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

Abstract

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.

Paramagnetic relaxation enhancement‐assisted structural characterization of a partially disordered conformation of ubiquitin

“…We have focused on the structural characterization of the high‐energy conformations of proteins using high‐pressure nuclear magnetic resonance (NMR) spectroscopy . Elevated pressure can shift a population of protein conformers from the basic folded conformer to a fully unfolded conformer because the former has a larger partial molar volume .…”

“…We previously reported that an alternatively folded conformation (N 2 ) accounted for ∼20% of the population of wild‐type ubiquitin at ambient pressure (298 K and pH 7.2), but ∼70% of the population of the ubiquitin Q41N variant (hereafter referred to as Q41N) at ambient pressure, and accounted for 97% of the population at 2500 bar (298 K and pH 7.2) . Because there were only a couple of missing signals in N 2 , we were able to determine the NOE‐based structure of the conformation using high‐pressure NMR …”

High-Pressure NMR Spectroscopy Reveals Functional Sub-states of Ubiquitin and Ubiquitin-Like Proteins

“…We conducted an NOE-based structural determination of the Q41N mutant at 2.5 kbar, where the N 2 state is 97 % populated (Kitazawa et al 2014). This allowed us to characterize the structure of the "pure" N 2 state of ubiquitin.…”

“…This allowed us to characterize the structure of the "pure" N 2 state of ubiquitin. To obtain high-quality structural constraints, we used a ceramic pressure-resistant cell (Daedalus Innovations), which has a tenfold higher signal-to-noise ratio than that , which is a model for N 1 , and Q41N variant at 2.5 kbar (right, PDB entry 2RU6), which is a model for N 2 (Kitazawa et al 2014). The program MOLMOL was used to visualize NMR structures (Koradi et al 1996) of our previous quartz capillary NMR cell.…”

“…Therefore, we strongly suggest that N 2 would be involved in the E1-E2-E3 cascade reaction commonly conserved in the ubiquitin-like post-translational modifiers. N 2 in the structural ensembles of these proteins would thus serve as a prerequisite for the binding of the proteins to their E1, and that the recognition of E1 by the proteins is best explained by conformational selection rather than by induced-fit motion (Kitazawa et al 2014).…”