Nonspecific yet decisive: Ubiquitination can affect the native-state dynamics of the modified protein

Abstract

Ubiquitination is one of the most common post-translational modifications of proteins, and mediates regulated protein degradation among other cellular processes. A fundamental question regarding the mechanism of protein ubiquitination is whether and how ubiquitin affects the biophysical nature of the modified protein. For some systems, it was shown that the position of ubiquitin within the attachment site is quite flexible and ubiquitin does not specifically interact with its substrate. Nevertheless, it was revealed that polyubiquitination can decrease the thermal stability of the modified protein in a site-specific manner because of alterations of the thermodynamic properties of the folded and unfolded states. In this study, we used detailed atomistic simulations to focus on the molecular effects of ubiquitination on the native structure of the modified protein. As a model, we used Ubc7, which is an E2 enzyme whose in vivo ubiquitination process is well characterized and known to lead to degradation. We found that, despite the lack of specific direct interactions between the ubiquitin moiety and Ubc7, ubiquitination decreases the conformational flexibility of certain regions of the substrate Ubc7 protein, which reduces its entropy and thus destabilizes it. The strongest destabilizing effect was observed for systems in which Lys48-linked tetra-ubiquitin was attached to sites used for in vivo degradation. These results reveal how changes in the configurational entropy of the folded state may modulate the stability of the protein's native state. Overall, our results imply that ubiquitination can modify the biophysical properties of the attached protein in the folded state and that, in some proteins, different ubiquitination sites will lead to different biophysical outcomes. We propose that this destabilizing effect of polyubiquitin on the substrate is linked to the functions carried out by the modification, and in particular, regulatory control of protein half-life through proteasomal degradation.

Keywords: molecular dynamics simulations; multidomain protein; native state dynamics; ubiquitination.

Figure 1

Ubiquitination sites on substrate protein Ubc7. Residues ubiquitinated in vivo (His94 and Cys89) are colored red. Other surface-exposed Lys residues capable of acting as ubiquitination sites are colored blue.

Figure 2

Characteristics of the flexibility of the Ub–Ubc7 interface. (a) Two angles, α and β, are defined to evaluate the rotation of Ub relative to the substrate (Ubc7). For each angle, two vectors connecting two residues are defined (one on Ubc7 and the other one on Ub). The vectors were placed opposite and in parallel with each other (α angle) or lined up on a single line (β angle). In the case of tetra-Ub, the vector is defined based on the first Ub protein that is directly linked to Ubc7. (b) Values of the angles α for the tetra-Ub (red) and mono-Ub (blue) attached at site 94 as a function of time in the three MD runs; (c) Histograms of the values of angles α and β (solid and dashed lines, respectively) for mono-Ub (blue) and tetra-Ub (red) attached at site 94. (d) Variation in the value of angles α and β (i.e., standard deviation of the distribution of the angle values) for mono-Ub (blue) and tetra-Ub (red) at all the attachment sites examined.

Figure 3

Structural and energetic characterization of the Ub–Ubc7 interface. (a) Distance (Å) between the centers of mass of Ubc7 and the attached Ub moiety at different positions. Mono-Ub and tetra-Ub are designated by blue triangles and red circles, respectively; (b) Ratio of electrostatics (columbic) to van der Waals (vdW) interactions within the Ub–Ubc7 interface at each ubiquitination site. The average ratio is also given for six representative protein–protein complexes from (PDB codes: 1acb, 1dvf, 1mct, 1tgs, 2sni, and 3sgb; solid gray line).

Figure 4

Dynamics of the interfaces in the transient complexes of Ub–Ubc7 (top raw) and in stable complexes (bottom raw). The x- and y- axes of each panel correspond to the interfacial residues in the two proteins of each complex (ordered based on their residue index). These interfacial residues were defined using the modeled structured for the ubiquitinated Ubc7 complexes or using the crystal structures for the protein complexes (PDB IDs 1dvf, 1tgs, 2sni, and 1mct). The numbers in brackets estimated the hydrophobicity of the corresponding patch (estimated by the fraction of hydrophobic residues A, V, L, I, M, F, W, and Y). The matrices show the value of the standard deviation of the pairwise distance between residue i and j from the two proteins that comprise the complexes. The more reddish the color is the more fluctuating is the corresponding pairwise distance.

Figure 5

Geometry and hydration of the Ub–Ubc7 interface: (a) Area (Ų) of the interface between Ubc7 and the tetra-Ub (red circles) or mono-Ub (blue triangles) attached to it at different sites. (b) Number of water molecules per 1000 Ų interfacial area for tetra-Ub –Ubc7 (red circles) and for mono-Ub–Ubc7 (blue triangles). For comparison with the interfaces of other protein–protein interfaces, panels (a) and (b) indicate the interfacial area and hydration found in a bioinformatics survey of protein complexes. In (a) and (b), slanted stripes represent data for interfaces in protein–protein complexes and gray represents interfaces found in crystal packing (see main text). The vertical stripes in (b) represent data for the interfacial area in protein–protein complexes following a molecular dynamic simulation.

Figure 6

Water molecules in the interface between Ubc7 and Lys48-linked tetra-Ub at Lys94 are shown as blue spheres. The image in (b) is rotated 90° relative to the image in (a). Tetra-Ub is depicted in red and Ubc7 is in gray. We defined interfacial water molecules as those within 4.5 Å of the interface atoms of both Ubc7 and tetra-Ub, where interface atoms were those that had lost at least 50% of the value of their accessible surface area.

Figure 7

Top panel: Effect of ubiquitination with tetra-Ub on the internal dynamics of Ubc7. Difference distance matrices (Ubc7^{ubiquitinated} - Ubc7^unmodified) for Lys48-linked tetra-Ub attached at: (a) Lys18; (b) Lys89; (c) Lys94. Bottom panel: Differences between ubiquitinated and unmodified Ubc7 with respect to the standard deviation of their mean inter-residue distances. Lys48-linked tetra-Ub attached at: (d) Lys18; (e) Lys89; (f) Lys94. In both panels, color indicates the difference distances (variations in distance) in Å, with blue representing the compression (reduced variation) and red representing the expansion (higher variation) of some parts of ubiquitinated Ubc7 relative to the unmodified system. Short horizontal and vertical bars indicate the location (residue number) of the ubiquitination site.

Figure 8

Similar to Figure 7 but for ubiquitination with mono-Ub.

Figure 9

Structure and conformational dynamics of ubiquitinated Ubc7. (a) Unmodified Ubc7. (b) Mono-Ub at Lys94. (c) Lys48-linked tetra-Ub at Lys94. Each system is represented by 31 aligned snapshots from three 100 ns simulations sampled every 10 ns from each simulation. Ubc7 is depicted in gray, loop residues 95–105 are depicted in green. The Lys94 residue of Ubc7 and the C-terminal Gly76 of the attached Ub moiety are depicted in orange. The initial position of Ub is shown in the cartoon representation (mono-Ub, blue; Lys48-tetra-Ub, red). Other positions of Ub during the simulation are shown in the surface representation (as blue and red “clouds”).

Figure 10

Comparison of the configurational entropic contributions to the free energy (normalized by the number of residues in the substrate, kcal/mol) in ubiquitinated and unmodified Ubc7. (ΔTS_conf=TS_conf^{ubiquitinated} − TS_conf^unmodified). The configurational entropy was estimated using the Schlitter approximation.