FIGURE 2.

Interaction of the Ubl domain with Ubc9. a, crystal structure of the NEDD8 E2 (Ubc12) in complex with the Ubl domain is compared with the crystal structure of the Ubl domain of SUMO E1. Both Ubl domains are shown in a similar orientation to illustrate the conserved binding surface for E2. Residues 484 and 485 of the SAE2 subunit, where the Gly-Gly-Gly sequence was inserted, are highlighted in yellow, and the equivalent residues in the NEDD8 E1 are also highlighted in yellow. The Asp-484 side chain, which was deleted, is directed away from the E2-binding surface. The segment that undergoes folding-unfolding equilibrium is indicated in magenta. b, expanded region of the HSQC spectra showing the titration result of unlabeled Ubc9 with the ¹⁵N-labeled SAE2 Ubl domain. Assignments for the major (black letters) and minor (magenta letters) conformations are indicated. The spectra of the complexes of the two proteins with various ratios are overlaid with those of the free Ubl domain (black). Minimal changes were observed when the Ubl:E2 ratio was 3:1 (red). Upon addition of more E2 (2:1 Ubl:E2 ratio, green), there was a significant loss of intensity of the cross-peaks from the major conformer. At a Ubl:E2 ratio of 1:1 (blue), new resonances corresponding to the complex were observed. A further increase in the E2 concentration (magenta) did not alter the spectra but caused greater precipitation.