Nucleotide Dependent Motion and Mechanism of Action of p97/VCP

Abstract

The AAA (ATPases associated with a variety of cellular activities) family of proteins bind, hydrolyze, and release ATP to effect conformational changes, assembly, or disassembly upon their binding partners and substrate molecules. One of the members of this family, the hexameric p97/valosin-containing protein p97/VCP, is essential for the dislocation of misfolded membrane proteins from the endoplasmic reticulum. Here, we observe large motions and dynamic changes of p97/VCP as it proceeds through the ATP hydrolysis cycle. The analysis is based on crystal structures of four representative ATP hydrolysis states: APO, AMP-PNP, hydrolysis transition state ADP·AlF₃, and ADP bound. Two of the structures presented herein, ADP and AMP-PNP bound, are new structures, and the ADP·AlF₃ structure was re-refined to higher resolution. The largest motions occur at two stages during the hydrolysis cycle: after, but not upon, nucleotide binding and then following nucleotide release. The motions occur primarily in the D2 domain, the D1 α-helical domain, and the N-terminal domain, relative to the relatively stationary and invariant D1α/β domain. In addition to the motions, we observed a transition from a rigid state to a flexible state upon loss of the γ-phosphate group, and a further increase in flexibility within the D2 domains upon nucleotide release. The domains within each protomer of the hexameric p97/VCP deviate from strict 6-fold symmetry, with the more flexible ADP state exhibiting greater asymmetry compared to the relatively rigid ADP·AlF₃ state, suggesting a mechanism of action in which hydrolysis and conformational changes move about the hexamer in a processive fashion.