An empirical energy landscape reveals mechanism of proteasome in polypeptide translocation

The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits (Erzberger and Berger, 2006; Puchades et al., 2020). How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is unknown. In this study, we developed a novel approach for delineating the nucleotide-dependent free-energy landscape (FEL) of the proteasome's heterohexameric ATPase complex based on complementary structural and kinetic measurements. We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties. The FEL model predictions are consistent with a wide range of experimental observations in this and previous studies and suggested novel mechanistic features of the proteasomal ATPases. We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding status. ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex. eLife digest In cells, many biological processes are carried out by large complexes made up of different proteins. These macromolecules act like miniature machines, flexing and moving their various parts to perform their cellular roles. One such complex is the 26S proteasome, which is responsible for recycling other proteins in the cell. The proteasome consists of approximately 31 subunits, including a ring of six ATPase enzymes that provide the complex with the energy it needs to mechanically unfold proteins. To understand how the proteasome and other large complexes work, researchers need to be able to monitor how their structure changes over time. These dynamics are challenging to probe directly with experiments, but can be assessed using computer simulations which track the movement of individual molecules and atoms. However, currently available computer systems do not have enough power to simulate the dynamics of large protein assemblies, like the 26S proteasome: for example, it would take longer than a thousand years to model how each atom in the complex moves over a timescale in which a biological change would happen (roughly 100ms). Here, Fang, Hon et al. have developed a new approach to simulate the structural dynamics of the proteasome's ring of ATPase enzymes. Different known structures of the proteasome were used to identify the range of possible movements and shapes the complex can make. Fang, Hon et al. then used this data to calculate the energy level of each structure - also known as the 'free energy landscape' - and the rate of transition between them. This made it possible to simulate how the different ATPase enzymes move within the ring under a wide range of conditions. The simulated ATPase movements predicted how the proteasome machine would behave during various tasks, including degrading other proteins. Fan, Hon et al. carefully examined these predictions and found that they were consistent with experimental observations, validating their new simulation method. This work demonstrates the feasibility of simulating the actions of a large protein complex based on its free energy landscape. The results offer important insights into the functional mechanics of the 26S proteasome and related protein machines. Further work may help to simplify this process so the approach can be used to investigate the dynamics of other protein assemblies.