skip to main content


Title: Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility
Abstract

Endogenous biomolecular condensates, composed of a multitude of proteins and RNAs, can organize into multiphasic structures with compositionally distinct phases. This multiphasic organization is generally understood to be critical for facilitating their proper biological function. However, the biophysical principles driving multiphase formation are not completely understood. Here we use in vivo condensate reconstitution experiments and coarse-grained molecular simulations to investigate how oligomerization and sequence interactions modulate multiphase organization in biomolecular condensates. We demonstrate that increasing the oligomerization state of an intrinsically disordered protein results in enhanced immiscibility and multiphase formation. Interestingly, we find that oligomerization tunes the miscibility of intrinsically disordered proteins in an asymmetric manner, with the effect being more pronounced when the intrinsically disordered protein, exhibiting stronger homotypic interactions, is oligomerized. Our findings suggest that oligomerization is a flexible biophysical mechanism that cells can exploit to tune the internal organization of biomolecular condensates and their associated biological functions.

 
more » « less
NSF-PAR ID:
10491908
Author(s) / Creator(s):
; ; ; ; ; ;
Publisher / Repository:
Nature Publishing Group
Date Published:
Journal Name:
Nature Chemistry
Volume:
16
Issue:
7
ISSN:
1755-4330
Format(s):
Medium: X Size: p. 1073-1082
Size(s):
p. 1073-1082
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract Background

    Biomolecular condensates are non-stoichiometric assemblies that are characterized by their capacity to spatially concentrate biomolecules and play a key role in cellular organization. Proteins that drive the formation of biomolecular condensates frequently contain oligomerization domains and intrinsically disordered regions (IDRs), both of which can contribute multivalent interactions that drive higher-order assembly. Our understanding of the relative and temporal contribution of oligomerization domains and IDRs to the material properties of in vivo biomolecular condensates is limited. Similarly, the spatial and temporal dependence of protein oligomeric state inside condensates has been largely unexplored in vivo.

    Methods

    In this study, we combined quantitative microscopy with number and brightness analysis to investigate the aging, material properties, and protein oligomeric state of biomolecular condensates in vivo. Our work is focused on condensates formed by AUXIN RESPONSE FACTOR 19 (ARF19), a transcription factor integral to the auxin signaling pathway in plants. ARF19 contains a large central glutamine-rich IDR and a C-terminal Phox Bem1 (PB1) oligomerization domain and forms cytoplasmic condensates.

    Results

    Our results reveal that the IDR amino acid composition can influence the morphology and material properties of ARF19 condensates. In contrast the distribution of oligomeric species within condensates appears insensitive to the IDR composition. In addition, we identified a relationship between the abundance of higher- and lower-order oligomers within individual condensates and their apparent fluidity.

    Conclusions

    IDR amino acid composition affects condensate morphology and material properties. In ARF condensates, altering the amino acid composition of the IDR did not greatly affect the oligomeric state of proteins within the condensate.

     
    more » « less
  2. Abstract

    Numerous biological systems contain vesicle‐like biomolecular compartments without membranes, which contribute to diverse functions including gene regulation, stress response, signaling, and skin barrier formation. Coacervation, as a form of liquid–liquid phase separation (LLPS), is recognized as a representative precursor to the formation and assembly of membrane‐less vesicle‐like structures, although their formation mechanism remains unclear. In this study, a coacervation‐driven membrane‐less vesicle‐like structure is constructed using two proteins, GG1234 (an anionic intrinsically disordered protein) and bhBMP‐2 (a bioengineered human bone morphogenetic protein 2). GG1234 formed both simple coacervates by itself and complex coacervates with the relatively cationic bhBMP‐2 under acidic conditions. Upon addition of dissolved bhBMP‐2 to the simple coacervates of GG1234, a phase transition from spherical simple coacervates to vesicular condensates occurred via the interactions between GG1234 and bhBMP‐2 on the surface of the highly viscoelastic GG1234 simple coacervates. Furthermore, the shell structure in the outer region of the GG1234/bhBMP‐2 vesicular condensates exhibited gel‐like properties, leading to the formation of multiphasic vesicle‐like compartments. A potential mechanism is proposed for the formation of the membrane‐less GG1234/bhBMP‐2 vesicle‐like compartments. This study provides a dynamic process underlying the formation of biomolecular multiphasic condensates, thereby enhancing the understanding of these biomolecular structures.

     
    more » « less
  3. Abstract

    Biomolecular condensates, protein-rich and dynamic membrane-less organelles, play critical roles in a range of subcellular processes, including membrane trafficking and transcriptional regulation. However, aberrant phase transitions of intrinsically disordered proteins in biomolecular condensates can lead to the formation of irreversible fibrils and aggregates that are linked to neurodegenerative diseases. Despite the implications, the interactions underlying such transitions remain obscure. Here we investigate the role of hydrophobic interactions by studying the low-complexity domain of the disordered ‘fused in sarcoma’ (FUS) protein at the air/water interface. Using surface-specific microscopic and spectroscopic techniques, we find that a hydrophobic interface drives fibril formation and molecular ordering of FUS, resulting in solid-like film formation. This phase transition occurs at 600-fold lower FUS concentration than required for the canonical FUS low-complexity liquid droplet formation in bulk. These observations highlight the importance of hydrophobic effects for protein phase separation and suggest that interfacial properties drive distinct protein phase-separated structures.

     
    more » « less
  4. Abstract

    Phase separation provides intracellular organization and underlies a variety of cellular processes. These biomolecular condensates exhibit distinct physical and material properties. Current strategies for engineering condensate formation include using intrinsically disordered domains and altering protein surface charge by chemical supercharging or site-specific mutagenesis. We propose adding to this toolbox designer peptide tags that provide several potential advantages for engineering protein phase separation in bacteria. Herein, we demonstrate the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates and provide a foundational study for their development as tools for condensate engineering. Using a panel of GFP variants, we demonstrate how cationic tag and globular domain charge contribute to intracellular phase separation inE. coliand observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active.

     
    more » « less
  5. Across bacteria, protein-based organelles called bacterial microcompartments (BMCs) encapsulate key enzymes to regulate their activities. The model BMC is the carboxysome that encapsulates enzymes for CO2fixation to increase efficiency and is found in many autotrophic bacteria, such as cyanobacteria. Despite their importance in the global carbon cycle, little is known about how carboxysomes are spatially regulated. We recently identified the two-factor system required for the maintenance of carboxysome distribution (McdAB). McdA drives the equal spacing of carboxysomes via interactions with McdB, which associates with carboxysomes. McdA is a ParA/MinD ATPase, a protein family well studied in positioning diverse cellular structures in bacteria. However, the adaptor proteins like McdB that connect these ATPases to their cargos are extremely diverse. In fact, McdB represents a completely unstudied class of proteins. Despite the diversity, many adaptor proteins undergo phase separation, but functional roles remain unclear. Here, we define the domain architecture of McdB from the model cyanobacteriumSynechococcus elongatusPCC 7942, and dissect its mode of biomolecular condensate formation. We identify an N-terminal intrinsically disordered region (IDR) that modulates condensate solubility, a central coiled-coil dimerizing domain that drives condensate formation, and a C-terminal domain that trimerizes McdB dimers and provides increased valency for condensate formation. We then identify critical basic residues in the IDR, which we mutate to glutamines to solubilize condensates. Finally, we find that a condensate-defective mutant of McdB has altered association with carboxysomes and influences carboxysome enzyme content. The results have broad implications for understanding spatial organization of BMCs and the molecular grammar of protein condensates.

     
    more » « less