The crystal structure of Lon Protease: Molecular Architecture of Gated Entry to A Sequestered Degradation Chamber

Abstract

The ATP-dependent Lon protease has orthologs distributed in all kingdoms of life and is required for survival when cells are subjected to various stress conditions. Lon is a tandem fusion of a molecular chaperone belonging to the AAA+ family and a protease with a serine-lysine catalytic dyad. Here, we report the 2.0 Å resolution crystal structure of the Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three-tiered hexagonal cylinder with a sequestered internal proteolytic chamber accessible through a restricted channel through the AAA+ domains. Conserved axial loops together with an insertion domain containing the membrane anchor comprise a domain positioned on the apical surface of the AAA+ ring that serves as a gate to regulate substrate access to the internal unfolding and degradation chambers. Alternating AAA+ domains in the hexameric ring exist in tight- and weak-binding nucleotide states, displaying different domain orientations and intersubunit contacts, reflective of the ATP hydrolysis-coupled motions driving protein unfolding and translocation. The bowl-shaped proteolytic chamber is contiguous with the chamber formed by the AAA+ domains, and consequently internalized proteins can directly access the proteolytic sites with no further gating restrictions. The structure suggests a model by which Lon can degrade unfolded proteins and small folded proteins or prot