Minireview: Dynamic Structures of Nuclear Hormone Receptors: New Promises and Challenges

Abstract Therapeutic targeting of nuclear receptors (NRs) is presently restricted due to 2 constraints: 1) a limited knowledge of the structural dynamics of intact receptor when complexed to DNA and coregulatory proteins; and 2) the inability to more selectively modulate NR actions at specific organ/gene targets. A major obstacle has been the current lack of understanding about the function and structure of the intrinsically disordered N-terminal domain that contains a major regulatory transcriptional activation function (AF1). Current studies of both mechanism of action and small molecule-selective receptor modulators for clinical uses target the structured pocket of the ligand-binding domain to modulate coregulatory protein interactions with the other activation function AF2. However, these approaches overlook AF1 activity. Recent studies have shown that highly flexible intrinsically disordered regions of transcription factors, including that of the N-terminal domain AF1 of NRs, not only are critical for several aspects of NR action but also can be exploited as drug targets, thereby opening unique opportunities for endocrine-based therapies. In this review article, we discuss the role of structural flexibilities in the allosteric modulation of NR activity and future perspectives for therapeutic interventions.

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The N-terminal domain of unknown function (DUF959) in collagen XVIII is intrinsically disordered and highly O-glycosylated

Biochemical Journal ◽

10.1042/bcj20180405 ◽

2018 ◽

Vol 475 (22) ◽

pp. 3577-3593 ◽

Cited By ~ 3

Author(s):

Inderjeet Kaur ◽

Salla Ruskamo ◽

Jarkko Koivunen ◽

Ritva Heljasvaara ◽

Jarkko J. Lackman ◽

...

Keyword(s):

Protein Interactions ◽

Unknown Function ◽

Mammalian Cells ◽

Post Translational Modifications ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Terminal Domain ◽

X Ray Scattering ◽

Collagen Xviii ◽

Domain Of Unknown Function

Collagen XVIII (ColXVIII) is a non-fibrillar collagen and proteoglycan that exists in three isoforms: short, medium and long. The medium and long isoforms contain a unique N-terminal domain of unknown function, DUF959, and our sequence-based secondary structure predictions indicated that DUF959 could be an intrinsically disordered domain. Recombinant DUF959 produced in mammalian cells consisted of ∼50% glycans and had a molecular mass of 63 kDa. Circular dichroism spectroscopy confirmed the disordered character of DUF959, and static light scattering indicated a monomeric state for glycosylated DUF959 in solution. Small-angle X-ray scattering showed DUF959 to be a highly extended, flexible molecule with a maximum dimension of ∼23 nm. Glycosidase treatment demonstrated considerable amounts of O-glycosylation, and expression of DUF959 in HEK293 SimpleCells capable of synthesizing only truncated O-glycans confirmed the presence of N-acetylgalactosamine-type O-glycans. The DUF959 sequence is characterized by numerous Ser and Thr residues, and this accounts for the finding that half of the recombinant protein consists of glycans. Thus, the medium and long ColXVIII isoforms contain at their extreme N-terminus a disordered, elongated and highly O-glycosylated mucin-like domain that is not found in other collagens, and we suggest naming it the Mucin-like domain in ColXVIII (MUCL-C18). As intrinsically disordered regions and their post-translational modifications are often involved in protein interactions, our findings may point towards a role of the flexible mucin-like domain of ColXVIII as an interaction hub affecting cell signaling. Moreover, the MUCL-C18 may also serve as a lubricant at cell–extracellular matrix interfaces.

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β-Catenin Binds to the Activation Function 2 Region of the Androgen Receptor and Modulates the Effects of the N-Terminal Domain and TIF2 on Ligand-Dependent Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.23.5.1674-1687.2003 ◽

2003 ◽

Vol 23 (5) ◽

pp. 1674-1687 ◽

Cited By ~ 116

Author(s):

Liang-Nian Song ◽

Roger Herrell ◽

Stephen Byers ◽

Salimuddin Shah ◽

Elizabeth M. Wilson ◽

...

