Cryo-EM reveals disrupted human p97 allosteric activation by disease mutations and inhibitor binding

The human AAA ATPase p97, a potential cancer target, plays a vital role in clearing misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders. Here, we present cryo-EM structural analyses of four disease mutants p97R155H, p97R191Q, p97A232E, p97D592N, as well as p97E470D, implicated in resistance to the drug CB-5083. These structures demonstrate that the mutations affect nucleotide-driven p97 allosteric activation by predominantly interfering with either the coupling between the D1 and N-terminal domains (p97R155H and p97R191Q), the inter-protomer interactions (p97A232E), or the coupling between D1 and D2 nucleotide domains (p97D592N, p97E470D). We also show that binding of the competitive inhibitor CB-5083 to the D2 domain prevents conformational changes similar to that seen for mutations that affect coupling between D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations. ### Competing Interest Statement SS is Founder and CEO of Gandeeva Therapeutics Inc., a drug discovery company based in Vancouver.