Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
The role of central pore residues of p97/VCP on substrate unfolding and translocation: A computational model
The p97/VCP nanomachine, a double ring member of the AAA+ superfamily, is involved in substrate protein unfolding within the proteasomal degradation pathway (1). Currently, it is unclear how p97/VCP interacts with its substrate. Revealing the underlying mechanism of p97 could lead to better understanding of its bacterial homologues ClpA and ClpB.
P97/VCP has a homo-hexameric structure that encloses a central pore. Within each subunit, there are two nucleotide binding domains, D1 and D2, and the N domain, which is connected to D1 and is known to interact with p97/VCP’s adaptors. ATP hydrolysis leads to large scale conformational changes in D2 domain, which affects the topology of its pore (2,3).
Conserved loops at the entrance of D2 pore are suggested to enable substrate propagation through the pore via ATP-driven paddling motion of Trp551 and Phe552 residues. Two other essential residues inside the D2 pore, Arg586 and Arg599, contribute to the p97 function (4). We propose that the substrate, which enters through the D1 pore, binds to the Arg599 sites on the D2 cavity lining. Repetitive ATP-driven cycles of p97 mediate the complete translocation of the substrate protein into the D2 pore via the paddling motion of the D2 loops (centered onto Trp551 and Phe552).
To test this hypothesis, we perform implicit solvent simulations of the SsrA-SsrA peptide threading through p97/VCP. Our results confirm the role of Arg599 as binding sites. These simulations reveal that these Arginines interact with the substrate primarily via hydrogen-bonds formed with the peptide backbone, indicating a non-specific interaction type.
Using the results from implicit solvent simulations, we develop a coarse-grained model that extends our simulations to biologically relevant timescales. We investigate the unfolding mechanism of a four helix bundle protein fused with the SsrA peptide coupled to ATP-driven conformational changes in the D2 domain of p97 (fig. 1). Our simulations show that complete unfolding and translocation is due to the collaboration between Arginine residues and the critical residues at the paddling D2 loop, such that substrate is held by the Arginine residues of adjacent subunits and the force exerted by the D2 loops pull the substrate through the central pore of p97.
Fig. 1 A snapshot of unfolding and translocation of the four helix bundle (purple) and the SsrA peptide (orange) by the p97/VCP nanomachine (N-D1 yellow, D2 green). To show the location of the D2 loops (blue) and the Arginines (brown), the two front subunits are not presented in the figure.
This research has been supported by a grant from the American Heart Association and by the National Science Foundation CAREER grant to G. S. and a University Research Council fellowship at the University of Cincinnati to M.J.
Department of Chemistry,
University of Cincinnati,
Cincinnati OH 45221