Fig 1.
Schematic, NMR-derived docked model, and crystal structure of the Ube2S-ubiquitin conjugate.
(A) Schematic of the critical step in ubiquitin linkage formation by E2 enzymes. The C-terminal carbonyl group of a donor ubiquitin that is thioesterified with the E2 active site cysteine undergoes a nucleophilic attack by a primary amino group of an acceptor ubiquitin. As a result, an isopeptide bond between donor and acceptor ubiquitin is formed. (B) NMR-derived docked model of the Ube2S-donor ubiquitin complex [6]. The catalytic UBC domain of Ube2S (yellow) and ubiquitin (blue) are shown in cartoon representation. The C-terminal carbonyl group of ubiquitin and the catalytic cysteine side chain of Ube2S are displayed in ball-and-stick mode. (C) Crystal structure of Ube2S C118M (orange) disulfide-linked to ubiquitin G76C (light green). The cysteine side chains forming the disulfide linkage are highlighted in ball-and-stick mode. Note that contacts between the two proteins (in cis) are limited to the vicinity of the disulfide linkage. In the crystal, however, Ube2S forms an interface with a second ubiquitin molecule (dark green) in trans. (D) The crystallographic complex formed in trans between Ube2S (orange) and a neighboring ubiquitin molecule (green) is superposed with the NMR-derived docked model of Ube2S (yellow) and donor ubiquitin (blue) [6]. The catalytic cysteine side chains are shown in ball-and-stick rendition. The angle by which the axes of helix α1 of ubiquitin are pivoted with respect to each other in the two configurations is indicated.
Table 1.
Structure determination and refinement.
Fig 2.
Comparison of donor recognition between the crystal structure and the NMR-derived docked model.
(A) Open-book view of the interaction “footprint” on the surface of Ube2S and ubiquitin (residues 1–71) in the crystal structure (top) and in the docked model (bottom), respectively, as defined by residues that become ≥ 10% buried at the interface. The C-terminal tail of ubiquitin (residues 72–76) was omitted in this analysis and representation. (B) Details of the Ube2S-donor ubiquitin interfaces seen in the crystal structure (top) and the docked model (bottom), respectively. Key interfacial residues are displayed as balls-and-sticks. Note that Lys 117 of Ube2S and Thr 66 of ubiquitin do not make contacts in the docked model and are displayed for comparison only. Cys 118 is replaced by methionine in the crystal structure.
Fig 3.
Effect of Lys 117 of Ube2S on activity.
(A) Detail of the structural superposition of the docked model and the crystal structure provided in Fig 1D. The Ube2S molecules are shown in yellow and orange, respectively; the ubiquitin molecules in blue and green, respectively. In the crystal structure the sidechain amino group of Lys 117 of Ube2S forms a hydrogen bond with the backbone oxygen atom of Leu 8 of ubiquitin (as indicated by the dotted line), while Lys 117 is removed from the donor interface in the docked model (see Fig 2B). (B) In vitro activity assays monitoring diubiquitin formation by full-length Ube2S (left) and the UBC domain of Ube2S (right). We compared the corresponding wildtype proteins with the K117A variants in the absence (-) and presence (+) of ATP. The K117A substitution results in decreased diubiquitin formation.
Fig 4.
Molecular dynamics simulations.
(A) Five independent trajectories (colored differently) of the crystal structure over 100 ns each. For each simulation the Ube2S molecules were aligned and the Cα RMSD values for ubiquitin (in Å) with respect to the crystal structure are plotted over the time. (B) Eight independent trajectories (colored differently) of the docked model over 100 ns each. For each trajectory the Cα RMSD values for ubiquitin (in Å) with respect to the starting model after aligning the Ube2S molecules are plotted. (C) For each trajectory of the docked model (see (B)), the Cα RMSD values for ubiquitin (in Å) with respect to the crystal structure after aligning the Ube2S molecules are plotted. In three trajectories (colored in red, dark blue, and green) the model converges to a configuration that is very similar to the crystal structure. (D) Superposition of the docked model after 100 ns of simulation time (see (B) and (C), green simulation run) with the crystal structure.