Conformational Switching in Proteasomal ATPases Allows for Proper Substrate Processing

Virtually every cellular process relies on properly regulated protein degradation, and thus, improper regulation of this degradation can lead to a wide range of human diseases (e.g. Neurodegeneration, Cancers, Cardiomyopathies). The ubiquitin‐proteasome system is responsible for the majority of targeted degradation within eukaryotes, so it is critical to understand how this system functions to degrade proteins. However, due in part to the size and complexity of the proteasome, we still do not understand how it functions at a molecular level. The 26S proteasome from eukaryotes is a 2.5 megadalton macromolecular complex composed of 2 subcomplexes: a hollow 20S core particle with protease sites sequestered on its interior, and a 19S regulatory particle that caps one or both ends of the 20S. Proteins destined for degradation (e.g. polyubiquitinated proteins) bind and are recognized by the 19S regulatory particle, and then in an ATP‐dependent manner are unfolded and injected into the 20S core particle, where proteins are then degraded. The 19S ATPase is a heterohexamer, and although each of the 6 subunits are highly similar to one another, recent cryo‐EM structures reveal that that these subunits form an asymmetric “lockwasher” conformation. However the structural elements that give rise to this asymmetric conformation are unknown, and may come from the hetero‐oligomeric subunit arrangement of the ATPase ring. Furthermore, cryo‐EM structures reveal different conformations of the 19S ATPases in the ADP (i.e. ATPh) or ATPgS‐bound states. In the present study, we employ an exhaustive crosslinking approach to show that Proteasome Activating Nucleotidase (PAN), a homolog of the 19S ATPases, adopts similar conformations as the 19S ATPases in the ATP‐ and ADP‐bound states, even though PAN is composed of 6 identical subunits (i.e. a homohexamer). Thus, the asymmetrical state of the proteasomal ATPases appears to be an inherent property of the ATPase subunit and its allosteries that arise from oligomerization rather than the heteroligomeric nature of the 19S ATPases. Most interestingly, in the conformationally restricted variants that we designed we observed functional defects when the subunits are prevented from switching out of the different nucleotide‐bound conformations, suggesting that the asymmetrical subunit switching observed in the cryo‐EM structures is part of the proteasomal ATPase's normal mechanochemical cycle that allows for proper processing of substrates. Support or Funding Information NIH‐R01GM107129, NIH‐F31GM115171