ATPases associated with various cellular activities (AAA+) form a superfamily of ring-shaped motor proteins that utilize cyclical allosteric motions to remodel or translocate substrate proteins (SP) through a narrow central pore. The p97 ATPase is a homohexameric, double-ring member of this superfamily that encloses a central channel with nonuniform width. A narrow compartment is present within the D1 ring and a larger cavity within the D2 ring, separated by a constriction formed by six His amino acids. We use molecular dynamics simulations to probe the interaction between p97 and an extended peptide substrate. Mechanical pulling of the substrate through the p97 pore reveals that smaller work is required for translocation from the D1 toward the D2 compartment than in the opposite direction. These distinct energetic requirements originate in structural aspects and chemical properties of the pore lining. Whereas van der Waals interactions are dominant within the D1 pore, interaction within the D2 pore are strongly electrostatic. Two charged amino acids in the D2 pore, Arg599 and Glu554, provide the largest contribution to the interaction and hinder translocation from the D2 pore. SP threading requires smaller forces when the SP is pulled from the D1 side due to lower barrier to rotation of the His side chains in the direction of the D2 pore. Based on additional simulations of SP binding to two allosteric conformations of p97, we propose that transient binding and release of SP from the pore involves a lever mechanism. Binding to the open pore conformation of p97 occurs primarily at the Arg599 side chain, where the SP backbone is engaged through electrostatic interactions and hydrogen bonds. ATP driven conformational transitions within the D2 ring alter the chemical environment inside the p97 cavity in the closed pore state. In this state, Glu554 side chains project further into the pore and interacts strongly through van der Waals contacts with the SP backbone. Based on mutations at the two sites in each of the states we identify a specific requirement of these side chains for interaction with the substrate.