D1 Ring Is Stable and Nucleotide-independent, whereas D2 Ring Undergoes Major Conformational Changes during the ATPase Cycle of p97-VCP

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.

Hereditary Inclusion Body Myopathy-Linked p97/VCP Mutations in the NH2 Domain and the D1 Ring Modulate p97/VCP ATPase Activity and D2 Ring Conformation

“…Our results indicate that the Arg 155 and Ala 232 mutations influence distant residues within the p97 structure, a fact consistent with the dynamic nature of p97 interdomain communication (21,22). Considering that Trp 476 fluorescence has been used to report ATP-dependent conformational changes within the D2 ring (27), our data suggest that p97 R155P and p97 A232E display different D2 ring conformations than p97…”

“…3 and Table 2). In this assay, the change in protein fluorescence is inversely related to the hydrophobicity of tryptophan 476 (Trp 476 ) within the D2 ring (27). In the absence of ATP binding, only p97…”

Lysine methylation of VCP by a member of a novel human protein methyltransferase family

“…The selected interaction domain is located in the D1 ATPase domain and appears inaccessible in the VCP hexamer structure; therefore, a VCP deletion mutant (VCP∆D2) that lacks the D2 ATPase domain (Fig. 2b) and forms a less stable hexamer 13,14 was also tested. Indeed, both VCP and VCP∆D2 were methylated by METTL21D in vitro, but considerably higher levels of methylation were obtained in the case of VCP∆D2 (Fig.…”

Valosin-Containing Protein Cleavage by Granzyme K Accelerates an Endoplasmic Reticulum Stress Leading to Caspase-Independent Cytotoxicity of Target Tumor Cells

“…The VCP molecule consists of an N-terminal domain and two ATPase domains named D1 and D2, and these two ATPase domains have different roles in ATPase activity. The D2 domain accounts for the major ATPase activity (15,28). Residue Arg 713 in the GzmK cleavage site is located in a disordered loop (705-731) of the D2 domain.…”

“…VCP has two ATPase activity domains named D1 and D2 (15). VCP is a highly evolutionarily conserved protein (16,17).…”