Abstract
IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97R155H with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97R155H mutant all show up configurations in ADP- or ATPγS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97R155H ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97R155H.
Highlights
Human p97/VCP belongs to the type II AAA+ (ATPase Associated with various cellular Activities) protein family, which has two AAA+ ATPase domains [1,2]
The orthologs are known as Cdc48 in Saccharomyces cerevisiae and transitional endoplasmic reticulum (TER) ATPase in archaebacteria and eukaryotes. p97 acts as a molecular hub, interacting with various cofactors to perform a wide variety of cellular functions, including autophagy, cell-cycle regulation, ubiquitin-dependent proteostasis, and reassembly of Golgi and nuclear membranes [3,4,5,6,7]. p97 is abundant in the cytosol, comprising 1% of the cytosolic proteins [8,9], and carries out ATP hydrolysis to gain energy to fuel the conformational change underlying its activities [10,11,12,13]
Single amino acid mutations in p97 have long been linked to diseases, including IBMPFD and amyotrophic lateral sclerosis (ALS) [71], and these disease mutants alter the p97 ATPase activity and cofactor binding on the N-terminal domain (NTD) [45]
Summary
Human p97/VCP (valosin-containing protein) belongs to the type II AAA+ (ATPase Associated with various cellular Activities) protein family, which has two AAA+ ATPase domains [1,2]. Previous cryogenic electron microscopic (cryo-EM) structures of the AAA+ ATPases revealed that the substrate processing by the Cdc ATPase results in a broken ring and an asymmetric structure of the six p97 monomers These structures have been used to suggest a hand-over-hand model for the substrate’s induced motion [30,31,32]. The substrate movement along the central channel is dependent on the D1 nucleotide state and is mainly driven by the ATP hydrolysis on the D2 domain [33,34] This movement would call for cooperation between domains within the AAA+ ATPase complex and cofactor bindings [10,35,36]. It is not certain whether the hand-over-hand model applies to other p97 functions, such as membrane fusion
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