Mechanism of Transient Binding and Release of Substrate Protein during the Allosteric Cycle of the p97 Nanomachine Sam Tonddast-Navaei and George Stan* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221, United States * S Supporting Information ABSTRACT: 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. ■ INTRODUCTION Members of the AAA+ (ATPases associated with diverse cellular activities) superfamily 1,2 are ubiquitous proteins found in prokaryotic cells as well as in multicellular eukaryotes. They participate in a broad range of cellular functions, including DNA replication, membrane fusion, and protein degradation, 3 which renders them essential for cell survival. Most AAA+ proteins effect protein remodeling by promoting protein folding, unfolding and translocation, and assembly and disassembly of complexes. The distinguishing building block of this class is a highly conserved nucleotide binding domain, with ≃200−250 amino acids, known as AAA domain. Subunits containing one (type I) or two (type II) AAA domains assemble into toroidal structures with narrow central channels. p97 (also called valosin-containing protein or Cdc48) is an eukaryotic type II member of the AAA+ superfamily found abundantly in mammals and yeasts. 4−8 As part of the protein quality control system, it has a critical role in substrate protein (SP) unfolding within the ubiquitin-dependent degradation pathway. 4,9 Each p97 subunit includes two AAA domains, D1 and D2, and the N domain, which is covalently linked to D1 and is involved in interaction with cofactors. The functional structure of p97 is a homohexamer with two stacked rings that comprise the D1 and D2 domains (Figure 1a). Distinct roles of the two rings are indicated by asymmetric structure and catalytic activity. 10−13 The D1 ring encloses a narrow central pore and it is attributed heat-induced activity in ATP hydrolysis, 5 whereas the D2 ring includes a large cavity and it has the major ATPase activity, under physiological conditions, coupled to its greater conformational flexibility. 10,14,15 The two ring compartments are separated by a constriction with diameter ≃4 Å formed by the six His317 side chains. Experimental studies indicating unfolding activity of each of the p97 rings, 16,17 proteolytic activity of the Cdc48·20S complex, 18 and unfoldase action of an archaeal homologue of p97 19 suggest that p97 threads the SP through the His317 gate. Irrespective of the possible substrate transfer between ring compartments, p97 has been shown to interact with SPs in an ATP-dependent manner. 17,20 A set of amino acids lining the pore in the D2 ring, Trp551, Phe552, Arg586, and Arg599 (Figure 1b), has been identified as critical for this interaction. 17 The two bulky amino acids, Trp551 and Phe552, are part of loops possessing the Gly-aromatic-hydrophobic-Gly motif Received: April 23, 2013 Published: September 5, 2013 Article pubs.acs.org/JACS © 2013 American Chemical Society 14627 dx.doi.org/10.1021/ja404051b | J. Am. Chem. Soc. 2013, 135, 14627−14636