Abstract
The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders, such as MultiSystem Proteinopathy 1 (MSP-1) and Familial Amyotrophic Lateral Sclerosis (ALS). However, the structural basis of its role in such diseases remains elusive. 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, a potent p97 inhibitor. Our cryo-EM structures demonstrate that these mutations affect nucleotide-driven allosteric activation across the three principal p97 domains (N, D1, and D2) by predominantly interfering with either (1) the coupling between the D1 and N-terminal domains (p97R155H and p97R191Q), (2) the interprotomer interactions (p97A232E), or (3) 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 those seen for mutations that affect coupling between the 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 and have potential implications for the design of novel allosteric compounds that can modulate p97 function.
Highlights
P97 is a member of the classic clade of AAA+ ATPases and is an essential cellular enzyme. p97 is composed of six protomers forming a homo-hexamer with an N-terminal domain (NTD) and two tandem ATPase domains D1 and D2 [1]. p97 is an essential protein in regulating cellular homeostasis from membrane fusion, Endoplasmic Reticulum-Associated Degradation, Mitochondrial-Associated Degradation (MAD), Chromatin-Associated Degradation to NF-κB activation [2]
The majority of p97 mutations are associated with a rare disease called Multi System Proteinopathies-1 (MSP-1), which is characterized by a cluster of muscular, bone, and neurological illnesses known as Inclusion Body Myopathy; Paget’s Disease of the Bone (PDB); and FrontoTemporal Dementia (FTD) [11]
The ADP-bound conformation of the D1 domain displays many of the structural signatures observed only upon ATPγS binding to p97WT, suggesting that these mutations shift the conformational equilibrium toward promoting ATP binding
Summary
P97 is a member of the classic clade of AAA+ ATPases and is an essential cellular enzyme. p97 is composed of six protomers forming a homo-hexamer with an N-terminal domain (NTD) and two tandem ATPase domains D1 and D2 [1]. p97 is an essential protein in regulating cellular homeostasis from membrane fusion, Endoplasmic Reticulum-Associated Degradation, Mitochondrial-Associated Degradation (MAD), Chromatin-Associated Degradation to NF-κB activation [2]. Critical functions for p97 include the translocation and restructuring of proteins from large cellular structures such as organelle membranes and extraction of ubiquitinated client proteins to facilitate their degradation through the ubiquitin-proteasome system In this role, p97 recruits ubiquitin-binding cofactors to denature ubiquitinated substrates by pulling the polypeptide through the central pore [3, 4]. As these mutations tend to cluster in the Nand D1-domains of the p97 protein and the majority of p97 cofactors bind the N-terminus of the protein, there is likely cofactor involvement in the mechanism of disease These N-D1 mutants are commonly characterized by an increase in basal ATPase activity [12] and higher substrate processing relative to wild-type p97 [13], these effects have not been shown to be directly correlated. We have examined full-length forms of five p97 mutants: three were chosen for their relatively high incidence in the p97-related neurodegenerative disorder MSP-1 (R155H, R191Q, A232E), one for its relatively unusual position in the D2 domain in p97 and its role in familial ALS (D592N), and one for its identification in resistance to the drug CB-5083 (E470D) [15]
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