Exploring the linkage dependence of polyubiquitin conformations using molecular modeling

Abstract

Posttranslational modification of proteins by covalent attachment of a small protein ubiquitin (Ub) or a polymeric chain of Ub molecules (called polyubiquitin) is involved in controlling a vast variety of processes in eukaryotic cells. The question of how different polyubiquitin signals are recognized is central to understanding the specificity of various types of polyubiquitination. In polyubiquitin, monomers are linked to each other via an isopeptide bond between the C-terminal glycine of one Ub and a lysine of the other. The functional outcome of polyubiquitination depends on the particular lysine involved in chain formation and appears to rely on linkage-dependent conformation of polyubiquitin. Thus, K48-linked chains, a universal signal for proteasomal degradation, under physiological conditions adopt a closed conformation where functionally important residues L8, I44, and V70 are sequestered at the interface between two adjacent Ub monomers. By contrast, K63-linked chains, which act as a nonproteolytic regulatory signal, adopt an extended conformation that lacks hydrophobic interubiquitin contact. Little is known about the functional roles of the so-called "noncanonical" chains (linked via K6, K11, K27, K29, or K33, or linked head-to-tail), and no structural information on these chains is available, except for information on the crystal structure of the head-to-tail-linked diubiquitin (Ub(2)). In this study, we use molecular modeling to examine whether any of the noncanonical chains can adopt a closed conformation similar to that in K48-linked polyubiquitin. Our results show that the eight possible Ub(2) chains can be divided into two groups: chains linked via K6, K11, K27, or K48 are predicted to form a closed conformation, whereas chains linked via K29, K33, or K63, or linked head-to-tail are unable to form such a contact due to steric occlusion. These predictions are validated by the known structures of K48-, K63-, and head-to-tail-linked chains. Our study also predicts structural models for Ub(2) chains linked via K6, K11, or K27. The implications of these findings for linkage-selective recognition of noncanonical polyubiquitin signals by various receptors are discussed.

Figure 1

Cartoon representations of the best structures of the best cluster for di-ubiquitin chains which are able to form close contacts between the neighboring Ub units. In all these structures, the distal domain is colored blue, the proximal domain is red, the G76-KX linkage between the two Ubs is colored gray. The hydrophobic patch residues L8, I44, and V70 are shown in ball-and-stick, colored yellow.

Figure 2

Cartoon representations of the best structures of the best cluster for di-ubiquitin chains which are not able to form close contacts between the neighboring Ub units. The K48* structure was generated without any interdomain constraints between the hydrophobic patches. In all these structures, the distal domain is colored blue, the proximal domain is red, the G76-KX or G76-M1 linkage between the two Ubs is colored gray. The hydrophobic patch residues L8, I44, and V70 are shown in ball-and-stick, colored yellow. HT indicates the head-to-tail linked chain. Note that these are merely computer-generated models under specific set of restraints, not bona fide structures.

Figure 3

Experimental validation of the HADDOCK-based approach to generate Ub₂ structures used in this study. Shown is the overlay of the crystal structure of K48-linked Ub₂ (red, PDB code 1AAR) with the structures (blue) of this chain generated with (a, b) and without (c, d) constraints that mimic the hydrophobic contact between the L8-I44-V70 patches of the two Ub units. All these structures were aligned by the distal Ub only. Panels b and d show the structures from panels a and c, respectively, rotated by 90° about the vertical axis, to facilitate their comparison. Both the relative position and orientation of the two Ub units in the generated Ub₂ chain in panels a, b are in good agreement with the crystal structure. By contrast, in the K48*-Ub₂ chain (c, d) the interdomain orientation is twisted by 45° compared to the experimental structure.

Figure 4

Principal component analysis of the generated structures. Shown are the loading plots (a, c) and the score plots (b, d). Analysis has been applied to the 12 descriptors (Table 2) averaged over the 10 best structures of the best cluster for each generated chains. The loading plots represent coordinates of each descriptor in the principal axes system of the selected PC components, whereas the score plots represent coordinates of the original data in the PC principal axes system (see Supplementary Material). The score plots clearly show a separation of the data along the PC1 axis, allowing the clustering of Ub₂ chains into two groups. The meaning of the different labels is as follows: E_inter: intermolecular energy; E_vDw: van der Waals energy; E_elec: electrostatic energy; BSA: buried surface area; E_rest: total restraints energy; E_unamb: unambiguous-restraints energy; E_amb: ambiguous-restraints energy; H_bonds: number of interdomain hydrogen bonds at the interface; H_ydro: number of hydrophobic contacts involving residues V8, I44, V70; E_bind: binding free energy; H_score: HADDOCK score; ASA_hp: accessible surface area of residues L8, I44, and V70. HT indicates the head-to-tail linked chain. The solid circle (radius = 1) in panels a and c encompasses the area that includes all the data, while the dashed circle includes 70% of the data. In the geometric sense, the circumferences of these two circles represent projections of a vector that is in-plane or tilted 30° away from the projection plane, respectively. It is usually assumed that variables lying between the two circles are well represented in the corresponding plane.