Research ArticleBIOPHYSICS

The dynamics of linear polyubiquitin

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Science Advances  14 Oct 2020:
Vol. 6, no. 42, eabc3786
DOI: 10.1126/sciadv.abc3786
  • Fig. 1 Schematic illustrations linear polyubiquitins.

    Cartoon representation of linear tetraubiquitin; the ubiquitin domains are numbered from the N terminus to the C terminus from 1 to 4. The inset shows an atomistic and coarse-grained (CG) (Martini) representation of the hydrophobic patch (Ile44, Val70, and Leu8) of the ubiquitin domain.

  • Fig. 2 Characterization of the dynamics of linear diubiquitin.

    (A to C) Free energy landscapes (in kilojoules per mole) as a function of the distance between the center of mass of the two ubiquitin domains and their relative orientation (measured as the torsion angle between two axes defined using the first and second half of the sequence of each ubiquitin; see Methods). The dots represent the coordinates associated with the available diubiquitin crystal structures. On top is shown the probability distribution of the distance between the centers of the two ubiquitin domains. (D) Experimental and from-simulation calculated Kratky plot. The shaded area represents the error range.

  • Fig. 3 Characterization of the dynamics of linear tri- and tetraubiquitin.

    (A) Experimental and from-simulations calculated Kratky plot for tri- and tetraubiquitin. The shaded area represents the error range. (B) Distribution of the radius of gyration from the ensembles with and without M&M.

  • Fig. 4 Intramolecular interactions of polyubiquitin.

    (A) Minimum distance distribution between two neighboring ubiquitin cores (residues 1 to 70, residues 77 to 146, and so forth). Structures with a minimal distance larger than 0.6 nm are defined as open. (B) Minimum distance distribution between two non-neighboring ubiquitin cores. Structures with a minimal distance larger than 0.6 nm are defined as open. (C) Probability of finding contacts between two amino acids of neighboring ubiquitin cores. (D) Interaction surface of two neighboring ubiquitins. Residues from the blue marked surface (first ubiquitin, left) are interacting with residues of the orange marked surface (middle) or red marked surface (right) of the second ubiquitin. (E) Average end-to-end distance of a linear polyubiquitin chain.

  • Fig. 5 Effect of chain length on the binding of NEMO.

    (A) and (B) SAXS experiments for different ratios of NEMO and Ub3 (A) and Ub4 (B). (C and D) ITC measurement of the interaction of NEMO with Ub3 (C) and Ub4 (D). NEMO was titrated into the polyubiquitin solutions. The experiment was repeated three times. DP, differential power. (E) MW determination. SAXS and SEC in combination with SLS were used to determine the MW of NEMO, Ub3, Ub4, NEMO:Ub3, and NEMO:Ub4. The conditions were 50 mM tris HCl (pH 8) and 300 mM NaCl. (F) ITC measurement of the NEMO interaction with Ub2, Ub3, and Ub4. NEMO was titrated into the ubiquitin solutions in 50 mM sodium phosphate (pH 7) and 50 mM NaCl. Values are averages ± SEs from three measurements. The individual ITC curves are shown in fig. S7. *Experiments taken from Vincendeau et al. (23). A stoichiometry of N = 2 corresponds to one NEMO dimer binding to one polyubiquitin protein.

  • Fig. 6 Comparison between free and NEMO-bound polyubiquitin ensembles.

    (A) Free energy landscapes (in kJ/mol) as a function of the distance between the centers of the two ubiquitin domains and their relative orientation for Ub2 bound to NEMO. The dots represent the coordinates associated with the available crystal structures with Ub2 bound to different proteins. (B and C) Conformational space of free (B) and NEMO-bound (C) ubiquitin pairs in Ub2. The blue area represents the first ubiquitin, while the red area shows the conformation of the second ubiquitin relative to the first one. (D) Conformational space of third (gray area) and forth (orange area) ubiquitin in Ub4 with the first Ub pair being in a NEMO-bound conformation. (E and F) Probability of free Ub2, Ub3, and Ub4 of being in a NEMO-bound conformation for one (E) or two (F) NEMO dimers. The transparent bars show the likelihood of the individual pairs being in the NEMO-bound conformation (root mean square deviation <6 A compared to the average Ub2 structure in the NEMO-bound simulation). The dark bars show the probability of being in the NEMO-bound conformation, excluding structures with an overlap between the nonbound ubiquitins and NEMO.

Supplementary Materials

  • Supplementary Materials

    The dynamics of linear polyubiquitin

    Alexander Jussupow, Ana C. Messias, Ralf Stehle, Arie Geerlof, Sara M. Ø. Solbak, Cristina Paissoni, Anders Bach, Michael Sattler, Carlo Camilloni

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    • Figs. S1 to S10
    • Tables S1 and S2

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