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Probing Cooperativity of N‐Terminal Domain Orientations in the p97 Molecular Machine: Synergy Between NMR Spectroscopy and Cryo‐EM
Author(s) -
Huang Rui,
Ripstein Zev A.,
Rubinstein John L.,
Kay Lewis E.
Publication year - 2020
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202009767
Subject(s) - cooperativity , atp hydrolysis , nuclear magnetic resonance spectroscopy , structural biology , chemistry , mutant , crystallography , biophysics , protein subunit , biochemistry , stereochemistry , enzyme , biology , atpase , gene
The hexameric p97 enzyme plays an integral role in cellular homeostasis. Large changes to the orientation of its N‐terminal domains (NTDs), corresponding to NTD‐down (p97‐ADP) or NTD‐up (p97‐ATP), accompany ATP hydrolysis. The NTDs in a series of p97 disease mutants interconvert rapidly between up and down conformations when p97 is in the ADP‐bound state. While the populations of up and down NTDs can be determined from bulk measurements, information about the cooperativity of the transition between conformations is lacking. Here we use cryo‐EM to determine populations of the 14 unique up/down NTD states of the homo‐hexameric R95G disease‐causing p97 ring, showing that NTD orientations do not depend on those of neighboring subunits. In contrast, NMR studies establish that inter‐protomer cooperativity is important for regulating the orientation of NTDs in p97 particles comprising mixtures of different subunits, such as wild‐type and R95G, emphasizing the synergy between cryo‐EM and NMR in establishing how the components of p97 function.