Figure 1.
A schematic illustration of the ubiquitin-proteasome system (UPS).
(A) Overview of the ubiquitin protein modification pathway. (B) The Ub-E2-E3-substrate machinery.
Figure 2.
Structure superimposition of substrate-binding proteins.
(A) Superposition of Elongin C (purple, violet) and VHL-box/SOCS-box of pVHL (Cyan) (PDB code 1lm8) and SOCS2 (pink) (PDB code 2c9w). The substrate binding domain of pVHL and SOCS2 are different, but their substrate binding sites (blue and red as circled) overlap. (B) Superposition of Skp1 (blue, red, orange or green) and F-box of Skp2 (Cyan) (PDB code 2ast), Fbw7 (pink) (PDB code 2ovq), Cdc4 (yellow) (PDB code 1nex) and Fbs1 (lime) (PDB code 2e31). Their substrate binding sites (blue, red, orange or green) overlap.
Figure 3.
Comparison of snapshots from the simulations.
(A) Skp2; (B) Fbw7; (C) β-TrCP1; (D) Cdc4; (E) Fbs1; (F) TIR1; (G) pVHL; (H) SOCS2; (I) SOCS4. The unbound form trajectory 1(left), trajectory 2 (middle) and bound form (right) comparison is shown in each figure. Structural snapshots of the F-box, VHL-box and SOCS-box domains are superimposed. The snapshots are taken at 0 ns (orange for substrate binding proteins and blue for adaptor Skp1, ASK1 or Elongin C) and maximum rotation angle (green). The rotation angles of the substrate binding domain are shown.
Figure 4.
Rotation angles of Skp2 unbound (black) and bound (red) forms during the simulations.
Figure 5.
Conserved prolines in the linker region.
(A) Skp2 proline puckering up and down is coupled with backbone conformational change. Two snapshots from simulations with prolines puckering up and down were superimposed and the backbone rotations are shown. (B) Superposition of pVHL (Cyan), SOCS2 (pink), and SOCS4 (orange) box domain with prolines at the linker. (C) Superposition of Skp2 (Cyan), Fbw7 (pink), β-TrCP1 (orange), Cdc4 (yellow), Fbs1 (purple) and TIR1 (green) with prolines at the linker. (D) Sequence alignment of pVHL, SOCS2 and SOCS4. (E) Sequence alignment of Skp2, Fbw7, β-TrCP1, Cdc4, Fbs1 and TIR1.
Table 1.
The ratio of down/up proline pucker in the unbound versus bound simulations.
Figure 6.
Model of the E2-Rbx1-Cul1-Skp1-Skp2 complex.
E2 (purple, PDB code 1fbv) is docked to Rbx1(gray)-Cul1(blue)-Skp1(red)-Skp2 F-box (yellow) complex (PDB code 1ldk). Skp2 snapshots at 0 ns (orange) and 20 ns (green) for unbound form and 20 ns (cyan) for bound form are superimposed on the crystal structure using the F-box domain as the pivot.
Figure 7.
A scheme of the proposed pathway.
(A) Prior to binding to other E3 modules, the linker is flexible. (B) In the favored E3-bound conformation the substrate binding domain is rotated on the linker to the optimal position. (C) The strong correlations in the motions between the linker and the substrate binding domain in the bound state, suggest allosteric effects with the linker further rotating the substrate binding domain following substrate binding for optimal ubiquitin transfer position. (D) The linker rotates to facilitate additional ubiquitin transfer. (E) The linker rotation facilitates the poly-ubiquitin-labeled substrate dissociation from the E3 ligase.