Joshua A. Riback, Jorine M. Eeftens, Daniel S. W. Lee, Sofia A. Quinodoz, Lien Beckers, Lindsay A. Becker, Clifford P. Brangwynne
TL;DR: Nucleoli are the factories of ribosomal RNA, but the way rRNA is processed and expelled from the nucleoli — once assembled into ribosomes — is not well understood. The authors implemented an RNA tracking method that shows how rRNA is expelled thanks to a directional flow, and not only by diffusion.
Ribosomal RNA (rRNA), a key component of the ribosomes, is known to be transcribed and assembled in the nucleoli, and then expelled to reach the rough endoplasmic reticulum. What is not known is how the nucleolar material properties play a role in such a dynamical process. It has been recently shown that the nucleolus is a multiphasic, liquid, membraneless organelle made of many different biomolecules. In first approximation, within the nucleolus there are several centers where the rRNA is transcribed (the FC and DFC), immersed in a connected medium (the GC) where the rRNA meets the ribosomal protein subunits and is assembled.
Riback and colleagues study the rRNA motion relative to the different components within the nucleolus, and they find that that the rRNA spreads not only by diffusion, but with an advective component (slow directed motion outwards ~0.1nm/s). This motion is driven by the continuous polymerization of new rRNA at the interface of the FCs and DFCs, that underlines also the material properties of these regions (that are mainly elastic given the nascent entangled network of rRNA). Within ~1h from its transcription, the rRNA starts to be assembled and therefore it also becomes more folded and compacted. This determines a gradual transformation of the material properties of the nucleoli that from elastic and solid, they become more viscous and liquid-like. The processing of the rRNA fluidizes the nucleolar periphery therefore favoring the release of the ribosomal particles.
Clifford P. Brangwynne is June K. Wu ‘92 Professor of Engineering and Professor of Chemical and Biological Engineering at Princeton University, USA.
Pétur O. Heidarsson, Davide Mercadante, Andrea Sottini, Daniel Nettels, Madeleine B. Borgia, Alessandro Borgia, Sinan Kilic, Beat Fierz, Robert B. Best and Benjamin Schuler
TL;DR: The dissociation kinetics of the linker histone H1 from the nucleosome can be mediated by the protein ProTα via the formation of a transient ternary complex. Disorder and multivalent interactions play an important role in such ‘competition substitution’ mechanism.
Linker histones are essential in regulating chromatin function. The highly positively charged human linker histone H1.0 (H1) was previously identified to form an ultrahigh affinity disordered complex with the highly negatively charged human prothymosin α (ProTα). In this work, the authors integrated single-molecule FRET and molecular simulations to characterize the binding affinity, kinetics, and dynamics of the H1-nucleosome-ProTα interaction. They discovered a remarkable competition mechanism in which ProTα accelerates the dissociation of H1 from the nucleosome via the formation of a transient ternary complex.
The authors performed single-molecular FRET experiments on both freely diffusive and immobilized molecules to measure the binding affinity (KD) and the association and dissociation rate coefficients (kon and koff) of H1 binding to the nucleosome. kon was near diffusion limit, suggesting association is downhill in free energy. KD and koff were highly dependent on the salt concentration. Extrapolation of koff to physiological salt concentration suggested a 3-hour dwell time for H1 bound to the nucleosome, which contradicts the in vivo dwell time of minutes. Remarkably, the dissociation rate was increased by an order of magnitude in the presence of ProTα. Such a phenomenon could not be explained by the simple mechanism in which H1 forms a mutually exclusive binary complex with the nucleosome or ProTα. In the binary complex mechanism, H1 must spontaneously dissociate from the nucleosome before it binds to ProTα, and therefore the dissociation rate should not depend on ProTα. The dependence of the dissociation rate on the ProTα concentration indicated the formation of a ternary complex. The author further performed single-molecule FRET with nanosecond fluorescence correlation spectroscopy to probe the 100 ns dynamics of H1 on the nucleosome and constructed a coarse-grained model to capture the process and the free energy profile of such ‘competitive substitution’. Disorder and multivalent interactions are showed to be essential for accelerated dissociation of H1 from the nucleosome – together they allow ProTα to access the partially bound H1-nucleosome complex and lower the dissociation barrier by reducing the number of H1-nucleosome contacts. The mechanism of accelerated dissociation via transient ternary complex formation is likely to be prevalent in cellular regulation.
Pétur Orri Heiðarsson is an Associate Professor of Biochemistry at University of Iceland.
Robert B. Best is a Senior Investigator at the National Institutes of Health.
Benjamin Schuler is a Professor of Molecular Biophysics at University of Zurich.
