Structural and functional analysis of p47 cofactor binding on the p97 disease mutant.

Abstract

Human p97/VCP, an AAA+ ATPase, plays a pivotal role in the regulation of multiple cellular activities by interacting with various cofactor proteins. p97 is involved in the ubiquitin-dependent protein quality control and regulation of membrane-fusion in the Golgi apparatus in the presence of cofactor p47. Heterozygous missense mutations of p97 have been implicated in neurodegenerative diseases, such as IBMPFD (Inclusion body myopathy with early-onset Paget's disease and frontotemporal dementia) and ALS. The single amino-acid mutation of R155H on the N-domain is the highest mutated sites, leading to a rare degenerative disease MSP1, and resulting in abnormal ATPase activity and cofactor dysregulation. Our study aims to characterize the unknown protein interactions between p97R155H and p47 cofactor and identify the key complex structure to answer disease relevance of the p97R155H . Biochemical characterization along with single-particle cryo-electron microscopy (cryo-EM). Performed ATPase activity assays and fluorescent labeling to detect the temperature-related intensity change signals for quantitative determination of binding affinities of p97R155H-p47. First cryo-EM structures of the full-length p97R155H-p47 complex in different nucleotide-binding states. Without nucleotides, p97R155H is stable either as a hexamer or stacked as p97R155H-dodecamer. The p97R155H-dodecamer revealed a highly ordered C-terminal tail density and does not bind to p47 or nucleotides. It bears close resemblance to the inhibitor bound CB-5083:p97 structure, implying that the dodecameric form is inactive. In the full-length p97R155H-p47 complex structure without nucleotides, four N-domain densities and one p47UBX domain were identified interacting in an asymmetric manner implying a dynamic equilibrium. For the nucleotide-bound states, the flexibility of the p47UBX bound N-domains were less for the p97R155H|ATP𝛾S-p47, whereas fragmented in p97R155H|ADP-p47. Superimposition of the D1 and D2 domains of the three nucleotide states revealed that the D1-domains exhibit negligible structural change, whereas the D2-HTH motifs of the complexes with nucleotides are tilted downward. The C-terminal tails of the p97R155H|ATP𝛾S-p47 structure points in an opposite direction than that of p97R155H-p47/p97R155H|ADP-p47. The D1 and D2 arginine fingers play a critical role for the elevated p97R155H ATPase activity. These recent findings give us an insight into the disease mechanism of the R155H p97 mutation regarding its role in its pathological functions.