Abstract

p97/VCP is a type II AAA ATPase that forms a homohexamer with each protomer containing an N-terminal domain, two ATPase domains, D1 and D2, and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease Inclusion Body Myopathy associated with Paget Disease of Bone and Frontotemporal Dementia (IBMPFD), the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N- domain with D1. Here we have tested ten major IBMPFD-linked mutants for ATPase activity and found that all have increased activity over wild type, with one mutant, p97A232E, having three times higher activity. Further mutagenesis of p97A232E shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a co-planar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97A232E suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N- domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive.