◽

Bruno Rizzuti

Keyword(s):

Computational Modelling ◽

Terminal Region ◽

Physiological Conditions ◽

Intrinsically Disordered ◽

Disordered Protein ◽

C Terminus ◽

Conformational Preferences ◽

Self Association ◽

Association Equilibrium ◽

Post Stress

The 191-residue-long LrtA protein of Synechocystis sp. PCC 6803 is involved in post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family, intervening in protein synthesis. The protein consists of two domains: The N-terminal region (N-LrtA, residues 1101), which is common to all the members of the HPF, and seems to be well-folded; and the C-terminal region (C-LrtA, residues 102-191), which is hypothesized to be disordered. In this work, we studied the conformational preferences of isolated C-LrtA in solution. The protein was disordered, as shown by computational modelling, 1D-1H NMR, steady-state far- UV circular dichroism (CD) and chemical and thermal denaturations followed by fluorescence and far-UV CD. Moreover, at physiological conditions, as indicated by several biochemical and hydrodynamic techniques, isolated C-LrtA intervened in a self-association equilibrium, involving several oligomerization reactions. Thus, C-LrtA was an oligomeric disordered protein.

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A CON-based NMR assignment strategy for pro-rich intrinsically disordered proteins with low signal dispersion: the C-terminal domain of histone H1.0 as a case study

Journal of Biomolecular NMR ◽

10.1007/s10858-018-0213-2 ◽

2018 ◽

Vol 72 (3-4) ◽

pp. 139-148 ◽

Cited By ~ 4

Author(s):

Belén Chaves-Arquero ◽

David Pantoja-Uceda ◽

Alicia Roque ◽

Inmaculada Ponte ◽

Pedro Suau ◽

...

Keyword(s):

Nmr Assignment ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Terminal Domain ◽

Assignment Strategy

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Progressive Phosphorylation Modulates the Self-Association of a Variably Modified Histone H3 Peptide

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.698182 ◽

2021 ◽

Vol 8 ◽

Author(s):

George V. Papamokos ◽

George Tziatzos ◽

Dimitrios G. Papageorgiou ◽

Spyros Georgatos ◽

Efthimios Kaxiras ◽

...

Keyword(s):

Intrinsically Disordered Proteins ◽

Histone H3 ◽

Intramolecular Interactions ◽

Disordered Proteins ◽

Post Translational Modifications ◽

Intrinsically Disordered ◽

Self Association ◽

Dynamics Simulations ◽

Peptide Charge

Protein phosphorylation is a key regulatory mechanism in eukaryotic cells. In the intrinsically disordered histone tails, phosphorylation is often a part of combinatorial post-translational modifications and an integral part of the “histone code” that regulates gene expression. Here, we study the association between two histone H3 tail peptides modified to different degrees, using fully atomistic molecular dynamics simulations. Assuming that the initial conformations are either α-helical or fully extended, we compare the propensity of the two peptides to associate with one another when both are unmodified, one modified and the other unmodified, or both modified. The simulations lead to the identification of distinct inter- and intramolecular interactions in the peptide dimer, highlighting a prominent role of a fine-tuned phosphorylation rheostat in peptide association. Progressive phosphorylation appears to modulate peptide charge, inducing strong and specific intermolecular interactions between the monomers, which do not result in the formation of amorphous or ordered aggregates, as documented by experimental evidence derived from Circular Dichroism and NMR spectroscopy. However, upon complete saturation of positive charges by phosphate groups, this effect is reversed: intramolecular interactions prevail and dimerization of zero-charge peptides is markedly reduced. These findings underscore the role of phosphorylation thresholds in the dynamics of intrinsically disordered proteins. Phosphorylation rheostats might account for the divergent effects of histone modifications on the modulation of chromatin structure.

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