(Summary by Monica Rieth)
Vahid Sheikhhassani, Barbara Scalvini, Julian Ng, Laurens W. H. J. Heling, Yosri Ayache, Tom M. J. Evers, Eva Estébanez-Perpiña, Iain J. McEwan, Alireza Mashaghi
TL;DR: This paper presents an approach not previously used to study molecular dynamics and topological conformations of an intrinsically disordered protein region, the N-terminal region of the human androgen receptor. Results from this work suggest a regulatory mechanism of the disordered N-terminal and disordered portion of the C-terminal region through their interactions with the DNA binding domain of the protein.
The human androgen receptor is an important nuclear transcription factor that interacts with hormones such as testosterone. It belongs to a larger family of steroid hormone nuclear receptors and binds to DNA in a ligand-dependent manner, regulating transcription. The receptor is expressed in numerous cell types and tissues. The N-terminal transactivation domain (NTD) of the receptor is variable while the DNA-binding domain (DBD) is highly conserved. The ligand-binding domain (LBD) is situated toward the C-terminal domain (CTD) of the receptor (225-538). In this paper, the authors investigate the topological dynamics and structure of the critically important intrinsically-disordered N-terminal domain (NTD). One long-term goal is to use this structural information to develop drug treatments that target this region.
Predicting the topology of intrinsically disordered proteins is difficult because of their flexibility and large conformational space. In this paper, the authors use a combination of molecular dynamics (MD) simulations and circuit topology (CT) analysis to probe the dynamics of the long, disordered N-terminal region (NR) of the human androgen receptor. The NR is 224 residues in length. Flexible-disordered regions of both N- and C-terminal regions (CR), revealed from this analysis, compete for binding to the DBD on the receptor, a suggested mechanism of regulation. A new small molecule inhibitor binding site within the CR was also identified.
Authors began by initially using I-TASSER to predict a 3D structure of the protein (full length). The structure was refined using the MD simulation program, GROMACS. A series of additional computational and bioinformatics programs were used to verify the accuracy of the predicted model structure. Next, dynamics were performed using GROMACS beginning with the minimized energy-predicted structure, refined from the I-TASSER pipeline. Noteworthy, I-TASSER was able to generate better predictions, in this case, compared to AlphaFold and Rosetta based on confidence metrics and experimental data.
Molecular docking experiments were employed to study binding interactions of the NTD (synonymous to NR) with LBD and DBD. CT analysis was performed to map the residue contact points of the NR and CR to better understand the evolution of any folded structures. CT analysis shows the NR is truly dynamic and conformational “folding” is random, while the CR less so. The latter is capable of assuming multiple “sub-structures” indicating less dynamic flexibility in relation to evolutionary fold.
Link to paper
Corresponding author:
Alireza Mashaghi, Medical Systems Biophysics and Bioengineering, Leiden Academic Centre for Drug Research, Faculty of Science, Leiden University, Leiden, The Netherlands
(Summary by Sara Mingu)
Alain Ibáñez de Opakua, James A. Geraets, Benedikt Frieg, Christian Dienemann, Adriana Savastano, Marija Rankovic, Maria-Sol Cima-Omori, Gunnar F. Schröder & Markus Zweckstetter
TL;DR: Nup98 is a nucleoporin with a long intrinsically disordered region rich in FG motifs. The FG interactions in Nup98 were probed using NMR and cryo-EM. Liquid-like and reversible amyloid-like interactions were observed. Cross-beta structures were found to contain asparagine and glutamine residues.
Nup98 is one of the nucleoporins lining the inner channel of the nuclear pore complex and it contributes to the formation of the permeability barrier. Besides its roles in transport, Nup98 has also been implicated in Alzheimer’s disease, as well as in acute myeloid leukemias, where it is found fused to various transcription factors, forming transcriptional condensates.
Nup98 contains a long N-terminal intrinsically disordered domain highly enriched in phenylalanine-glycine (FG) motifs. Mutagenesis studies have consistently demonstrated the importance of the FG repeats in Nup98's roles in both nucleocytoplasmic transport and leukemia progression. However, the nature of these molecular interactions is largely unknown. In this study, de Opakua et al. combine NMR spectroscopy with cryo-EM to give detailed insights about the interactions between FG motifs within transient liquid-like or stable FG interactions.
The results suggest that Nup98 condensate formation may be a balance between amyloid-like and liquid-like interactions. In support of this, the authors show that Nup98 shows different self-association propensities along its FG-rich disordered region, with 3 segments of 30-40 residues showing the strongest interactions, which were captured by cryo-EM. On the other hand, liquid-like FG interactions resulted in highly dynamic FG-repeat domain and the formation of transient beta structures, as well as a stable pre-formed helix (the GLEBS domain). The amyloid-like interactions were reversible, suggesting that the two types of interactions may be continuous and present even within a single FG-region of Nup98. The cross-beta structures were confirmed by ThT staining, and found to implicate asparagine and glutamine residues. Indeed, these residues were also found in the cryo-EM resolved structures of the stable interactions.
In summary, this study offers new insights on the nature of FG interactions in Nup98. These interactions are essential to Nup98 (dys)function in a variety of settings, including the nuclear pore complex permeability barrier and acute myeloid leukemia.
Link to paper
Markus Zweckstetter is a Honorary Professor at the University of Goettingen. He is also the head of the senior research group “Structural Biology in Dementia” at the German Center for Neurodegenerative Diseases (DZNE).
