Interaction between the AAA+ ATPase p97 and its cofactor ataxin3 in health and disease: Nucleotide-induced conformational changes regulate cofactor binding

Abstract

p97 is an essential ATPase associated with various cellular activities (AAA) that functions as a segregase in diverse cellular processes, including the maintenance of proteostasis. p97 interacts with different cofactors that target it to distinct pathways; an important example is the deubiquitinase ataxin3, which collaborates with p97 in endoplasmic reticulum-associated degradation. However, the molecular details of this interaction have been unclear. Here, we characterized the binding of ataxin3 to p97, showing that ataxin3 binds with low-micromolar affinity to both wild-type p97 and mutants linked to degenerative disorders known as multisystem proteinopathy 1 (MSP1); we further showed that the stoichiometry of binding is one ataxin3 molecule per p97 hexamer. We mapped the binding determinants on each protein, demonstrating that ataxin3's p97/VCP-binding motif interacts with the inter-lobe cleft in the N-domain of p97. We also probed the nucleotide dependence of this interaction, confirming that ataxin3 and p97 associate in the presence of ATP and in the absence of nucleotide, but not in the presence of ADP. Our experiments suggest that an ADP-driven downward movement of the p97 N-terminal domain dislodges ataxin3 by inducing a steric clash between the D1-domain and ataxin3's C terminus. In contrast, MSP1 mutants of p97 bind ataxin3 irrespective of their nucleotide state, indicating a failure by these mutants to translate ADP binding into a movement of the N-terminal domain. Our model provides a mechanistic explanation for how nucleotides regulate the p97-ataxin3 interaction and why atypical cofactor binding is observed with MSP1 mutants.

The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

“…Here, we report that p97 and ATX3 (Rao et al, 2017) form a constitutive physical complex with RNF8. The p97-ATX3 complex safeguards the soluble pool of RNF8 under physiological conditions.…”

A strategy to study intrinsically mixed folded proteins: The structure in solution of ataxin-3

“…In conclusion, the ability to study the structural behaviour of this protein paves the way to the investigation of interactions with other proteins. An obvious candidate is the interaction with the valosin-containing protein which was mapped in the basic motif RKRR in the C-terminal tail (38). Our work will also potentially be important for the advance of ataxin-3 aggregation since a number of studies have shown that the regions immediately N-and C-terminal to the polyQ repeats play an important role in modulating protein self-assembly (39)(40)(41)(42)(43)(44).…”

The Structural Properties in Solution of the Intrinsically Mixed Folded Protein Ataxin-3

“…The intrinsically unfolded nature of the C-terminal tail may help tune the affinities of the interaction. The helicity of the region preceding the polyQ tract will instead promote interactions with other proteins such as valosin-containing protein, whose binding site is supposed to be located in the region that includes the basic motif RKRR in the C-terminal tail (72).…”