Intrinsically disordered N-terminal domain of the Helicoverpa armigera Ultraspiracle stabilizes the dimeric form via a scorpion-like structure

Nuclear receptors (NRs) are a family of ligand-dependent transcription factors activated by lipophilic compounds. NRs share a common structure comprising three domains: a variable N-terminal domain (NTD), a highly conserved globular DNA-binding domain and a ligand-binding domain. There are numerous papers describing the molecular details of the latter two globular domains. However, very little is known about the structure-function relationship of the NTD, especially as an intrinsically disordered fragment of NRs that may influence the molecular properties and, in turn, the function of globular domains. Here, we investigated whether and how an intrinsically disordered NTD consisting of 58 amino acid residues affects the functions of the globular domains of the Ultraspiracle protein from Helicoverpa armigera (HaUsp). The role of the NTD was examined for two well-known and easily testable NR functions, i.e., interactions with specific DNA sequences and dimerization. Electrophoretic mobility shift assays showed that the intrinsically disordered NTD influences the interaction of HaUsp with specific DNA sequences, apparently by destabilization of HaUsp-DNA complexes. On the other hand, multi-angle light scattering and sedimentation velocity analytical ultracentrifugation revealed that the NTD acts as a structural element that stabilizes HaUsp homodimers. Molecular models based on small-angle X-ray scattering indicate that the intrinsically disordered NTD may exert its effects on the tested HaUsp functions by forming an unexpected scorpion-like structure, in which the NTD bends towards the ligand-binding domain in each subunit of the HaUsp homodimer. This structure may be crucial for specific NTD-dependent regulation of the functions of globular domains in NR

The Significance of the Intrinsically Disordered Regions for the Functions of the bHLH Transcription Factors

The bHLH proteins are a family of eukaryotic transcription factors regulating expression of a wide range of genes involved in cell differentiation and development. They contain the Helix-Loop-Helix (HLH) domain, preceded by a stretch of basic residues, which are responsible for dimerization and binding to E-box sequences. In addition to the well-preserved DNA-binding bHLH domain, these proteins may contain various additional domains determining the specificity of performed transcriptional regulation. According to this, the family has been divided into distinct classes. Our aim was to emphasize the significance of existing disordered regions within the bHLH transcription factors for their functionality. Flexible, intrinsically disordered regions containing various motives and specific sequences allow for multiple interactions with transcription co-regulators. Also, based on in silico analysis and previous studies, we hypothesize that the bHLH proteins have a general ability to undergo spontaneous phase separation, forming or participating into liquid condensates which constitute functional centers involved in transcription regulation. We shortly introduce recent findings on the crucial role of the thermodynamically liquid-liquid driven phase separation in transcription regulation by disordered regions of regulatory proteins. We believe that further experimental studies should be performed in this field for better understanding of the mechanism of gene expression regulation (among others regarding oncogenes) by important and linked to many diseases the bHLH transcription factors

Metal Ions Induce Liquid Condensate Formation by the F Domain of <i>Aedes aegypti</i> Ecdysteroid Receptor. New Perspectives of Nuclear Receptor Studies

The superfamily of nuclear receptors (NRs), composed of ligand-activated transcription factors, is responsible for gene expression as a reaction to physiological and environmental changes. Transcriptional machinery may require phase separation to fulfil its role. Although NRs have a similar canonical structure, their C-terminal domains (F domains) are considered the least conserved and known regions. This article focuses on the peculiar molecular properties of the intrinsically disordered F domain of the ecdysteroid receptor from the Aedes aegypti mosquito (AaFEcR), the vector of the world’s most devastating human diseases such as dengue and Zika. The His-Pro-rich segment of AaFEcR was recently shown to form the unique poly-proline helix II (PPII) in the presence of Cu2+. Here, using widefield microscopy of fluorescently labeled AaFEcR, Zn2+- and Cu2+-induced liquid-liquid phase separation (LLPS) was observed for the first time for the members of NRs. The perspectives of this finding on future research on the F domain are discussed, especially in relation to other NR members

Getting Closer to Decrypting the Phase Transitions of Bacterial Biomolecules

Liquid&ndash;liquid phase separation (LLPS) of biomolecules has emerged as a new paradigm in cell biology, and the process is one proposed mechanism for the formation of membraneless organelles (MLOs). Bacterial cells have only recently drawn strong interest in terms of studies on both liquid-to-liquid and liquid-to-solid phase transitions. It seems that these processes drive the formation of prokaryotic cellular condensates that resemble eukaryotic MLOs. In this review, we present an overview of the key microbial biomolecules that undergo LLPS, as well as the formation and organization of biomacromolecular condensates within the intracellular space. We also discuss the current challenges in investigating bacterial biomacromolecular condensates. Additionally, we highlight a summary of recent knowledge about the participation of bacterial biomolecules in a phase transition and provide some new in silico analyses that can be helpful for further investigations

Insight into the Unfolding Properties of Chd64, a Small, Single Domain Protein with a Globular Core and Disordered Tails.

Two major lipophilic hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH), govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64) and Tribolium castaneum (TcChd64). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH) domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV) circular dichroism (CD) spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC) revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG)-like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners

The Role of Intrinsically Disordered Proteins in Liquid&ndash;Liquid Phase Separation during Calcium Carbonate Biomineralization

Some animal organs contain mineralized tissues. These so-called hard tissues are mostly deposits of calcium salts, usually in the form of calcium phosphate or calcium carbonate. Examples of this include fish otoliths and mammalian otoconia, which are found in the inner ear, and they are an essential part of the sensory system that maintains body balance. The composition of ear stones is quite well known, but the role of individual components in the nucleation and growth of these biominerals is enigmatic. It is sure that intrinsically disordered proteins (IDPs) play an important role in this aspect. They have an impact on the shape and size of otoliths. It seems probable that IDPs, with their inherent ability to phase separate, also play a role in nucleation processes. This review discusses the major theories on the mechanisms of biomineral nucleation with a focus on the importance of protein-driven liquid&ndash;liquid phase separation (LLPS). It also presents the current understanding of the role of IDPs in the formation of calcium carbonate biominerals and predicts their potential ability to drive LLPS

Heurs et malheurs de la décentralisation en France, de la fin de l’Ancien Régime à la troisième République

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to regulate insect development. Recently,two proteins, a calponin-like Chd64 and immunophilin FKBP39 have been found to play a pivotal role in the cross-talkbetween 20E and JH, although the molecular basis of interaction remains unknown. The aim of this work was to identify thestructural features that would provide understanding of the role of Chd64 in multiple and dynamic complex that cross-linksthe signaling pathways. Here, we demonstrate the results of in silico and in vitro analyses of the structural organization ofChd64 from Drosophila melanogaster and its homologue from Tribolium castaneum. Computational analysis predicted theexistence of disordered regions on the termini of both proteins, while the central region appeared to be globular, probablycorresponding to the calponin homology (CH) domain. In vitro analyses of the hydrodynamic properties of the proteinsfrom analytical size-exclusion chromatography and analytical ultracentrifugation revealed that DmChd64 and TcChd64 hadan asymmetrical, elongated shape, which was further confirmed by small angle X-ray scattering (SAXS). The Kratky plotindicated disorderness in both Chd64 proteins, which could possibly be on the protein termini and which would give rise tospecific hydrodynamic properties. Disordered tails are often involved in diverse interactions. Therefore, it is highly possiblethat there are intrinsically disordered regions (IDRs) on both termini of the Chd64 proteins that serve as platforms formultiple interaction with various partners and constitute the foundation for their regulatory function