Figure 2.
Interactions between the Uba1SCCH_ALT and NSC624206. A, middle, the Uba1SCCH_ALT structure is shown as a surface representation in two orientations, and the NSC624206 compound noncovalently bound to the IAD and the 2-(decylamino)ethanethiol fragment covalently bound to the UFD via disulfide bond are shown as sticks. The domains are colored and labeled as in Fig. 1. Left, magnified view of the 2-(decylamino)ethanethiol fragment of NSC624206, shown as sticks, bound to the UFD. Omit 2Fo − Fc electron density for the fragment is shown in green. The UFD and AAD are shown as surface representations, and residues that form contacts with the fragment are colored and labeled. Second from left, magnified view of the 2-(decylamino)ethanethiol fragment, shown as sticks, bound to Cys994 of the UFD, shown as a ribbon diagram. Side chains of residues that form contacts with the fragment are shown as sticks and labeled. The AAD is shown as a surface representation. Second from right, magnified view of the NSC624206 compound, shown as sticks, bound to the IAD, shown as a ribbon diagram. Side chains of residues that form contacts with the compound are shown as sticks and labeled. The IAD and SCCH are shown as surface representations. Right, magnified view of the NSC624206 compound, shown as sticks, bound to the IAD. Omit 2Fo − Fc electron density for the compound is shown in green. The IAD is shown as a surface representation, and residues that form contacts with the fragment are colored and labeled. B, chemical structures of the NSC624206 compound and the 2-(decylamino)ethanethiol fragment. C, crystal contacts with the NSC624206 compound. Top, the Uba1SCCH_ALT structure is shown as a ribbon diagram in two different orientations, and the domains are colored and labeled as in A. Symmetry (sym) mates that form contacts with the 2-(decylamino)ethanethiol fragment, shown as sticks, bound to the UFD (left) and to the NSC624206 compound, shown as sticks, bound to the IAD (right) are also shown as semitransparent ribbon diagrams with the domains colored and labeled. Bottom, magnified views of the 2-(decylamino)ethanethiol fragment bound to the UFD (left) and NSC624206 compound bound to the IAD (right). The side chains of residues from Uba1SCCH_ALT and the symmetry mates that form contacts with the fragment or compound are shown as sticks and labeled. The .x indicates residues residing in symmetry-related Uba1 molecules. D, biochemical analysis of the effect of 2-(decylamino)ethanethiol modification on UFD Cys994. An E1-E2 single turnover thioester transfer assay from Uba1 to Ubc4 was performed with different concentrations of NSC624206 or DMSO (see “Experimental procedures” for details). E, biochemical analysis of the inhibitory effect of NSC624206 on WT and mutant Uba1 activity. Uba1∼Ub thioester formation assays were performed as readouts of Uba1 catalytic activity with different concentrations of NSC624206 or DMSO (see “Experimental procedures” for details).