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.