Mechanism of Transient Binding and Release of Substrate Protein during the Allosteric Cycle of the p97 Nanomachine

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

Unfolding and translocation pathway of the <i>α</i>/<i>β</i> protein by allosteric ClpY.

<p>(A) The <i>α</i>/<i>β</i> model protein (magenta) fused at the C-terminus with the SsrA degradation tag (yellow) binds to the central channel loops (blue) of ClpY (green). The dimensions of ClpY and pore size are indicated. For clarity, two ClpY subunits are not shown and two are shown in gray. (B) The central channel loops engage the substrate protein. Iterative pulling results in unfolding of the substrate to <i>Q</i>_<i>N</i> ≃ 0.7. (C) Following several allosteric cycles, in the absence of the ClpQ peptidase co-factor, the substrate is partially translocated (<i>Q</i>_<i>N</i> ≃ 0.5). (D) The harmonic restraints in the distal region (<math><mrow><mi>z</mi><mo>-</mo><msub><mi>z</mi><mo>¯</mo><mrow><mi>l</mi><mi>o</mi><mi>o</mi><mi>p</mi><mi>s</mi></mrow></msub><mo>></mo><mn>14</mn><mo>.</mo><mn>5</mn><mo>Å</mo></mrow></math>) are added to account for the ClpQ assistance. The allosteric motions of ClpYQ result in complete translocation and refolding of the tagged substrate protein. Molecular images in this paper are created using Visual Molecular Dynamics [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004675#pcbi.1004675.ref058" target="_blank">58</a>].</p

Interactions of SP variants with ClpY central channel loops.

<p>Probability density distribution of the interaction energy <i>E</i> between the central central loops and (A) the SsrA degradation tag, (B) <i>β</i>4 strand and (C) the <i>β</i>3 strand. The curves shown represent results obtained for Wild Type (black), Mut1 (red) and Mut2 (green) sequences.</p

Coarse-Grained Simulations of Topology-Dependent Mechanisms of Protein Unfolding and Translocation Mediated by ClpY ATPase Nanomachines

<div><p>Clp ATPases are powerful ring shaped nanomachines which participate in the degradation pathway of the protein quality control system, coupling the energy from ATP hydrolysis to threading substrate proteins (SP) through their narrow central pore. Repetitive cycles of sequential intra-ring ATP hydrolysis events induce axial excursions of diaphragm-forming central pore loops that effect the application of mechanical forces onto SPs to promote unfolding and translocation. We perform Langevin dynamics simulations of a coarse-grained model of the ClpY ATPase-SP system to elucidate the molecular details of unfolding and translocation of an <i>α</i>/<i>β</i> model protein. We contrast this mechanism with our previous studies which used an all-<i>α</i> SP. We find conserved aspects of unfolding and translocation mechanisms by allosteric ClpY, including unfolding initiated at the tagged C-terminus and translocation via a power stroke mechanism. Topology-specific aspects include the time scales, the rate limiting steps in the degradation pathway, the effect of force directionality, and the translocase efficacy. Mechanisms of ClpY-assisted unfolding and translocation are distinct from those resulting from non-allosteric mechanical pulling. Bulk unfolding simulations, which mimic Atomic Force Microscopy-type pulling, reveal multiple unfolding pathways initiated at the C-terminus, N-terminus, or simultaneously from both termini. In a non-allosteric ClpY ATPase pore, mechanical pulling with constant velocity yields larger effective forces for SP unfolding, while pulling with constant force results in simultaneous unfolding and translocation.</p></div

Unfolding and translocation by mechanical pulling with a constant force through a non-allosteric ClpY pore.

<p>Probability density map of the fraction of native contacts and radius of gyration for simulations of mechanical pulling through a non-allosteric pore with constant force <i>F</i> = 125 pN.</p

Structural details of <i>α</i>/<i>β</i> proteins.

<p>(A.) Model protein utilized in this study. (B.) Protein L. The <i>β</i>-strands are shown in green, <i>α</i>-helix in blue, and loop regions in magenta. The C-terminus is indicated by a red sphere and the N-terminus by a blue sphere. (C.) BLN-model sequence (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004675#sec012" target="_blank">Methods</a>) of the <i>α</i>/<i>β</i> model SP and variants with contiguous stretches of non-attractive amino acids (red).</p

Translocase action of the allosteric ClpY pore.

<p>(A) Contour length of the polypeptide translocated through the pore as a function of time. After 50 <i>τ</i> harmonic restraints are applied in the distal region to mimic the interaction between the SP and ClpQ. (B) Probability density distribution of the end-to-end extension of the translocated segment.</p

Unfoldase and translocase activity of allosteric ClpY.

<p>(A) Probability density maps of the fraction of native contacts <i>Q</i>_<i>N</i> and the radius of gyration <i>R</i>_<i>g</i> for trajectories which result in translocation without assistance from ClpQ. (Inset) Probability density map of the ClpYQ combined action. (B) Map for ClpY trajectories which do not result in translocation. (C) Probability density distribution of <i>Q</i>_<i>N</i> after 50 <i>τ</i> for trajectories which result in translocation (black) and trajectories which do not result in translocation (red). (D) Reaction kinetics for unfolding and translocation.</p