Close Identity between Alternatively Folded State N₂ of Ubiquitin and the Conformation of the Protein Bound to the Ubiquitin-Activating Enzyme

We present the nuclear Overhauser effect-based structure determination of the Q41N variant of ubiquitin at 2500 bar, where the alternatively folded N₂ state is 97% populated. This allows us to characterize the structure of the “pure” N₂ state of ubiquitin. The N₂ state shows a substantial change in the orientation of strand β₅ compared to that of the normal folded N₁ state, which matches the changes seen upon binding of ubiquitin to ubiquitin-activating enzyme E1. The recognition of E1 by ubiquitin is therefore best explained by conformational selection rather than induced-fit motion