Keyword(s):

Androgen Receptor ◽

Hormone Receptors ◽

Nuclear Translocation ◽

Retinoic Acid Receptor ◽

Activation Function ◽

Steroid Hormone Receptors ◽

Binding Assays ◽

Peptide Motifs ◽

Terminal Domain ◽

Helix 12

ABSTRACT β-Catenin is a multifunctional molecule that is activated by signaling through WNT receptors. β-Catenin can also enhance the transcriptional activity of some steroid hormone receptors such as the androgen receptor and retinoic acid receptor α. Androgens can affect nuclear translocation of β-catenin and influence its subcellular distribution. Using mammalian two-hybrid binding assays, analysis of reporter gene transcription, and coimmunoprecipitation, we now show that β-catenin binds to the androgen receptor ligand-binding domain (LBD) and modulates the transcriptional effects of TIF2 and the androgen receptor N-terminal domain (NTD). In functional assays, β-catenin bound to androgen receptor only in the presence of ligand agonists, not antagonists. β-Catenin binding to the androgen receptor LBD was independent of and cooperative with the androgen receptor NTD and the p160 coactivator TIF2, both of which bind to the activation function 2 (AF-2) region of the androgen receptor. Different mutations of androgen receptor helix 3 amino acids disrupted binding of androgen receptor NTD and β-catenin. β-Catenin, androgen receptor NTD, and TIF2 binding to the androgen receptor LBD were affected similarly by a subset of helix 12 mutations, but disruption of two sites on helix 12 affected only binding of β-catenin and not of TIF2 or the androgen receptor NTD. Mutational disruption of each of five LXXLL peptide motifs in the β-catenin armadillo repeats did not disrupt either binding to androgen receptor or transcriptional coactivation. ICAT, an inhibitor of T-cell factor 4 (TCF-4), and E-cadherin binding to β-catenin also blocked binding of the androgen receptor LBD. We also demonstrated cross talk between the WNT and androgen receptor signaling pathways because excess androgen receptor could interfere with WNT signaling and excess TCF-4 inhibited the interaction of β-catenin and androgen receptor. Taken together, the data show that β-catenin can bind to the androgen receptor LBD and modulate the effects of the androgen receptor NTD and TIF2 on transcription.

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Transient DNA Binding Induces RNA Polymerase II Compartmentalization During Herpesviral Infection Distinct From Phase Separation

10.1101/375071 ◽

2018 ◽

Cited By ~ 2

Author(s):

David T McSwiggen ◽

Anders S Hansen ◽

Hervé Marie-Nelly ◽

Sheila Teves ◽

Alec B Heckert ◽

...

Keyword(s):

Phase Separation ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Protein Interactions ◽

Viral Dna ◽

Herpes Simplex Virus 1 ◽

Pol Ii ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Simplex Virus

SummaryDuring lytic infection, Herpes Simplex Virus 1 generates replication compartments (RCs) in host nuclei that efficiently recruit protein factors, including host RNA Polymerase II (Pol II). Pol II and other cellular factors form hubs in uninfected cells that are proposed to phase separate via multivalent protein-protein interactions mediated by their intrinsically disordered regions. Using a battery of live cell microscopic techniques, we show that although RCs superficially exhibit many characteristics of phase separation, the recruitment of Pol II instead derives from nonspecific interactions with the viral DNA. We find that the viral genome remains nucleosome-free, profoundly affecting the way Pol II explores RCs by causing it to repetitively visit nearby binding sites, thereby creating local Pol II accumulations. This mechanism, distinct from phase separation, allows viral DNA to outcompete host DNA for cellular proteins. Our work provides new insights into the strategies used to create local molecular hubs in cells.

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Peptide-based Interaction Proteomics

Molecular & Cellular Proteomics ◽

10.1074/mcp.r120.002034 ◽

2020 ◽

Vol 19 (7) ◽

pp. 1070-1075 ◽

Cited By ~ 3

Author(s):

Katrina Meyer ◽

Matthias Selbach

Keyword(s):

Protein Interactions ◽

Synthetic Peptides ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Interaction Partners ◽

Linear Motifs ◽

Cellulose Membranes ◽

Interaction Proteomics ◽

Proteins Interactions

Protein-protein interactions are often mediated by short linear motifs (SLiMs) that are located in intrinsically disordered regions (IDRs) of proteins. Interactions mediated by SLiMs are notoriously difficult to study, and many functionally relevant interactions likely remain to be uncovered. Recently, pull-downs with synthetic peptides in combination with quantitative mass spectrometry emerged as a powerful screening approach to study protein-protein interactions mediated by SLiMs. Specifically, arrays of synthetic peptides immobilized on cellulose membranes provide a scalable means to identify the interaction partners of many peptides in parallel. In this minireview we briefly highlight the relevance of SLiMs for protein-protein interactions, outline existing screening technologies, discuss unique advantages of peptide-based interaction screens and provide practical suggestions for setting up such peptide-based screens.

