A new crystal form of Lys48-linked diubiquitin

Abstract

Lys48-linked polyubiquitin chains are recognized by the proteasome as a tag for the degradation of the attached substrates. Here, a new crystal form of Lys48-linked diubiquitin (Ub2) was obtained and the crystal structure was refined to 1.6 A resolution. The structure reveals an ordered isopeptide bond in a trans configuration. All three molecules in the asymmetric unit were in the same closed conformation, in which the hydrophobic patches of both the distal and the proximal moieties interact with each other. Despite the different crystallization conditions and different crystal packing, the new crystal structure of Ub2 is similar to the previously published structure of diubiquitin, but differences are observed in the conformation of the flexible isopeptide linkage.

Figure 1

Crystal structure of Lys48-linked Ub₂. (a) Cartoon representation of a Ub₂ molecule in the crystal structure. The proximal and distal moieties are coloured magenta and cyan, respectively. The atoms forming the isopeptide bond as well as the interface residues Ile44 and Val70 are shown as sticks. Residues labelled with primes belong to the distal moiety. (b) Close-up view of the residues forming the interface between the distal and proximal subunits. The molecular surface of the proximal subunit is displayed in transparent white. (c) Cross-eye stereoview ribbon display of the overlaid Ub₂ crystal structures. The three chains in the new crystal structure are shaded yellow, blue and red for A–B, C–D and E–F, respectively. The previously reported crystal structure of Ub₂ is shaded in magenta (PDB code 1aar; Cook et al., 1992 ▶). Residues that have different conformations in different subunits are labelled. The disordered C-termini of the proximal moieties are labelled ‘C’.

Figure 2

Conformation of the isopeptide bond in the crystal structure of Ub₂. (a) Cross-eye stereoview of the σ_A-weighted 2F _o − F _c electron-density map at the isopeptide linkage contoured in blue at 0.35 e Å⁻³. The atomic model is drawn as sticks. Water molecules are drawn as red spheres. (b) The three Lys48-linked Ub₂ molecules in one asymmetric unit are coloured yellow for chains A–B, blue for chains C–D and red for chains E–F. Distal (A, C and E) and proximal (B, D and F) ubiquitin moieties are distinguished by pale and dark shades, respectively. Chains C′ and D′ are from an adjacent asymmetric unit and are labelled in pale and dark cyan, respectively. The isopeptide linkages are shown as spheres coloured by atom type (white, carbon; blue, nitrogen; red, oxygen). (c) Cross-eye stereoview of the isopeptide bond and its interactions. Residues labelled with primes belong to a distal moiety. Hydrogen bonds are shown as dashed lines. C atoms of chains A–B and E–F are shown in yellow and salmon red, respectively.

Figure 3

Comparison of loop conformations in different Ub₂ crystal structures. (a) Comparison of the isopeptide-bond conformation in the two Ub₂ crystal structures. Chains A–B of the new crystal structure are coloured yellow and the previous structure (PDB code 1aar; Cook et al., 1992 ▶) is coloured magenta. Residues labelled with primes belong to a distal moiety. The conformation of the isopeptide bond in chains C–D and E–F is similar to that in chains A–B. (b) Comparison of the β1–β2 loop conformation in chain B (yellow) and the previous crystal structure (magenta). The conformation of this loop in chains C–D and E–F of the new structure is similar to that shown in magenta.