The molecular basis of proteolytic inactivation in the human LONP1 protease via long-timescale molecular dynamics.

Abstract

The ATP-dependent protease LONP1 plays a central role in protecting mitochondrial health by degrading misfolded proteins. Dysregulation of LONP1 has been implicated in numerous disease pathways, including neurodegenerative disorders and cancer. Several conformations of LONP1 have been resolved by cryo electron-microscopy, revealing distinct operational modes. However, the mechanism by which LONP1 is activated and uses ATP hydrolysis to power substrate translocation and degradation has remained unclear. In order to elucidate this mechanism, we modelled the full hexameric motor protein, consisting of the ATPase and protease domains, and performed more than 100µs of molecular dynamics simulations using Anton. The results of our simulations suggest a stochastic mechanism of proteolysis. In the absence of substrate, we observe random inactivation of individual proteolytic sites mediated by the formation of a helix and a hydrogen bond, which occlude the site both sterically and chemically. These results suggest both that activation is dependent on the presence of bound substrate, and that individual subunits may be regulated independently. Additionally, we observe nucleotide flips within the ATPase domain, resulting in distinct poses that are stable for several microseconds. Our simulations show that the transitions between these poses allosterically induce conformational changes at the interface of the ATPase and protease domains. These findings provide crucial insight into the mechanism of LONP1 proteolytic activation and regulation, which will ultimately aid in the development of novel therapeutics to protect against mitochondrial dysfunction.