Figure 1.
Ubiquitin conjugation cascade.
UbA1 consists of four domains: the Adenylation domain (AD), the First Catalytic Cysteine Half-domain (FCCH), the Second Catalytic Cysteine Half-domain (SCCH) and the Ubiquitin Folding Domain (UFD). I) UbA1 catalyzes the adenylation of the Ub C-terminus in an ATP-dependent process in the AD domain; II) the activated Ub forms a thioester bond with a conserved catalytic cysteine in the SCCH domain of UbA1 [Ub(T)]; III) UbA1 is loaded with a second Ub molecule in the AD domain, followed by its C-terminal adenylation [Ub(A)]; IV) the ternary UbA1∼Ub(T)-Ub(A) thioester complex recruits E2 (e.g. UbcH10); V) the thioester-linked Ub is transferred to a conserved E2 cysteine (transthioesterification); VI) E3 mediates the binding of Ub to the target lysine ε-amino groups.
Table 1.
Structural models of hUbA1.
Figure 2.
A) Comparison of the best two binding modes of Ub resulting from HADDOCK calculation, Ha and Hb, shown in yellow and orange, respectively. Terminal Ub-Gly76 and catalytic UbA1-Cys632 are highlighted in spheres coloured by atom type. B) Detail of hUbA1∼Ub(T) interactions in the lowest energy MD frame (time 9.6 ns). Apolar hydrogens were omitted for sake of clarity. Colour code: hUbA1, grey; Ub(T), yellow; van der Waals interactions are highlighted with transparent Connolly surfaces. Carbons are in cyan, nitrogen in blue, oxygens in red, sulphur in yellow and hydrogens in white.
Figure 3.
Model of hUbA1-Ub(T)-Ub(A) complex.
A) Superimposition of the hUbA1∼Ub(T)-Ub(A) model C_Ha on the crystal structure 3CMM_C; B) Detail of the main interactions of Ub(A) and AMP in the hUbA1 loop pocket; C) Detail of the main interactions between Ub(A) and hUbA1: hydrophobic and polar interface. AMP is highlighted in CPK. Apolar hydrogens were omitted for sake of clarity. Colour code: hUbA1, grey; Ub(T) yellow; Ub(A), orange; scUbA1, blue; scUb(A), green.
Table 2.
Comparison of structural and energy data for selected docking results of the quaternary complex.
Table 3.
Alanine scanning.
Figure 4.
A) SIE values (kcal/mol) determined for the E1-E2 interaction along the trajectory (averaged for 20 ns windows). B) Contribution of key residues as derived from alanine scanning in UbcH10 N-terminal helix and the hUbA1 UFD region during the MD simulation of the model C_Ha_R. Colours: Glu1037, orange; Asp1047, violet; Glu1049, light green; Lys33′, bordeaux: Gln36′, blue; Gln37′, dark green.
Figure 5.
Model of the hUbA1-Ub(T)-Ub(A)-UbcH10 complex.
A) Average structure of last 20 ns of MD simulation of the C_Ha_R model. The catalytic cysteines, the thioester bond and AMP were highlighted in spheres. Detail of interactions between B) UbcH10 and SCCH region, C) UbcH10 and Ub(T) and D) UbcH10 and UFD region. Catalytic cysteins and the thioester bond were highlighted in CPK. Apolar hydrogens were omitted for the sake of clarity. Colour code: hUbA1, grey; scUbA1, blue; Ub(T) yellow; Ub(A), orange; UbcH10, violet. The van der Waals interactions are highlighted with transparent Connolly surfaces.
Figure 6.
Final refined model of the tetrameric complex.
A) Average structure of the last 20 ns of MD simulation of the model after SMD. B) Detail of the UbcH10-Cys-cap loop interactions. C) Detail of UbcH10-Cys region involved in hydrophobic interactions; D) Detail of the UbcH10-Cys region involved in polar interactions; E) Detail of the hydrophobic interactions between hUbA1 UFD and UbcH10. F) Detail of the polar interactions between hUbA1 UFD and UbcH10. Colour code: hUbA1, grey; Ub(T) yellow; Ub(A), orange; UbcH10, violet. Catalytic cysteins were highlighted in spheres. Apolar hydrogens were omitted for the sake of clarity. The van der Waals interactions are highlighted with transparent Connolly surfaces.
Figure 7.
Rotation of the Ub(T) induced by UbcH10 interaction in the final quaternary model with respect to the UbA1∼Ub(T) model.
Superimposition was made on the Cα atoms of the SCCH and FCCH domains. Colour code: Final quaternary complex model: hUbA1, grey; UbA1∼Ub(T) model: UbA1, black; Ub(T), magenta; Arg74 in the two models is shown as licorice. Apolar hydrogens were omitted for sake of clarity.
Table 4.
Sequence and binding assays results of the designed peptides.