Howard Flack Crystallographic Lectures Series 2021/5

The mitochondrial Lon protease homolog (LonP1) hexamer regulates mitochondrial health by digesting proteins from the mitochondrial matrix that are damaged or must be removed. Here, we describe nine different, nucleotide dependent conformational states that we determined by cryo-EM. We show that LonP1 has an additional, previously unidentified Thr-type proteolytic center. Although resolutions locally extended to 3.6 Å for the best ordered states, the N-terminal domains were poorly ordered in all conformations. These N-domains formed an assembly with threefold symmetry and the orientation of its threefold axis depended on the conformational state. For most states we could infer the nucleotide occupancy. Our data indicate how LonP1 recognizes, binds, translocates and digests substrate protein. We propose that translocation requires a sixfold rotational binding change mechanism with threefold rocking motions of the N-domain assembly. The rocking motions of the N-domain assembly, together with its flexibility, may assist thermal unfolding of the substrate protein. Our data suggest that ATP hydrolysis prevents translocation reversal, and we propose LonP1 functions as a Brownian ratchet.