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Structural Dynamic Heterogeneity of Polyubiquitin Subunits Affects Phosphorylation Susceptibility

  • Daichi Morimoto
    Daichi Morimoto
    Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan
  • Erik Walinda
    Erik Walinda
    Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan
    More by Erik Walinda
  • Shingo Takashima
    Shingo Takashima
    Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan
  • Mayu Nishizawa
    Mayu Nishizawa
    Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan
  • Kazuhiro Iwai
    Kazuhiro Iwai
    Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan
  • Masahiro Shirakawa
    Masahiro Shirakawa
    Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan
  • , and 
  • Kenji Sugase*
    Kenji Sugase
    Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan
    *Phone: 075-383-2535. Fax: 075-383-2541. Email: [email protected]
    More by Kenji Sugase
Cite this: Biochemistry 2021, 60, 8, 573–583
Publication Date (Web):February 22, 2021
https://doi.org/10.1021/acs.biochem.0c00619
Copyright © 2021 American Chemical Society

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    Abstract

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    Polyubiquitin is a multifunctional protein tag formed by the covalent conjugation of ubiquitin molecules. Due to the high rigidity of the ubiquitin fold, the ubiquitin moieties in a polyubiquitin chain appear to be structurally equivalent to each other. It is therefore unclear how a specific ubiquitin moiety in a chain may be preferentially recognized by some proteins, such as the kinase PINK1. Here we show that there is structural dynamic heterogeneity in the two ubiquitin moieties of K48-linked diubiquitin by NMR spectroscopic analyses. Our analyses capture subunit-asymmetric structural fluctuations that are not directly related to the closed-to-open transition of the two ubiquitin moieties in diubiquitin. Strikingly, these newly identified heterogeneous structural fluctuations may be linked to an increase in susceptibility to phosphorylation by PINK1. Coupled with the fact that there are almost no differences in static tertiary structure among ubiquitin moieties in a chain, the observed subunit-specific structural fluctuations may be an important factor that distinguishes individual ubiquitin moieties in a chain, thereby aiding both efficiency and specificity in post-translational modifications.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.0c00619.

    • Supplementary Methods section; Tables S1–S4 showing inter-subunit rotation angles and distances in reported structures of K48-linked diubiquitin, the main chain root-mean-square deviation between monoubiquitin and a ubiquitin moiety in diubiquitin, estimated standard enthalpy and entropy changes between the ground and alternative states by chemical shift temperature dependence analysis, and exchange rate constants kex values of Ubdis and Ubprox estimated by R2 relaxation dispersion experiments; and Figures S1–S21 showing the per-residue buried surface area between a ubiquitin moiety and a UBP, correlation of amide proton chemical shift temperature gradients with hydrogen bond length for monoubiquitin, correlation of differences in amide proton chemical shift temperature gradients with differences in amide proton chemical shifts between monoubiquitin and each subunit of K48-linked diubiquitin, temperature dependence of amide proton chemical shifts of the residues in the ubiquitin-intrinsic group, temperature dependence of amide proton chemical shifts of the residues in the diubiquitin-specific group, temperature dependence of amide proton chemical shifts of the residues in the Ubdis-specific group, temperature dependence of the amide proton chemical shifts of the residues in the Ubprox-specific group, location of the residues of monoubiquitin showing curvature in chemical shift temperature dependence, temperature-dependent amide proton chemical shift changes for monoubiquitin mutants used in this study, temperature dependence of amide proton chemical shifts of the monoubiquitin mutants used in this study, comparison of the temperature dependence of amide proton chemical shifts among Ub1, UbK48R, and UbG75A/G76A, thermodynamic analysis of curved temperature-dependent chemical shift changes of the residues in the diubiquitin-specific group, thermodynamic analysis of curved temperature-dependent chemical shift changes of the residues in the Ubdis- and Ubprox-specific groups, estimated standard enthalpy and entropy changes between the ground and alternative states, on-resonance 15N R relaxation dispersion profiles of K48-linked Ub2 at 298 K, 15N R2 relaxation dispersion profiles of monoubiquitin at 277 K, 15N R2 relaxation dispersion profiles of monoubiquitin and the two subunits in K48-linked diubiquitin at 277 K, comparison of the regions showing structural fluctuations with the surfaces recognized by K48-linked diubiquitin-specific UBPs, and implications of subunit-specific conformational fluctuations on post-translational modification of K48-linked diubiquitin (PDF)

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    Cited By

    This article is cited by 3 publications.

    1. Tomoki Sorada, Erik Walinda, Masahiro Shirakawa, Kenji Sugase, Daichi Morimoto. An integrated approach of NMR experiments and MD simulations visualizes structural dynamics of a cyclic multi‐domain protein. Protein Science 2023, 32 (10) https://doi.org/10.1002/pro.4768
    2. Erik Walinda, Kenji Sugase, Naoki Ishii, Masahiro Shirakawa, Kazuhiro Iwai, Daichi Morimoto. Solution structure of the HOIL-1L NZF domain reveals a conformational switch regulating linear ubiquitin affinity. Journal of Biological Chemistry 2023, 299 (9) , 105165. https://doi.org/10.1016/j.jbc.2023.105165
    3. Tomoki Sorada, Daichi Morimoto, Erik Walinda, Kenji Sugase. Molecular recognition and deubiquitination of cyclic K48-linked ubiquitin chains by OTUB1. Biochemical and Biophysical Research Communications 2021, 562 , 94-99. https://doi.org/10.1016/j.bbrc.2021.05.031

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