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The Disordered Cellular Multi-Tasker WIP and Its Protein–Protein Interactions: A Structural View

Biomolecules ◽

10.3390/biom10071084 ◽

2020 ◽

Vol 10 (7) ◽

pp. 1084 ◽

Cited By ~ 2

Author(s):

Chana G. Sokolik ◽

Nasrin Qassem ◽

Jordan H. Chill

Keyword(s):

Protein Interactions ◽

Cell Biology ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

Actin Binding ◽

Structural Description ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

Terminal Domain ◽

Binding Partners

WASp-interacting protein (WIP), a regulator of actin cytoskeleton assembly and remodeling, is a cellular multi-tasker and a key member of a network of protein–protein interactions, with significant impact on health and disease. Here, we attempt to complement the well-established understanding of WIP function from cell biology studies, summarized in several reviews, with a structural description of WIP interactions, highlighting works that present a molecular view of WIP’s protein–protein interactions. This provides a deeper understanding of the mechanisms by which WIP mediates its biological functions. The fully disordered WIP also serves as an intriguing example of how intrinsically disordered proteins (IDPs) exert their function. WIP consists of consecutive small functional domains and motifs that interact with a host of cellular partners, with a striking preponderance of proline-rich motif capable of interactions with several well-recognized binding partners; indeed, over 30% of the WIP primary structure are proline residues. We focus on the binding motifs and binding interfaces of three important WIP segments, the actin-binding N-terminal domain, the central domain that binds SH3 domains of various interaction partners, and the WASp-binding C-terminal domain. Beyond the obvious importance of a more fundamental understanding of the biology of this central cellular player, this approach carries an immediate and highly beneficial effect on drug-design efforts targeting WIP and its binding partners. These factors make the value of such structural studies, challenging as they are, readily apparent.

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Glutamate promotes SSB protein–protein Interactions via intrinsically disordered regions

Journal of Molecular Biology ◽

10.1016/j.jmb.2017.07.021 ◽

2017 ◽

Vol 429 (18) ◽

pp. 2790-2801 ◽

Cited By ~ 23

Author(s):

Alexander G. Kozlov ◽

Min Kyung Shinn ◽

Elizabeth A. Weiland ◽

Timothy M. Lohman

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Disordered Regions

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tau4/tau c/AF-2 of the thyroid hormone receptor relieves silencing of the retinoic acid receptor silencer core independent of both tau4 activation function and full dissociation of corepressors.

Molecular and Cellular Biology ◽

10.1128/mcb.17.8.4259 ◽

1997 ◽

Vol 17 (8) ◽

pp. 4259-4271 ◽

Cited By ~ 15

Author(s):

A Baniahmad ◽

D Thormeyer ◽

R Renkawitz

Keyword(s):

Amino Acids ◽

Retinoic Acid ◽

Thyroid Hormone ◽

Transcriptional Activation ◽

Hormone Receptors ◽

Thyroid Hormone Receptor ◽

Retinoic Acid Receptor ◽

Activation Function ◽

Acid Receptor ◽

C Terminus

Members of the thyroid hormone (TR)-retinoic acid receptor (RAR) subfamily of nuclear hormone receptors silence gene expression in the absence of hormone. Addition of cognate ligands leads to dissociation of corepressors, association of coactivators, and transcriptional activation. Here, we used the hRAR alpha silencer core, which encompasses the ligand binding domain, including receptor regions D and E of RAR alpha without the activation function called tau4/tau c/AF-2 and without the F region, to analyze the mechanisms by which transcriptional silencing is relieved. Although the RAR silencer core is able to bind ligand, it acts as a constitutive transcriptional silencer. We have fused various small activation domains to the C terminus of the silencer core and analyzed hormone-dependent changes in receptor function. We show that nine amino acids derived from the hTRbeta are sufficient to transform the RAR silencer core into a hormone-dependent activator. Lengthening the linker between the silencer core and these nine amino acids is not critical for mediating ligand-induced relief of silencing and activation. In addition, we show that a transactivation function at the C terminus is not required for relief of silencing by the hormone, but it is required for transcriptional activation. Furthermore, we created functional silencer fusions which lose their repressive function upon addition of hormone, although the corepressors SMRT and N-CoR remain attached to the receptor.

