A Mighty "Protein Extractor" of the Cell: Structure and Function of the p97/CDC48 ATPase

Abstract

p97/VCP (known as Cdc48 in S. cerevisiae or TER94 in Drosophila) is one of the most abundant cytosolic ATPases. It is highly conserved from archaebacteria to eukaryotes. In conjunction with a large number of cofactors and adaptors, it couples ATP hydrolysis to segregation of polypeptides from immobile cellular structures such as protein assemblies, membranes, ribosome, and chromatin. This often results in proteasomal degradation of extracted polypeptides. Given the diversity of p97 substrates, this "segregase" activity has profound influence on cellular physiology ranging from protein homeostasis to DNA lesion sensing, and mutations in p97 have been linked to several human diseases. Here we summarize our current understanding of the structure and function of this important cellular machinery and discuss the relevant clinical implications.

Keywords: AAA ATPase; Cdc48; chaperones; neurodegenerative diseases; p97/VCP; protein denaturation; protein quality control.

Figure 1

Structure of p97/Cdc48. (A) Cartoon representation of the domain organization of p97/Cdc48. Color code reflects that for subunit A in (B,C). The ribbon structure shows the D1 domain of a single protomer bound by a ATPγS molecule (PDB:4KO8). The RecA-like domain is colored in light blue and the characteristic helical domain is in cyan. The nucleotide-binding site communicates with a neighboring subunit through the SRH (second region of homology, in red) motif, where a conserved Arg-finger residue R359 is in contact with the bound nucleotide. (B,C) Surface representation of the structure of hexameric p97 (PDB: 3CF2 in the ADP-bound form) (B) is a top view down the 6-fold symmetry axis showing the N-D1 ring. The six subunits are labeled in colors. The D1 domain and the N-domain are indicated with arrows and labeled for one of the six subunits. (C) is a side view of the p97 hexamer.

Figure 2

A nucleotide-dependent N-domain conformational change. (A) When ADP is bound to the D1 domain in ribbon diagram in cyan, the N-domain (in light-blue surface representation) assumes the Down-conformation (PDB: 1E32, wild type N-D1). (B) When ATP is bound to the D1 domain, the N-domain moves to the Up-conformation (PDB: 4KO8, R155H mutant N-D1). (C) A schematic model of the N-D1 conformational change upon D1 ATP hydrolysis. The p97 hexamer is represented as two concentric rings with D1 in blue and D2 in brown. The N-domains in the Down-conformation are shown as magenta balls and their cognate D1 domains are occupied with occluded ADP (labeled D). D1 domains with empty nucleotide-binding pockets are not labeled and their cognate N-domains are likely to be mobile (brown balls). ATP binding to the empty sites of the D1 domains will lead the N-domains to the Up-conformation. Occupation of ATP to the D1 domain renders the cognate D2 domain capable of hydrolyzing ATP, which is labeled with a red ^*. The D1 domain probably hydrolyzes ATP once a few D2 domains have been converted to the ADP bound state.

Figure 3

The domain structures of p97-interacting proteins. Known p97-interacting proteins are grouped by their p97-interacting domains, which are highlighted by colored boxes: UBX, ubiquitin like/ubiquitin regulatory X; UBA, ubiquitin associated; SEP, Shp1, Eyc and p47; VIM, VCP interacting motif; PUB, Peptide:N-glycanase/UBA or UBX-containing proteins; UIM, ubiquitin interacting motif; PAW, domain present in PNGases and other worm proteins; PUL (PLAP, UFD3 and Lub1) domain; PFU, PLAA family ubiquitin binding; RING, really interesting new gene; CUE (Coupling of Ubiquitin to ER-degradation) domain; UIM, ubiquitin interacting motif. The schematic representations are drawn to scale.

Figure 4

The atomic representation of p97 in complex with various representative interacting motifs. (A) The structure of the N-terminal domain of p97 shown as electrostatic potential surface. The positive potential is in blue, negative in red and neutral in white. (B) Structure of the p97 N-domain in complex with the UBX domain of FAF1 (PDB:3QC8). The N-domain, depicted as a molecular surface overlaid to a ribbon representation, has the N-terminal double Y-barrel domain colored green and C-terminal β-barrel domain colored red. The UBX domain of FAF1 is depicted as ribbon diagram in yellow. Critical residues for interaction are shown as ball-and-stick models and labeled. (C) Structure of the p97 N-domain in complex with the VIM motif of gp78 (PDB:3TIW). Here the VIM motif is shown as helix in yellow and its binding to the N-domain is mostly mediated by charged residues. (D) Structure of the p97 N-domain in complex with the Ufd1 derived SHP peptide (PDB:5C1B). Here the SHP peptide is shown as the stick model in yellow and it binds exclusively to the C-terminal β-barrel domain. (E) Structure of the N-terminal domain of PNGase in complex with a C-terminal peptide of p97 (PDB:2HPL). The PNGase N-terminal domain is shown in cartoon representation in yellow. The bound peptide is shown as a stick model with five residues (labeled) seen in the structure. The carbon atoms are colored in black, nitrogen in blue and oxygen in red. (F) Structure of the PUL domain of FLAA/Ufd3 in complex with a C-terminal peptide of p97 (PDB:3EBB). The PLAA PUL domain is shown in cartoon representation in yellow. The bound peptide is shown as a stick model with four residues visible in the structure. The carbon atoms are colored in black, nitrogen in blue and oxygen in red.

Figure 5

Location of pathogenic mutations in the structure of full-length p97. Surface representation of the structure of p97 is decorated with pathogenic mutations in p97 identified from patients of various muscular and neurological disorders. Each subunit is given a unique color with distinct shades for different domains. Interface mutations are colored in black and non-interface mutations in red and labeled. The non-interfacial mutations are mostly identified in ALS patients. (A) Top view. (B) Side view.

Figure 6

Structures of selected p97 inhibitors. Shown are the chemical structure of some well characterized p97 inhibitors. EerI inhibits p97 function by binding to its D1 domain. DBeQ was the first reversible inhibitor that blocks the D2 ATPase activity. CB-5083 is a derivative of DBeQ, but it is much more potent than DBeQ. CB-5083 and NMS-873 are the most potent and specific p97 inhibitor identified to date. UPCDC30245 is a recently identified inhibitor and its binding to p97 has been characterized by EM.