Fig. 3. Lon proteolytic active site forms cleft for substrates in Lon^ENZ and is autoinhibited in Lon^OFF.

(A) An axial view of the chamber of the sixfold symmetric protease ring from the substrate-bound Lon^ENZ structure is shown as a molecular surface with subunits are colored as in Fig. 1, with the six catalytic serine-containing loops (673 to 677) emphasized using a more saturated color. These loops organize into a ring-like assembly, generating a series of substrate-binding grooves. (B) A cutaway side view of the substrate-bound structure shown as a molecular surface. A close-up view of the cryo-EM density of the proteolytic active site is shown to the right as a gray mesh, with the atomic model showing the catalytic dyad (magenta) and serine-containing loop (purple stick representation). The serine-containing loop is likely stabilized in this extended conformation, in part, by hydrogen bonding between D676 (pink) and the peptide backbone of a nearby loop (light purple). (C) An axial view of the chamber of the protease ring from the Lon^OFF structure is shown and colored as in (A). The serine-containing loops are no longer interacting because of the separation of the protease domains in this conformation. (D) A cutaway side view of the substrate-free structure showing the location of the protease active sites in the open, exposed proteolytic chamber. A close-up view of the cryo-EM density of the proteolytic active site is shown as in (B), showing that in Lon^OFF, the catalytic serine-containing loop adopts a 3₁₀ helix that sterically occludes the proteolytic active site. D676 and K722 are within hydrogen-bonding distance, further limiting cleavage by the catalytic dyad. (E) A five–amino acid polyalanine peptide (orange) was modeled into the substrate-binding groove of Lon^ENZ (represented using a transparent space-filling representation of the atomic model), based on the position of bortezomib covalently bound to S679 (PDB: 4YPM) (57). This demonstrates how an unfolded peptide substrate putatively docks into the active site for proteolytic cleavage by Lon protease. (F) The rearrangement of the serine-containing loop during the transition from Lon^OFF (dark gray/purple) to Lon^ENZ (light gray/purple). This rearrangement is also shown in movie S2.