Summary by Monica Rieth
Anas Malki, Jean-Marie Teulon, Aldo R. Camacho-Zarco, Shu-wen W. Chen, Wiktor Adamski, Damien Maurin, Nicola Salvi, Jean-Luc Pellequer, and Martin Blackledge
TL;DR: Tardigrade-specific intrinsically disordered proteins (TDPs) are responsible for conferring survival to the organism under extreme harsh environment conditions. Authors use a variety of biophysical techniques to characterize the properties that drive conformational changes in the protein responsible for its protective properties.
The ability of tardigrades to withstand extreme environmental conditions ranging from dessication to high temperatures and pressures has baffled researchers for decades. The biology underlying this unique ability has remained a mystery until recently. In this article, the authors point to two intrinsically disordered proteins unique to the tardigrade species, Hypsibius exemplaris, called (cytosolic abundant heat-soluble (CAHS) proteins and secreted abundant heat-soluble (SAHS) proteins), which are known as tardigrade disordered proteins (TDPs). In this paper the CAHS-8 protein is characterized using 2D NMR, atomic force microscopy (AFM), small angle x-ray scattering (SAXS) and dynamic light scattering (DLS) to study its conformational properties in an effort to explain the mechanism by which these proteins protect tardigrades under extreme conditions.
2D NMR reveals that this protein forms a central two-helical domain structure flanked on the N- and C- termini by disordered regions. It further shows a propensity to oligomerize in a concentration-dependent manner, which was shown using SAX-generated curves to illustrate that it behaves similarly to fibrils at high concentrations rather than a monomeric species observed at lower concentrations. The formation of higher-order oligomers was also deduced at lower temperatures from C13 and N15-relaxation experiments to probe protein dynamics. DLS experiments reveal formation of a hydrogel phase at low temperatures and high concentration, which correlates qualitatively with the NMR and SAXS findings. Further, AFM confirmed the formation of these fibrils consistent with the characteristics of hydrogel formation. Over time a dense network of fibrillar mesh hydrogel could be observed with the disordered domains of the CAHS-8 protein remaining intact.
The protective nature of this TDP-generated hydrogel was tested by incorporating the soluble protein, ANP32a, an intrinsically disordered transcription factor. The protein was isotopically labeled so that dynamics and translational diffusion could be monitored in the dense hydrogel using H1-N15 NMR. The disordered domain of ANP32a remained unchanged within the hydrogel, maintaining its conformational integrity. However, diffusion was slowed compared to the buffer control. Other IDP proteins were tested in the TDP-hydrogel in addition to ANP32a with similar results.
The authors propose a mechanism for hydrogel formation whereby the two central helical regions of the TDP protein associate in a coiled-coil-like manner forming oligomers first, then eventually long fibrils leading to the hydrogel formation. They carefully suggest that the absence of water in these hydrogels may convey protective properties in the organism under such unfavorable environmental conditions.
Link to paper
Martin Blackledge is a researcher at the Institut de Biologie Structurale (IBS) in Grenoble, France.
(Summary by Sara Mingu)
Amy Yewdall, Alain A. M. André, Merlijn H. I. van Haren, Frank H. T. Nelissen, Aafke Jonker, Evan Spruijt
TL;DR: In model NPM1-rRNA condensates in vitro, Mg2+ concentration can affect the RNA dynamics. RNA-RNA interactions within RNA-protein condensates could impart viscoelastic properties to the condensate and affect its properties, as well as the partitioning of client proteins.
Heterotypic biomolecular condensates, such as the nucleolus, are made from protein and RNA. Although the morphology of such condensates sometimes suggests a gel-like state, the dynamics of their protein constituents suggest liquid-like properties.
In this study, Yewdall et al. focus on RNA-RNA interactions in RNA-protein condensates as a possible solution to this puzzling behavior. By probing the dynamics of NPM1-rRNA condensates in vitro under a variety of conditions, the authors were able to show that the RNA component, via multivalent RNA-RNA interactions, imparts viscoelastic properties onto protein-RNA condensates. With increasing Mg2+ concentration, the mobility of RNA molecules within the condensates decreased. In line with this, ATP (which strongly chelates Mg2+) was found to promote liquid-like behavior by relaxing the RNA-RNA network; enzymatic depletion of ATP also affected condensate properties. The authors suggest that RNA forms a viscoelastic network. This was also supported by evidence of gradual condensate aging, which varied non-linearly with temperature. On the other hand, the protein component could freely diffuse throughout the network. Importantly, this also affected the partition of client proteins with strong dependence on client interaction type. For the in vitro NPM1-rRNA condensates, the authors found that Mg2+ and ATP concentration affected 70S ribosome partitioning, pointing to possible mechanisms involved in ribosome formation.
This study highlights the importance of RNA-RNA interactions in imparting viscoelastic properties to the condensates. The dependence of RNA-RNA interactions on Mg2+ concentration could constitute an important regulatory mechanism for the cell, since Mg2+ concentration is strongly dependent on ATP levels and these, in turn, can be modulated by a variety of parameters such as the cell cycle or ATP-consuming enzymes. Besides controlling the liquid- or gel-like nature of the condensates, Mg levels could also affect the partitioning of client molecules, thus modulating condensate function.
Evan Sprujit is Associate Professor of physical organic chemistry at Radboud University.