Lon protease degrades stable substrates slowly but with enhanced processivity, redefining the model of a strong and robust AAA+ protease.

Abstract

AAA+ proteases function in all domains of life using an ATP-dependent unfoldase/polypeptide-translocase motor. Lon is a highly conserved AAA+ protease especially important for degrading oxidatively-damaged proteins. To address if Lon is a weak vs. robust unfoldase, as its substrates are often poorly folded, and elucidate whether its mechanism of translocation is similar to the previously-characterized AAA+ proteases, ClpXP and ClpAP, which have a distinct subunit assembly/architecture, we used a modified single-bead optical trapping assay. We find that Lon: (i) unfolds stable proteins using substantially longer pre-unfolding dwell times compared to ClpXP/ClpAP but (ii) has a higher probability of fully degrading a multi-domain substrate than the other enzymes. (iii) Although a slower translocase, Lon's fundamental step size is shared by ClpXP/ClpAP and its step-size distribution mirrors that of ClpAP. (iv) Finally, both ClpXP and Lon hydrolyze ∼1 ATP per translocation step. Thus, the mechanism of the ATP hydrolysis-driven ‘power stroke’ appears largely conserved between three distinct AAA+ proteases, regardless of their speed, processivity, or whether the ATPase and peptidase modules are physically linked (Lon) or assembled from distinct polypeptides (ClpXP and ClpAP). Lon therefore likely uses a common set of operating principles but is ‘geared differently’ than other AAA+ proteases.