BPS2026 – Allosteric mechanisms of the AAA+ ATPase VCP/p97 during nucleotide and substrate binding

The ATPase VCP/p97 is a mechanochemical protein that harnesses ATP hydrolysis to pull and unfold substrate proteins. Recent cryo-EM structures of both active and inactive states have enabled simulations of its conformational dynamics during substrate processing. VCP functions as a homo-hexamer, with each subunit comprising 806 amino acids organized into the N-terminal domain (NTD), D1 domain, and D2 domain. The D1 and D2 domains form stacked rings that create a central pore through which substrates are processed. The NTD undergoes conformational switching, adopting an up position when ATP is bound and a down position following hydrolysis. Protein substrate binding induces further conformational rearrangements, including a transition from the planar ring structure to a ladder-like configuration. We performed atomistic microsecond molecular dynamics simulations of full-length VCP to explore these transitions at high temporal resolution. Simulations captured the complete NTD up-down transition and revealed a previously uncharacterized metastable intermediate state. Network analysis of inter-residue communication identified key residues critical for allosteric regulation during the motion. In substrate-bound simulations, five D2 pore loops tightly engaged the substrate, preventing slippage during VCP’s pulling function. Furthermore, we discovered a previously unreported interaction between a flexible D2 loop and the D1 domain during substrate binding, suggesting a new sophisticated allosteric pathway that couples nucleotide state to substrate pulling. The computational findings are complemented with mutagenesis, NMR, and cryo-EM experiments that link molecular interaction networks to VCP function. Together, this study elucidates the conformational pathways of VCP and identifies key residues that coordinate nucleotide- and substrate-dependent allosteric regulation, providing atomistic insight into the mechanisms underlying its protein remodeling activity.