Priya R. Banerjee

Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA

The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus. DOI: http://dx.doi.org/10.7554/eLife.13571.001

Cataract-associated mutant E107A of human γD-crystallin shows increased attraction to α-crystallin and enhanced light scattering

Several point mutations in human γD-crystallin (HGD) are now known to be associated with cataract. So far, the in vitro studies of individual mutants of HGD alone have been sufficient in providing plausible molecular mechanisms for the associated cataract in vivo. Nearly all the mutant proteins in solution showed compromised solubility and enhanced light scattering due to altered homologous γ–γ crystallin interactions. In sharp contrast, here we present an intriguing case of a human nuclear cataract-associated mutant of HGD—namely Glu107 to Ala (E107A), which is nearly identical to the wild type in structure, stability, and solubility properties, with one exception: Its pI is higher by nearly one pH unit. This increase dramatically alters its interaction with α-crystallin. There is a striking difference in the liquid–liquid phase separation behavior of E107A–α-crystallin mixtures compared to HGD–α-crystallin mixtures, and the light-scattering intensities are significantly higher for the former. The data show that the two coexisting phases in the E107A–α mixtures differ much more in protein density than those that occur in HGD–α mixtures, as the proportion of α-crystallin approaches that in the lens nucleus. Thus in HGD–α mixtures, the demixing of phases occurs primarily by protein type while in E107A–α mixtures it is increasingly governed by protein density. Analysis of these results suggests that the cataract due to the E107A mutation could result from the instability caused by the altered attractive interactions between dissimilar proteins—i.e., heterologous γ–α crystallin interactions—primarily due to the change in surface electrostatic potential in the mutant protein.

Increase in Surface Hydrophobicity of the Cataract-Associated, P23T Mutant of Human GammaD-Crystallin is Responsible for Its Dramatically Lower, Retrograde Solubility †

The cataract-associated Pro23 to Thr (P23T) mutation in human gammaD-crystallin (HGD) has a variety of phenotypes and is geographically widespread. Therefore, there is considerable interest in understanding the molecular basis of cataract formation due to this mutation. We showed earlier [Pande, A., et al. (2005) Biochemistry 44, 2491-2500] that the probable basis of opacity in this case is the severely compromised, retrograde solubility and aggregation of P23T relative to HGD. The dramatic solubility change occurs even as the structure of the mutant protein remains essentially unchanged in vitro. We proposed that the retrograde solubility and aggregation of P23T were mediated by net hydrophobic, protein-protein interactions. On the basis of these initial findings for P23T and related mutants, and the subsequent finding that they show atypical phase behavior [McManus, J. J., et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 16856-16861], we concluded that the protein clusters formed in solutions of the mutant proteins were held together by net hydrophobic, anisotropic interactions. Here we show, using chemical probes, that the surface hydrophobicities of these mutants are inversely related to their solubility. Furthermore, by probing the isolated N-terminal domains of HGD and P23T directly, we find that the increase in the surface hydrophobicity of P23T is localized in the N-terminal domain. Modeling studies suggest the presence of sticky patches on the surface of the N-terminal domain that could be engaged in the formation of protein clusters via hydrophobic protein-protein interactions. This work thus provides direct evidence of the dominant role played by net hydrophobic and anisotropic protein-protein interactions in the aggregation of P23T.

Reentrant Phase Transition Drives Dynamic Substructure Formation in Ribonucleoprotein Droplets

Intracellular ribonucleoprotein (RNP) granules are membrane-less droplet organelles that are thought to regulate posttranscriptional gene expression. While liquid-liquid phase separation may drive RNP granule assembly, the mechanisms underlying their supramolecular dynamics and internal organization remain poorly understood. Herein, we demonstrate that RNA, a primary component of RNP granules, can modulate the phase behavior of RNPs by controlling both droplet assembly and dissolution in vitro. Monotonically increasing the RNA concentration initially leads to droplet assembly by complex coacervation and subsequently triggers an RNP charge inversion, which promotes disassembly. This RNA-mediated reentrant phase transition can drive the formation of dynamic droplet substructures (vacuoles) with tunable lifetimes. We propose that active cellular processes that can create an influx of RNA into RNP granules, such as transcription, can spatiotemporally control the organization and dynamics of such liquid-like organelles.

Ultrafast cooling reveals microsecond-scale biomolecular dynamics

The temperature-jump technique, in which the sample is rapidly heated by a powerful laser pulse, has been widely used to probe the fast dynamics of folding of proteins and nucleic acids. However, the existing temperature-jump setups tend to involve sophisticated and expensive instrumentation, while providing only modest temperature changes of ~10-15 °C, and the temperature changes are only rapid for heating, but not cooling. Here we present a setup comprising a thermally conductive sapphire substrate with light-absorptive nano-coating, a microfluidic device and a rapidly switched moderate-power infrared laser with the laser beam focused on the nano-coating, enabling heating and cooling of aqueous solutions by ~50 °C on a 1-μs time scale. The setup is used to probe folding and unfolding dynamics of DNA hairpins after direct and inverse temperature jumps, revealing low-pass filter behaviour during periodic temperature variations.

