Abstract
A computational study of p97/VCP ATPase using hybrid quantum mechanics/molecular mechanics (QM/MM) simulations is presented that explores the conformational landscape of the active site and hydrolysis-competent states of the crystallographic water molecules. Our investigation focuses on the reaction mechanism, particularly the events of the rate-determining first reaction step, which we study using extensive sampling with the path well-tempered metadynamics extended-system adaptive biasing force (WTM-eABF) enhanced sampling method. We identify the highly conserved glutamate (Glu305) from the Walker B motif as a catalytic base that activates the lytic water molecule for nucleophilic attack on the γ-phosphate in the first reaction step, while the final product is formed in a second step that involves proton transfer and rearrangements in the Mg(2+) coordination sphere. We show that phosphate bond formation and breakage occur concertedly in the first reaction step. The findings gained through versatile QM/MM approaches are validated against recent cryo-EM and NMR data for the post-hydrolysis protein state, elucidating the role of amino acids from conserved motifs across the AAA+ protein family. To the best of our knowledge, this is the first in silico exploration of ATP hydrolysis in p97/VCP or any other AAA+ protein.