Oligomerization of the Human Adenosine A2AReceptor is Driven by the Intrinsically Disordered C-Terminus

Abstract Heterochromatin, a transcriptionally silenced chromatin domain, is important for genome stability and gene expression. Histone 3 lysine 9 methylation (H3K9me) and histone hypoacetylation are conserved epigenetic hallmarks of heterochromatin. In fission yeast, RNA interference (RNAi) plays a key role in H3K9 methylation and heterochromatin silencing. However, how RNAi machinery and histone deacetylases (HDACs) are coordinated to ensure proper heterochromatin assembly is still unclear. Previously, we showed that Dpb4, a conserved DNA polymerase epsilon subunit, plays a key role in the recruitment of HDACs to heterochromatin during S phase. Here, we identified a novel RNA-binding protein Dri1 that interacts with Dpb4. GFP-tagged Dri1 forms distinct foci mostly in the nucleus, showing a high degree of colocalization with Swi6/Heterochromatin Protein 1. Deletion of dri1+ leads to defects in silencing, H3K9me, and heterochromatic siRNA generation. We also showed that Dri1 physically associates with heterochromatic transcripts, and is required for the recruitment of the RNA-induced transcriptional silencing (RITS) complex via interacting with the complex. Furthermore, loss of Dri1 decreases the association of the Sir2 HDAC with heterochromatin. We further demonstrated that the C-terminus of Dri1 that includes an intrinsically disordered (IDR) region and three zinc fingers is crucial for its role in silencing. Together, our evidences suggest that Dri1 facilitates heterochromatin assembly via the RNAi pathway and HDAC.

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Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry

Cellular and Molecular Life Sciences ◽

10.1007/s00018-020-03639-z ◽

2020 ◽

Author(s):

Lasse Staby ◽

Katherine R. Kemplen ◽

Amelie Stein ◽

Michael Ploug ◽

Jane Clarke ◽

...

Keyword(s):

Intrinsically Disordered Proteins ◽

Three Dimensional ◽

Life Time ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Order And Disorder ◽

C Terminus ◽

Close Relationship ◽

Stability Dynamics ◽

And Function

Abstract Understanding the interplay between sequence, structure and function of proteins has been complicated in recent years by the discovery of intrinsically disordered proteins (IDPs), which perform biological functions in the absence of a well-defined three-dimensional fold. Disordered protein sequences account for roughly 30% of the human proteome and in many proteins, disordered and ordered domains coexist. However, few studies have assessed how either feature affects the properties of the other. In this study, we examine the role of a disordered tail in the overall properties of the two-domain, calcium-sensing protein neuronal calcium sensor 1 (NCS-1). We show that loss of just six of the 190 residues at the flexible C-terminus is sufficient to severely affect stability, dynamics, and folding behavior of both ordered domains. We identify specific hydrophobic contacts mediated by the disordered tail that may be responsible for stabilizing the distal N-terminal domain. Moreover, sequence analyses indicate the presence of an LSL-motif in the tail that acts as a mimic of native ligands critical to the observed order–disorder communication. Removing the disordered tail leads to a shorter life-time of the ligand-bound complex likely originating from the observed destabilization. This close relationship between order and disorder may have important implications for how investigations into mixed systems are designed and opens up a novel avenue of drug targeting exploiting this type of behavior.

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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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In-Situ Flexibility of Both Redox-States of the Chloroplast Regulatory Protein CP12

10.20944/preprints202104.0048.v1 ◽

2021 ◽

Author(s):

Helene Launay ◽

Hui Shao ◽

Olivier Bornet ◽

Francois-Xavier Cantrelle ◽

Regine Lebrun ◽

...

Keyword(s):

Regulatory Protein ◽

Cell Extract ◽

Proton Exchange ◽

Conformational Exchange ◽

Amide Proton ◽

Cysteine Residues ◽

Intrinsically Disordered ◽

Amide Proton Exchange ◽

C Terminus ◽

Helical Turn

In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment imposed in the light via the electrons from the photosystems. In the dark these enzymes are inhibited, and this regulation is mainly mediated via oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges are simultaneous. In oxidized CP12, the C23-C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66-C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment that is, in cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66-C75 pair compared to the C23-C31 pair. Finally, the redox mid-potentials for the two cysteine pairs differ and are similar to those found for phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase, that we relate to the regulatory role of CP12.

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X-ray structure of the PHF core C-terminus: Insight into the folding of the intrinsically disordered protein tau in Alzheimer's disease

FEBS Letters ◽

10.1016/j.febslet.2007.11.067 ◽

2007 ◽

Vol 581 (30) ◽

pp. 5872-5878 ◽

Cited By ~ 12

Author(s):

Jozef Sevcik ◽

Rostislav Skrabana ◽

Radovan Dvorsky ◽

Natalia Csokova ◽

Khalid Iqbal ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Intrinsically Disordered Protein ◽

X Ray ◽

Protein Tau ◽

Intrinsically Disordered ◽

Disordered Protein ◽

C Terminus ◽

Insight Into

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DNA-binding regulates site-specific ubiquitination of IRF-1

Biochemical Journal ◽

10.1042/bj20121076 ◽

2013 ◽

Vol 449 (3) ◽

pp. 707-717 ◽

Cited By ~ 16

Author(s):

Vivien Landré ◽

Emmanuelle Pion ◽

Vikram Narayan ◽

Dimitris P. Xirodimas ◽

Kathryn L. Ball

Keyword(s):