Molecular Mechanism of the Chaperone Function of Mini-α-Crystallin,a 19-Residue Peptide of Human α-Crystallin

α-Crystallin is the archetypical chaperone of the small heat-shock protein family, all members of which contain the so-called “α-crystallin domain” (ACD). This domain and the N- and C-terminal extensions are considered the main functional units in its chaperone function. Previous studies have shown that a 19-residue fragment of the ACD of human αA-crystallin called mini-αA-crystallin (MAC) shows chaperone properties similar to those of the parent protein. Subsequent studies have confirmed the function of this peptide, but no studies have addressed the mechanistic basis for the chaperone function of MAC. Using human γD-crystallin (HGD), a key substrate protein for parent α-crystallin in the ocular lens, we show here that MAC not only protects HGD from aggregation during thermal and chemical unfolding but also binds weakly and reversibly to HGD (Kd ≈ 200–700 μM) even when HGD is in the native state. However, at temperatures favoring the unfolding of HGD, MAC forms a stable complex with HGD similar to parent α-crystallin. Using nuclear magnetic resonance spectroscopy, we identify the residues in HGD that are involved in these two modes of binding and show that MAC protects HGD from aggregation by binding to Phe 56 and Val 132 at the domain interface of the target protein, and residues Val 164 to Leu 167 in the core of the C-terminal domain. Furthermore, we suggest that the low-affinity, reversible binding of MAC on the surface of HGD in the native state is involved in facilitating its binding to both the domain interface and core regions during the early stages of the unfolding of HGD. This work highlights some structural features of MAC and MAC-like peptides that affect their chaperone activity and can potentially be manipulated for translational studies.

Asymmetric Modulation of Protein Order–Disorder Transitions by Phosphorylation and Partner Binding

As for many intrinsically disordered proteins, order-disorder transitions in the N-terminal oligomerization domain of the multifunctional nucleolar protein nucleophosmin (Npm-N) are central to its function, with phosphorylation and partner binding acting as regulatory switches. However, the mechanism of this transition and its regulation remain poorly understood. In this study, single-molecule and ensemble experiments revealed pathways with alternative sequences of folding and assembly steps for Npm-N. Pathways could be switched by altering the ionic strength. Phosphorylation resulted in pathway-specific effects, and decoupled folding and assembly steps to facilitate disorder. Conversely, binding to a physiological partner locked Npm-N in ordered pentamers and counteracted the effects of phosphorylation. The mechanistic plasticity found in the Npm-N order-disorder transition enabled a complex interplay of phosphorylation and partner-binding steps to modulate its folding landscape.

Two-Dimensional Crowding Uncovers a Hidden Conformation of α-Synuclein.

The intrinsically disordered protein (IDP), α-synuclein (αS), is well-known for phospholipid membrane binding-coupled folding into tunable helical conformers. Here, using single-molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction-folding landscape of αS can be tuned by two-dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS-membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher-affinity module remains membrane-bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane-binding IDPs with multiple binding modes.

Keeping it Disordered: A New Mechanism of Protein Quality Control?

Molecular chaperones are known for their function in maintaining a healthy proteome, partly by aiding globular folded proteins to remain in native regions of their folding landscapes. In contrast, although protein disorder has emerged as a significant component of the eukaryotic proteome, little is known about how intrinsically disordered protein (IDP) quality control is achieved. The cellular functions of IDPs are often precisely encoded by a complex landscape of coupled interaction and folding with their binding partners. A key question is therefore whether and how cellular protein chaperones can regulate the coupled interaction-folding events of IDPs, as part of maintaining a healthy protein homeostasis. To answer this question, we studied the effects of a stress-inducible, ATP-independent molecular chaperone, Hsp27, on the neuronal, Parkinsons diseases (PD)-associated IDP alpha-synuclein (aSyn), which served as an important and well-characterized model IDP system. Coupled membrane-binding and folding of aSyn has been suggested to play an important role in the putative function of aSyn in synaptic vesicle fusion, while aberrant interaction with lipid-bilayers have toxic effects. Here, using the strengths of single-molecule fluorescence to monitor mixtures of multiple conformational protein states in conjunction with complimentary ensemble experiments, we discovered that Hsp27 can modulate the membrane interaction and coupled-folding of aSyn. Intriguingly, we observed that this chaperone favors disorder rather than folding in this system, and that Hsp27 acts by directly interacting with the binding partner, rather than the IDP itself. Additional biophysical experiments suggest that by modulating the coupled interaction-folding landscape of aSyn, Hsp27 may directly regulate the role of aSyn in vesicle fusion. Analysis of our biophysical data point to a potential new model of chaperone regulation of IDP function and dysfunction via maintenance of the disordered state of the IDP.