Crystal structure of Lon protease: molecular architecture of gated entry to a sequestered degradation chamber

Lon proteases are distributed in all kingdoms of life and are required for survival of cells under stress. Lon is a tandem fusion of an AAA+ molecular chaperone and a protease with a serine‐lysine catalytic dyad. We report the 2.0‐Å resolution crystal structure of Thermococcus onnurineus NA1 Lon ( Ton Lon). The structure is a three‐tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight‐ and weak‐binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP‐driven protein unfolding and translocation. The bowl‐shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions. The prototypical ATP‐dependent protease Lon, like many other AAA+ ATPases, has proven notoriously resilient to crystallographic analysis. The first high‐resolution structure of the intact hexameric enzyme now unveils its closed degradation chamber and its gating.