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Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation

View ORCID ProfileTongyin Zheng, View ORCID ProfileCarlos A. Castañeda
doi: https://doi.org/10.1101/2021.02.22.432116
Tongyin Zheng
1Department of Chemistry, Syracuse University, Syracuse, NY 13244, USA
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Carlos A. Castañeda
2Departments of Biology and Chemistry, Syracuse University, Syracuse, NY 13244, USA
3Interdisciplinary Neuroscience Program, Syracuse University, Syracuse, NY 13244, USA
4BioInspired Institute, Syracuse University, Syracuse, NY 13244, USA
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  • For correspondence: cacastan@syr.edu
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Abstract

Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N-terminal ubiquitin-like (UBL) and C-terminal ubiquitin-associated (UBA) domains, respectively. Between these two folded domains are intrinsically disordered STI1-I and STI1-II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1-II region enables UBQLN2 to undergo liquid-liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically-disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using NMR spectroscopy. To our surprise, aside from well-studied canonical UBL:UBA interactions, there also exist moderate and weak interactions between the UBL and STI1-I/STI1-II domains, and between the UBA domain and the linker connecting the two STI1 regions, respectively. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or STI1 domains on the phase behavior and physiological functions of UBQLN2.

Impact of Work Statement Zheng and Castañeda show that interplay between the folded domains and intrinsically disordered regions regulates liquid-liquid phase separation behavior of UBQLN2, a protein quality control (PQC) shuttle protein. Despite their similar size, the folded UBL and UBA domains inhibit and promote phase separation, respectively, due to their previously uncharacterized, asymmetric interactions with the middle intrinsically-disordered region. These results strongly suggest that PQC components, including proteasomal receptors, are likely to modulate UBQLN2 phase separation behavior in cells.

Competing Interest Statement

The authors have declared no competing interest.

  • Abbreviations and symbols

    csat
    Saturation concentration
    CSP
    Chemical shift perturbation
    DSK2
    Ubiquitin domain-containing protein DSK2
    FUS
    Fused-in-sarcoma
    FL
    Full-length
    hnRNPA1
    Heterogeneous nuclear ribonucleoprotein A1
    HSP70
    Heat shock protein 70
    HSQC
    Heteronuclear single quantum coherence
    IDR
    Intrinsically disordered region
    Kd
    Binding dissociation constant
    LLPS
    Liquid-liquid phase separation
    NMR
    Nuclear magnetic resonance
    PRE
    Paramagnetic relaxation enhancement
    PQC
    Protein quality control
    PXX
    Proline-rich region
    STI1
    Stress induced protein 1 (STI1)-like domain
    TROSY
    Transverse-relaxation optimized spectroscopy
    UBA
    Ubiquitin-associating domain
    UBL
    Ubiquitin-like domain
    UBQLN2
    Ubiquilin-2
    Ub
    Ubiquitin
    UPS
    Ubiquitin-proteasome system
  • Copyright 
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    Posted February 22, 2021.
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    Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation
    Tongyin Zheng, Carlos A. Castañeda
    bioRxiv 2021.02.22.432116; doi: https://doi.org/10.1101/2021.02.22.432116
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    Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation
    Tongyin Zheng, Carlos A. Castañeda
    bioRxiv 2021.02.22.432116; doi: https://doi.org/10.1101/2021.02.22.432116

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