Dna Binding ◽

E3 Ligase ◽

Binding Domain ◽

Dna Binding Domain ◽

Docking Site ◽

Interacting Protein ◽

Site Specific ◽

E3 Ligases ◽

Intrinsically Disordered ◽

C Terminus

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

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Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the C-terminal domain of Par-4 (PAWR)

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14014691 ◽

2014 ◽

Vol 70 (9) ◽

pp. 1224-1227 ◽

Cited By ~ 4

Author(s):

Udaya Kumar Tiruttani Subhramanyam ◽

Jan Kubicek ◽

Ulf B. Eidhoff ◽

Joerg Labahn

Keyword(s):

Crystallographic Analysis ◽

X Ray Diffraction ◽

Unit Cell Parameters ◽

Data Set ◽

X Ray ◽

Cell Parameters ◽

Intrinsically Disordered ◽

Tumour Suppressor Function ◽

C Terminus ◽

Apoptotic Protein

Prostate apoptosis response-4 protein is an intrinsically disordered pro-apoptotic protein with tumour suppressor function. Par-4 is known for its selective induction of apoptosis in cancer cells only and its ability to interact with various apoptotic proteinsviaits C-terminus. Par-4, with its unique function and various interacting partners, has gained importance as a potential target for cancer therapy. The C-terminus of the rat homologue of Par-4 was crystallized and a 3.7 Å resolution X-ray diffraction data set was collected. Preliminary data analysis shows the space group to beP41212. The unit-cell parameters area=b= 115.351,c= 123.663 Å, α = β = γ = 90°.

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HN, N, Cα, Cβ and C′ assignments of the intrinsically disordered C-terminus of human adenosine A2A receptor

Biomolecular NMR Assignments ◽

10.1007/s12104-015-9618-y ◽

2015 ◽

Vol 9 (2) ◽

pp. 403-406 ◽

Cited By ~ 2

Author(s):

Helena Tossavainen ◽

Maarit Hellman ◽

Henni Piirainen ◽

Veli-Pekka Jaakola ◽

Perttu Permi

Keyword(s):

Adenosine A2a Receptor ◽

Intrinsically Disordered ◽

C Terminus ◽

A2a Receptor ◽

Adenosine A2a

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Protein-based condensation mechanisms drive the assembly of RNA-rich P granules

eLife ◽

10.7554/elife.63698 ◽

2021 ◽

Vol 10 ◽

Author(s):

Helen Schmidt ◽

Andrea Putnam ◽

Dominique Rasoloson ◽

Geraldine Seydoux

Keyword(s):

Intrinsically Disordered Protein ◽

P Granules ◽

C Elegans ◽

Intrinsically Disordered ◽

Disordered Protein ◽

The Core ◽

C Terminus ◽

Germ Granules

Germ granules are protein-RNA condensates that segregate with the embryonic germline. In C. elegans embryos, germ (P) granule assembly requires MEG-3, an intrinsically-disordered protein that forms RNA-rich condensates on the surface of PGL condensates at the core of P granules. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). We find that MEG-3 is modular protein that uses its IDR to bind RNA and its C-terminus to drive condensation. The HMGL motif mediates binding to PGL-3 and is required for co-assembly of MEG-3 and PGL-3 condensates in vivo. Mutations in HMGL cause MEG-3 and PGL-3 to form separate condensates that no longer co-segregate to the germline or recruit RNA. Our findings highlight the importance of protein-based condensation mechanisms and condensate-condensate interactions in the assembly of RNA-rich germ granules.

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Positive Charges in the Brace Region Facilitate the Membrane Disruption of MLKL-NTR in Necroptosis

Molecules ◽

10.3390/molecules26175194 ◽

2021 ◽

Vol 26 (17) ◽

pp. 5194

Author(s):

Yaqing Yang ◽

Encheng Xie ◽

Lingyu Du ◽

Yu Yang ◽

Bin Wu ◽

...

Keyword(s):

Cell Death ◽

Active Role ◽

Kinase Domain ◽

Dynamics Simulation ◽

Membrane Disruption ◽

Dependent Manner ◽

Intrinsically Disordered ◽

Membrane Rupture ◽

C Terminus ◽

Membrane Mimetic

Necroptosis is a type of programmed cell death executed through the plasma membrane disruption by mixed lineage kinase domain-like protein (MLKL). Previous studies have revealed that an N-terminal four-helix bundle domain (NBD) of MLKL is the executioner domain for the membrane permeabilization, which is auto-inhibited by the first brace helix (H6). After necroptosis initiation, this inhibitory brace helix detaches and the NBD can integrate into the membrane, and hence leads to necroptotic cell death. However, how the NBD is released and induces membrane rupture is poorly understood. Here, we reconstituted MLKL2–154 into membrane mimetic bicelles and observed the structure disruption and membrane release of the first brace helix that is regulated by negatively charged phospholipids in a dose-dependent manner. Using molecular dynamics simulation we found that the brace region in an isolated, auto-inhibited MLKL2–154 becomes intrinsically disordered in solution after 7 ns dynamic motion. Further investigations demonstrated that a cluster of arginines in the C-terminus of MLKL2–154 is important for the molecular conformational switch. Functional mutagenesis showed that mutating these arginines to glutamates hindered the membrane disruption of full-length MLKL and thus inhibited the necroptotic cell death. These findings suggest that the brace helix also plays an active role in MLKL regulation, rather than an auto-inhibitory domain.

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