Abstract
The 97-kDa valosin-containing protein (p97-VCP) belongs to the AAA (ATPases associated with various cellular activities) family and acts as a molecular chaperone in diverse cellular events, including ubiquitinproteasome-mediated degradation. We previously showed that VCP contains a substrate-binding domain, N, and two conserved ATPase domains, D1 and D2, of which D2 is responsible for the major enzyme activity. VCP has a barrel-like structure containing two stacked homo-hexameric rings made of the D1 and D2 domains, and this structure is essential for its biological functions. During ATPase cycles, VCP undergoes conformational changes that presumably apply tensions to the bound substrate, leading to the disassembly of protein complexes or unfolding of the substrate. How ATPase activity is coupled with the conformational changes in VCP complex and the D1 and D2 rings is not clear. In this report, we took biochemical approaches to study the structure of VCP in different nucleotide conditions to depict the conformational changes in the ATPase cycles. In contrast to many AAA chaperones that require ATP/ADP to form oligomers, both wild type VCP and ATP-binding site mutants can form hexamers without the addition of nucleotide. This nucleotide-independent hexamerization requires an intact D1 and the down-stream linker sequence of VCP. Tryptophan fluorescence and trypsin digestion analyses showed that ATP/ADP binding induces dramatic conformational changes in VCP. These changes do not require the presence of an intact ATP-binding site in D1 and is thus mainly attributed to the D2 domain. We propose a model whereby D1, although undergoing minor conformational changes, remains as a relatively trypsin-resistant hexameric ring throughout the ATPase cycle, whereas D2 only does so when it binds to ATP or ADP. After ADP is released at the end of the ATP hydrolysis, D2 ring is destabilized and adopts a relatively flexible and open structure.
Highlights
IntroductionAll these activities have been shown to be regulated, directly or indirectly, by the ubiquitin-proteasome degradation pathway
Site-specific mutations were introduced into the Walker A motif of each or both of the ATPase domains, in which a highly conserved lysine was changed to threonine (Fig. 1A)
Hexameric VCP was efficiently reassembled in both conditions [36], ATP/ADP was only detected in the hexamers reassembled in the presence of added nucleotide (Fig. 2C)
Summary
All these activities have been shown to be regulated, directly or indirectly, by the ubiquitin-proteasome degradation pathway This notion suggests that VCP may play a fundamental role in the degradation pathway that underlies all these seemingly unrelated functions. Previous reports have shown that the two ATPase domains of type II AAA proteins are different from each other with respect to sequence and function. A crystallography study of an ADP-bound N-D1 domain provided significant structural details of the D1 ring [34], very little was known about the D2 ring and the conformation of respective rings during the ATPase cycle. ATP/ADP binding induces dramatic conformational changes in D2 Both intrinsic Trp fluorescence and limited digestion studies suggest that D2 exhibits a relatively relaxed structure in the absence of nucleotide but forms a compact hexameric ring in the presence of ATP/ADP. In general agreement with their findings, our study, using an independent, biochemical approach, provides additional molecular insights to the conformations of VCP during ATPase cycle
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