Multi-state structure determination and dynamics analysis reveals a new ubiquitin-recognition mechanism in yeast ubiquitin C-terminal hydrolase

Despite accumulating evidence that protein dynamics is indispensable for understanding the structural basis of biological activities, it remains challenging to visualize the spatial description of the dynamics and to associate transient conformations with their molecular functions. We have developed a new NMR protein structure determination method for the inference of multi-state conformations using multiple types of NMR data, including paramagnetic NMR and residual dipolar couplings, as well as conventional NOEs. Integration of these data in the structure calculation permits delineating accurate ensemble structures of biomacromolecules. Applying the method to the protein yeast ubiquitin hydrolase 1 (YUH1), we find large dynamics of its N-terminus and crossover loop surrounding the active site for ubiquitin-recognition and proteolysis. The N-terminus gets into and out of the crossover loop, suggesting their underlying functional significance. Our results, including those from biochemical analysis, show that large motion surrounding the active site contributes strongly to the efficiency of the enzymatic activity.