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Affinity versus specificity in coupled binding and folding reactions

Protein Engineering Design and Selection ◽

10.1093/protein/gzz020 ◽

2019 ◽

Author(s):

Stefano Gianni ◽

Per Jemth

Keyword(s):

Protein Interactions ◽

Intrinsically Disordered Proteins ◽

Intrinsically Disordered Protein ◽

High Specificity ◽

Disordered Proteins ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Interaction Interface ◽

Disordered Regions

Abstract Intrinsically disordered protein regions may fold upon binding to an interaction partner. It is often argued that such coupled binding and folding enables the combination of high specificity with low affinity. The basic tenet is that an unfavorable folding equilibrium will make the overall binding weaker while maintaining the interaction interface. While theoretically solid, we argue that this concept may be misleading for intrinsically disordered proteins. In fact, experimental evidence suggests that interactions of disordered regions usually involve extended conformations. In such cases, the disordered region is exceptionally unlikely to fold into a bound conformation in the absence of its binding partner. Instead, these disordered regions can bind to their partners in multiple different conformations and then fold into the native bound complex, thus, if anything, increasing the affinity through folding. We concede that (de)stabilization of native structural elements such as helices will modulate affinity, but this could work both ways, decreasing or increasing the stability of the complex. Moreover, experimental data show that intrinsically disordered binding regions display a range of affinities and specificities dictated by the particular side chains and length of the disordered region and not necessarily by the fact that they are disordered. We find it more likely that intrinsically disordered regions are common in protein–protein interactions because they increase the repertoire of binding partners, providing an accessible route to evolve interactions rather than providing a stability–affinity trade-off.

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The ArathEULS3 Lectin Ends up in Stress Granules and Can Follow an Unconventional Route for Secretion

International Journal of Molecular Sciences ◽

10.3390/ijms21051659 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1659

Author(s):

Malgorzata Dubiel ◽

Tibo De De Coninck ◽

Vinicius Jose Silva Osterne ◽

Isabel Verbeke ◽

Daniël Van Van Damme ◽

...

Keyword(s):

Stress Granules ◽

Protein Translation ◽

Bioinformatics Analyses ◽

Lectin Domain ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Terminal Domain ◽

Unknown Structure ◽

Stressed Cells ◽

And Function

Stress granules are cytoplasmic compartments, which serve as mRNA storage units during stress, therefore regulating translation. The Arabidopsis thaliana lectin ArathEULS3 has been widely described as a stress inducible gene. This study aimed to examine in detail the localization of ArathEULS3 lectin in normal and stressed cells. Colocalization experiments revealed that the nucleo-cytoplasmic lectin ArathEULS3 relocates to stress granules after stress. The ArathEULS3 sequence encodes a protein with a EUL lectin domain and an N-terminal domain with unknown structure and function. Bioinformatics analyses showed that the N-terminal domain sequence contains intrinsically disordered regions and likely does not exhibit a stable protein fold. Plasmolysis experiments indicated that ArathEULS3 also localizes to the apoplast, suggesting that this protein might follow an unconventional route for secretion. As part of our efforts we also investigated the interactome of ArathEULS3 and identified several putative interaction partners important for the protein translation process.

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InterPep2: global peptide–protein docking using interaction surface templates

Bioinformatics ◽

10.1093/bioinformatics/btaa005 ◽

2020 ◽

Vol 36 (8) ◽

pp. 2458-2465 ◽

Cited By ~ 2

Author(s):

Isak Johansson-Åkhe ◽

Claudio Mirabello ◽

Björn Wallner

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

Structural Features ◽

Protein Docking ◽

Supplementary Information ◽

Peptide Ligand ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Improved Performance

Abstract Motivation Interactions between proteins and peptides or peptide-like intrinsically disordered regions are involved in many important biological processes, such as gene expression and cell life-cycle regulation. Experimentally determining the structure of such interactions is time-consuming and difficult because of the inherent flexibility of the peptide ligand. Although several prediction-methods exist, most are limited in performance or availability. Results InterPep2 is a freely available method for predicting the structure of peptide–protein interactions. Improved performance is obtained by using templates from both peptide–protein and regular protein–protein interactions, and by a random forest trained to predict the DockQ-score for a given template using sequence and structural features. When tested on 252 bound peptide–protein complexes from structures deposited after the complexes used in the construction of the training and templates sets of InterPep2, InterPep2-Refined correctly positioned 67 peptides within 4.0 Å LRMSD among top10, similar to another state-of-the-art template-based method which positioned 54 peptides correctly. However, InterPep2 displays a superior ability to evaluate the quality of its own predictions. On a previously established set of 27 non-redundant unbound-to-bound peptide–protein complexes, InterPep2 performs on-par with leading methods. The extended InterPep2-Refined protocol managed to correctly model 15 of these complexes within 4.0 Å LRMSD among top10, without using templates from homologs. In addition, combining the template-based predictions from InterPep2 with ab initio predictions from PIPER-FlexPepDock resulted in 22% more near-native predictions compared to the best single method (22 versus 18). Availability and implementation The program is available from: http://wallnerlab.org/InterPep2. Supplementary information Supplementary data are available at Bioinformatics online.

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