Luiza Mamigonian Bessa, Serafima Guseva, Aldo R. Camacho-Zarco, Nicola Salvi, Damien Maurin, Laura Mariño Perez, Maiia Botova, Anas Malki, Max Nanao, Malene Ringkjøbing Jensen, Rob W. H. Ruigrok, Martin Blackledge
TL:DR: Understanding the SARS-CoV-2 replication mechanism is an emerging topic during the pandemic which may lead to the discovery of novel medicine for ending the COVID-19 pandemic. Here, the author unveiled the structure nucleoprotein of SARS-CoV-2 (N) and its binding partner N-terminal component of nonstructural protein 3 (nsp3). Their research discovered two linear binding motifs and two interaction surfaces which can be potential active targets to combat COVID-19
Understanding the SARS-CoV-2 replication mechanism is an emerging topic during the pandemic which may lead to the discovery of novel medicine for ending the COVID-19 pandemic. The nucleoprotein of SARS-CoV-2 (N) is an essential cofactor of the replication machinery that colocalizes with replication-transcription complexes in beta coronaviruses. The N-terminal component of nonstructural protein 3 (nsp3) has been found crucial for this binding process in previous research. The author focus on the structural change of N protein upon the formation of transcription complex with nsp3.
The N protein has 3 intrinsically disordered regions (IDRs). The author uses NMR to quantify the structural change of the IDRs when forming a complex with nsp3. The author found the second IDR region on N protein N3 forming two a-helix regions after binding with nsp3 segment Ubl1. Further investigation of the data discovered two linear motifs on the N3 region that induce the binding by hydrophobic contact and induced a-helix forming through the binding-upon-folding mechanism. NMR data shows the binding is highly dynamic with the second binding sites exchanges between the folding and free state and will demonstrate a higher binding affinity with the existence of the first binding site indicating the dependency between two binding sites. To further investigate the dynamic binding complex, the author conducted NMR experiments using 15N dimeric N234 (a segment of N protein-containing N2, N3, N4) and unlabeled Ubl1. Under this construct, the N3 region still demonstrated the highest dynamic, and the previously observed interaction is maintained under this construct. Small-Angel X-ray scattering (SAXS) also support the NMR data. In addition, SAXS indicates the binding of Ubl1 induced a significant radius of gyration reduction on this new complex.
In conclusion, the author identified the structure of N protein and nsp3 which is closely related to viral replication and transcription. They also discovered two linear motifs and two interaction surfaces that provide a mechanism for the colocalization of N protein and can be potential active targets to combat COVID-19.
Dr. Martin Blackledge is a research director in Institut de Biologie Structurale, France
Priyanka Dogra#, Shruti Arya#, Avinash K. Singh, Anindya Datta, and Samrat Mukhopadhyay*
TL; DR: Solvent relaxation around an amyloid core governs the transition of an IDP into a fibril. Protein-water interactions involved in amyloid formation and phase transitions have remained elusive as current computational and experimental methods are mostly focused on the sequence and not the solvent determinants that govern the conformational heterogeneity of intrinsically disordered proteins (IDPs).
In this work, Dogra et al presented a framework based on steady-state and time-resolved fluorescence spectroscopy to probe the conformational and solvation dynamics of amyloidogenic IDPs using as an example the 130-amino acid repeat of the human melanosomal protein Pmel17 which forms naturally functional fibrils. First, they conjugated a fluorophore named acrylodan in two engineered cysteine mutations: one within the N-terminus disordered region (C350) and one within the C-terminus fibril region (C415). Fluorescence anisotropy data validated prior knowledge obtained using bioinformatic predictors (PONDR, CIDER) that C415 acrylodan-labeled cysteine has less rotational mobility because it’s part of an amyloid core region but also remains solvent exposed. Next, they probed the organization of water molecules around the two fluorophore positions by measuring the Time-Dependent Fluorescence Stokes Shift (TDFSS). This shift is associated with the mechanism of “solvent relaxation”; upon excitation of the fluorophore, the dipole moment changes causing the surrounding bulk water to reorganize or “relax” to compensate for the change in dipole moment until it reaches a new equilibrium configuration. The solvation correlation function can be obtained by collecting the fluorescence up-conversion transients; curves that reflect the librational/vibrational motions of water around acrylodan at different emission wavelengths. The fitting of the correlation function yielded 2 characteristic solvation times that correspond to different time scales of water (bulk, hydration) motion; the fast component (~1ps) represents bulk water and was similar at both fluorophore locations suggesting homogeneous solvation. However, the solvation time that stems from the component of the hydration water was higher at the amyloid C-terminus. This indicates more ordering of slow relaxing hydration water around the fibril. Taken together, these data highlight the importance of protein-solvent interactions. Ultrafast fluorescence spectroscopy could be a useful biophysical tool to probe the perturbation of real-time solvation dynamics in different buffer conditions that are often of interest in systems undergoing phase transitions (e.g., hexanediol, urea)
Dr. Samrat Mukhopadhyay is a Professor of Biology and Chemistry at the Indian Institute of Science Education and Research (IISER) Mohali