Multisystem Proteinopathy Mutations in VCP/p97 Increase NPLOC4·UFD1L Binding and Substrate Processing

Highlights

• p97 mutants exhibit accelerated NPLOC4·UFD1L (UN)-dependent substrate unfolding

• Mutations increase UN affinity but do not affect ATPase activity during unfolding

• p97 mutant N-terminal domain conformations are decoupled from nucleotide state

• Data support a gain-of-function model for p97·UN in multisystem proteinopathy

Summary

Valosin-containing protein (VCP)/p97 is an essential ATP-dependent protein unfoldase. Dominant mutations in p97 cause multisystem proteinopathy (MSP), a disease affecting the brain, muscle, and bone. Despite the identification of numerous pathways that are perturbed in MSP, the molecular-level defects of these p97 mutants are not completely understood. Here, we use biochemistry and cryoelectron microscopy to explore the effects of MSP mutations on the unfoldase activity of p97 in complex with its substrate adaptor NPLOC4⋅UFD1L (UN). We show that all seven analyzed MSP mutants unfold substrates faster. Mutant homo- and heterohexamers exhibit tighter UN binding and faster substrate processing. Our structural studies suggest that the increased UN affinity originates from a decoupling of p97's nucleotide state and the positioning of its N-terminal domains. Together, our data support a gain-of-function model for p97-UN-dependent processes in MSP and underscore the importance of N-terminal domain movements for adaptor recruitment and substrate processing by p97.