p97 is a protein complex of the AAA+ family. Although functions of p97 are well understood, the mechanism by which p97 performs its unfolding activities remains unclear. In this work, we present a novel way of applying normal mode analysis to study this six-fold symmetric molecular machine. By selecting normal modes that are axial symmetric and give the largest movements at D1 or D2 pore residues, we are able to predict the functional motions of p97, which are then validated by experimentally observed conformational changes. Our results shed light and provide new understandings on several key steps of the p97 functional process that were previously unclear or controversial, and thus are able to reconcile multiple previous findings. Specifically, our results reveal that (i) a venous valve-like mechanism is used at D2 pore to ensure a one-way exit-only traffic of substrates; (ii) D1 pore remains shut during the functional process; (iii) the “swing-up” motion of the N domain is closely coupled with the vertical motion of the D1 pore along the pore axis; (iv) because of the shut D1 pore and the one-way traffic at D2 pore, it is highly likely that substrates enter the chamber through the gaps at the D1/D2 interface. The limited chamber volume inside p97 suggests that a substrate may be pulling out from D2 while at the same time being pulling in at the interface; (v) lastly, p97 uses a series of actions that alternate between twisting and pulling to remove the substrate. Proteins 2016; 84:1823–1835.
All Science Journal Classification (ASJC) codes
- Structural Biology
- Molecular Biology