High-Speed Atomic Force Microscopic Observation of ATP-Dependent Rotation of the AAA+ Chaperone p97

Abstract

p97 (also called VCP and CDC-48) is an AAA+ chaperone, which consists of a substrate/cofactor-binding N domain and two ATPase domains (D1 and D2), and forms a homo-hexameric ring. p97 plays crucial roles in a variety of cellular processes such as the ubiquitin-proteasome pathway, the endoplasmic reticulum-associated protein degradation, autophagy, and modulation of protein aggregates. Mutations in human p97 homolog VCP are linked to neurodegenerative diseases. The key mechanism of p97 in these various functions has been proposed to be the disassembly of protein complexes. To understand the molecular mechanism of p97, we studied the conformational changes of hexameric CDC-48.1, a Caenorhabditis elegans p97 homolog, using high-speed atomic force microscopy. In the presence of ATP, the N-D1 ring repeatedly rotates ~23 ± 8° clockwise and resets relative to the D2 ring. Mutational analysis reveals that this rotation is induced by ATP binding to the D2 domain.

Nucleotide‐dependent conformational changes of the AAA+ ATPase p97 revisited

“…Extensive classification revealed that it is structurally homogeneous in the presence of ADP and its six-fold symmetrical, compact structure is essentially identical to that of full-length p97 determined by X-ray crystallography [7] and SAXS [12]. A relative rotation of the D1 and D2 domains, as described in AFM experiments in the presence of ATP or ADP [13], were not seen in any of the cryo-EM maps determined here.…”

“…Indeed, the AMP-PNP density shows a well-defined density segment (Fig. 3B), for which the fitted atomic model suggests that it corresponds to the very N-terminal residues (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. These N-terminal 20 residues are absent from all p97 crystal structures but one protomer of the ATPcS-bound IBMPFD mutant A232E, which includes residues 12-20 [10].…”

“…Low-resolution small-angle X-ray scattering (SAXS) [12], atomic force microscopy [13] and cryo-electron microscopy (EM) studies [14,15] all suggest much larger conformational differences between the different nucleotide states than observed in the full-length X-ray crystallographic structures. A likely source for the discrepancy between the scales of motion observed in the crystallographic and solution data are the extensive contacts between the D2 and N-domains of adjacent particles in the crystal lattices [7,8].…”

Filming Biomolecular Processes by High-Speed Atomic Force Microscopy

“…In fact, the 2D crystal surface has been used for HS-AFM imaging of actin polymerization processes at the plus and minus ends of an actin filament, 37 dynamic association and dissociation between GroEL and GroES, 37 Ca 2+ -induced conformational changes of calmodulin, 37 and ATP-invoked structural changes of an AAA ATPase, p97. 102 For monomeric proteins, the tethering to the surface through a single biotin binding site is locally fixed but can result in a rotational motion around the link. This mobility can however be reduced using reactive dibiotin compounds.…”

Control of p97 function by cofactor binding

“…For example, the radii of gyration of the nucleotide-free, AMP-PNP, ADP-AlF x and ADP states determined by SAXS are 61, 57, 55 and 58 Å, respectively. Furthermore, high speed atomic force microscopy revealed that ATP binding to the D2 domain induces forward clockwise and backward counterclockwise rotational movements (23 ± 8°) between the N-D1 and D2 hexameric rings [31]. The repeated back and forward rotational movements of p97 suggest a possible way of converting the chemical energy stored in ATP into mechanical energy required for the remodeling of substrates (see below).…”