Structure and the Mode of Activity of Lon Proteases from Diverse Organisms.

ABSTRACT

Lon proteases, members of the AAA+ superfamily of enzymes, are key components of the protein quality control system in bacterial cells, as well as in the mitochondria and other specialized organelles of higher organisms. These enzymes have been subject of extensive biochemical and structural investigations, resulting in 72 crystal and solution structures, including structures of the individual domains, multi-domain constructs, and full-length proteins. However, interpretation of the latter structures still leaves some questions unanswered. Based on their amino acid sequence and details of their structure, Lon proteases can be divided into at least three subfamilies, designated as LonA, LonB, and LonC. Protomers of all Lons are single-chain polypeptides and contain two functional domains, ATPase and protease. The LonA enzymes additionally include a large N-terminal region, and different Lons may also include non-conserved inserts in the principal domains. These ATP-dependent proteases function as homohexamers, in which unfolded substrates are translocated to a large central chamber where they undergo proteolysis by a processive mechanism. X-ray crystal structures provided high-resolution models which verified that Lons are hydrolases with the rare Ser-Lys catalytic dyad. Full-length LonA enzymes have been investigated by cryo-electron microscopy (cryo-EM), providing description of the functional enzyme at different stages of the catalytic cycle, indicating extensive flexibility of their N-terminal domains, and revealing insights into the substrate translocation mechanism. Structural studies of Lon proteases provide an interesting case for symbiosis of X-ray crystallography and cryo-EM, currently the two principal techniques for determination of macromolecular structures.